Nucleic acid molecules targeting the mitf gene and uses thereof

Abstract

Provided is a double-stranded nucleic acid molecule, in particular a small activating nucleic acid molecule, which can be a nucleic acid molecule targeting the MITF gene, comprising at least a first oligonucleotide strand. Also provided are compositions or formulations comprising the small activating nucleic acid molecule and uses thereof. The small activating nucleic acid molecule or the composition or formulation comprising the small activating nucleic acid molecule can be used to activate or upregulate the expression of the MITF gene in a cell, to treat a disease or condition associated with insufficient or reduced expression of the MITF protein.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid technology, specifically relating to a double-stranded nucleic acid molecule associated with gene activation, such as a small activating nucleic acid molecule, particularly a nucleic acid molecule targeting the MITF gene, and also relating to the application of the small activating nucleic acid molecule in activating / upregulating the microphthalmia-associated transcription factor (MITF) in gene transcription. This invention generally provides compounds, pharmaceutical compositions, and methods of use thereof; more specifically, this invention will benefit from its application in the treatment of diseases that increase MITF gene expression, such as vitiligo. Background Technology

[0002] Vitiligo is an acquired hypopigmentation disorder characterized by a reduction in melanocytes in the epidermis and hair follicles, resulting in irregular white patches. Statistics show that vitiligo affects approximately 0.5%-2% of the global population, with no significant gender difference (Picardo et al., 2015). Based on clinical characteristics, it can be divided into common vitiligo and segmental vitiligo. Common vitiligo is associated with further melanocyte damage due to autoimmune regulation or oxidative stress, while segmental vitiligo is related to genetic factors (Gauthier, Cario Andre, and Taieb 2003; Dell'anna and Picardo 2006).

[0003] Vitiligo is associated with factors such as family history, autoimmune regulation, oxidative stress, and functional melanocyte damage. The occurrence of vitiligo shows a clear familial clustering tendency, with a positive family history rate of approximately 6.25%–40% among vitiligo patients. The risk of developing the disease in first-degree relatives is 18 times higher than in the general population, and patients with a positive family history usually develop the disease earlier (Sehgaland Srivastava 2007). Vitiligo patients have elevated levels of cytokines (IL-6, TNF-α, IL-1β, IL-8) produced due to the immune response. In vitro experiments have shown that a large number of pro-inflammatory factors inhibit melanin synthesis and the expression of transcription factors related to melanin synthesis (Kotobuki et al., 2012). Anti-melanocyte antibodies can be detected in the serum of 50%–93% of vitiligo patients. These antibodies can lead to a reduction or disappearance of melanocytes through complement activation by immune complexes and / or antibody-dependent cell-mediated cytotoxicity (Kemp et al., 2007). Dell'Anna's laboratory proposed that the imbalance of oxidative stress in vitiligo patients may originate from abnormal cardiolipin in epidermal cells, which can lead to a decrease in the activity of the mitochondrial electron transport chain, induce excessive production of reactive oxygen species (ROS), and thus lead to melanocyte death (Dell'Anna et al., 2007).

[0004] The loss of melanocytes is a key factor in the pathogenesis of vitiligo. When melanocytes are damaged by physical or chemical means, they release antigens, stimulating the body to produce anti-melanocyte antibodies, leading to the inactivation of a large number of melanocytes and exacerbating epidermal depigmentation. Experiments have shown that melanin in skin lesions is damaged or lost when it crosses the epidermis, possibly due to defects in the adhesion of melanocytes to the basement membrane and keratinocytes (Kumar, Parsad, and Kanwar 2011). Melanocyte dysfunction is mainly manifested as a decrease in the number of melanocytes, disordered melanocyte adhesion and migration, excessive melanocyte apoptosis, and abnormal melanocyte development and differentiation.

[0005] The microphthalmia transcription factor encoded by the MITF gene is a transcription factor with a basic-helix-loop-helix-leucine zipper (bHLH-Zip) structure that binds to DNA in a dimer form. It plays a crucial role in the development of melanocytes, retinal pigment epithelial cells, osteoclasts, and mast cells. MITF, along with related factors transcription factor EB (TFEB), TFE3, and TFEC, constitutes the MiT family (Hemesath et al., 1994). MITF is the only factor in the MiT family that plays a vital role in normal melanocyte development (Levy, Khaled, and Fisher 2006). MITF is not only an essential regulator of melanocyte development, proliferation, and survival but also plays a crucial role in regulating the expression of related enzymes and melanosome proteins. The promoters of three major pigmentation-related enzymes—tyrosinase, TYRP1, and DCT—all contain a universal binding site for MITF (TCATGTG), making them target genes for MITF transcriptional regulation (Bentley, Eisen, and Goding 1994).

[0006] MITF mutations have been found to cause a variety of human diseases, including Waardenburg syndrome type 2A (WS2A), Tietz syndrome, and COMMAD syndrome, all of which present with severe hearing loss and pigmentation disorders. The first two are autosomal dominant.

[0007] While various treatments exist for vitiligo, a complete cure remains elusive. Simply improving appearance can be achieved by concealing the white patches with cosmetics. Other treatments include corticosteroids, ultraviolet (UV) irradiation, and surgery. UV-B irradiation can blur the borders of white patches, but phototherapy carries a risk of cancer. Early studies by Kwinter et al. using topical application of highly potent corticosteroids to treat 70 children with vitiligo showed an efficacy rate of 64% (Kwinter et al., 2007), but the recurrence rate was high. Clinically, psoralen-based drugs are commonly used as photosensitizers in combination with long-wave UV light for psoralen photochemotherapy (PUVA) to improve melanin synthesis and inhibit apoptosis in vitiligo lesions (Iannella et al., 2016). Afamelanotide is the only drug approved by the U.S. Food and Drug Administration (FDA) for preclinical trials, but it requires combination trials with narrow-band UVB phototherapy (NB-UVB). Because narrowband ultraviolet-B phototherapy (NB-UVB) is required to activate melanocorticoid receptor 1 (MCIR), afamelatin can bind to MCIR and produce melanin (Lim et al., 2015; Grimes et al., 2013). However, the specific duration of NB-UVB action and the dosage of the drug require further experimental research. Surgical treatments such as autologous scar removal epidermal transplantation, autologous micro-transplantation, autologous melanocyte transplantation, mixed epidermal transplantation, and micropigmentation also have good efficacy, but these procedures have strict contraindications and limited treatment options.

[0008] While these treatments each have some effectiveness, they are prone to relapse, have significant side effects, and the effective dosage of the drugs still needs to be further determined, which greatly limits clinical treatment. Summary of the Invention

[0009] To address one or more of the aforementioned problems, this invention provides a small activating RNA (saRNA) based on the RNA activation process, which treats diseases or conditions caused by MITF protein deficiency or insufficiency or by MITF monoallelic mutations, such as vitiligo, Waardenburg syndrome type 2A, or Titzer syndrome, by activating / upregulating MITF gene expression.

[0010] One objective of this invention is to provide small activating nucleic acid molecules based on RNA activation processes that increase the expression of MITF protein by activating / upregulating MITF gene transcription, or small activating nucleic acid molecules for the preparation of medicaments for treating diseases or conditions associated with MITF protein deficiency or insufficiency.

[0011] Another objective of the present invention is to provide compositions or formulations comprising small activating nucleic acid molecules.

[0012] Another objective of the present invention is to provide the use of small activating nucleic acid molecules or compositions or formulations containing them in the preparation of medicaments for activating / upregulating the expression of the MITF gene in cells.

[0013] Another objective of this invention is to provide a method for activating / upregulating the expression of the MITF gene in cells.

[0014] Another objective of this invention is to provide a method for activating / upregulating the expression of the MITF gene in cells, which can be used to treat diseases caused by MITF monoallelic mutations, such as Waldenberg syndrome type 2A and Tize syndrome.

[0015] Another objective of the present invention is to provide the use of small activating nucleic acid molecules or compositions or formulations containing them in the preparation of treatments for diseases or conditions associated with MITF protein deficiency or insufficiency, such as vitiligo.

[0016] Another objective of the present invention is to provide isolated small activating nucleic acid molecular target sites for the MITF gene, wherein the target sites comprise any sequence of 16-35 consecutive nucleotides selected from any one of the sequences of SEQ ID NO:299-SEQ ID NO:305 or sequences having at least 75%, for example at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% homology with the sequence composed of the aforementioned any consecutive 16-35 nucleotides.

[0017] In one aspect of the invention, a small activating nucleic acid molecule, such as a small activating RNA (saRNA), is provided, said small activating nucleic acid molecule comprising at least a first oligonucleotide chain having at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in length in the human MITF gene promoter region. Further, the human MITF gene promoter region is preferably a region extending 500 nucleotides upstream of the transcription start site (TSS) of the MITF gene (SEQ ID NO:1), wherein the first oligonucleotide chain has at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in length in the region extending 500 nucleotides upstream of the transcription start site (TSS) of the MITF gene (SEQ ID NO:1).

[0018] In one aspect of the invention, a small activating nucleic acid molecule, such as a small activating RNA (saRNA), is provided that activates or upregulates the expression of the MITF gene in cells. One strand of the small activating nucleic acid molecule has at least 75% homology or complementarity with any nucleic acid sequence of 16-35 nucleotides in length in the promoter region of the MITF gene, wherein the promoter region refers to 500 nucleotides (the sequence of which is SEQ ID NO:1) upstream of the transcription start site, thereby activating or upregulating the expression of the MITF gene. Specifically, one strand of the small activating nucleic acid molecule includes regions selected from the transcription start site in the MITF gene promoter: -500 to -408 (region H1, SEQ ID NO:299), -403 to -351 (region H2, SEQ ID NO:300), -342 to -262 (region H3, SEQ ID NO:301), -181 to -140 (region H4, SEQ ID NO:302), -250 to -193 (region W1, SEQ ID NO:303), -123 to -89 (region W2, SEQ ID NO:304), and -62 to -36 (region W3, SEQ ID NO:304). The 16-35 consecutive nucleotides in (SEQ ID NO:305) have at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% homology or complementarity with the nucleic acid sequence, with hotspot and sub-hotspot regions as shown in Table 5. More specifically, one strand of the small activating nucleic acid molecule of the present invention has at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity with any nucleotide sequence selected from SEQ ID NO:8-104. In one specific embodiment, one strand of the small activating nucleic acid molecule of the present invention comprises a nucleic acid sequence having at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity with any nucleotide sequence selected from SEQ ID NO:8-104. In another embodiment, one strand of the small activating nucleic acid molecule of the present invention comprises a nucleic acid sequence having at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity with any nucleotide sequence selected from SEQ ID NO: 8-104. In yet another embodiment, one strand of the small activating nucleic acid molecule of the present invention is a nucleic acid sequence having at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity with any nucleotide sequence selected from SEQ ID NO: 8-104.

[0019] The small activating nucleic acid molecule of the present invention includes a double-stranded small activating nucleic acid molecule, such as a small activating RNA (saRNA), targeting the promoter region of the MITF gene. This molecule comprises a first oligonucleotide chain and a second oligonucleotide chain. The first oligonucleotide chain is coupled to regions in the MITF gene promoter at distances from the transcription start site: -500 to -408 Å (region H1, SEQ ID NO: 299), -403 to -351 Å (region H2, SEQ ID NO: 300), -342 to -262 Å (region H3, SEQ ID NO: 301), -181 to -140 Å (region H4, SEQ ID NO: 302), -250 to -193 Å (region W1, SEQ ID NO: 303), -123 to -89 Å (region W2, SEQ ID NO: 304), and -62 to -36 Å (region W3, SEQ ID NO: 304). The 16-35 consecutive nucleotides in NO:305 have at least 75%, for example at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity, and the first oligonucleotide chain and the second oligonucleotide chain can form a double-stranded nucleic acid structure through complementarity, which can activate the expression of the MITF gene in the cell.

[0020] The first and second oligonucleotide chains of the small activating nucleic acid molecule of the present invention can exist on two different nucleic acid chains or on the same nucleic acid chain. When the first and second oligonucleotide chains are located on two separate chains, at least one chain of the small activating nucleic acid molecule can have overhangs (or dangling ends) at the 5' and / or 3' ends, for example, 0-6 nucleotide overhangs at the 3' end, such as 0, 1, 2, 3, 4, 5, or 6 nucleotide overhangs. Preferably, both chains of the small activating nucleic acid molecule of the present invention have overhangs; more preferably, both chains of the small activating nucleic acid molecule can have 0-6 nucleotide overhangs at the 3' end, for example, 0, 1, 2, 3, 4, 5, or 6 nucleotide overhangs; most preferably, 2 or 3 nucleotide overhangs. Preferably, the nucleotide type of the overhang can be dT (thymine deoxynucleotide, or T). Preferably, the overhangs at the 5' and / or 3' ends are dTdT or dTdTdT.

[0021] The small activating nucleic acid molecule of the present invention may also include a small activating nucleic acid molecule capable of forming a double-stranded hairpin structure, such as a single-stranded small activating RNA molecule. In one embodiment, the small activating nucleic acid molecule of the present invention includes a single-stranded small activating RNA molecule targeting the promoter region of the MITF gene, wherein the single-stranded small activating nucleic acid molecule can form a double-stranded hairpin structure. Preferably, when the first oligonucleotide chain and the second oligonucleotide chain are present on the same nucleic acid chain, the small activating nucleic acid molecule of the present invention can be a hairpin-type single-stranded nucleic acid molecule, wherein the first oligonucleotide chain and the second oligonucleotide chain have complementary regions capable of forming a double-stranded nucleic acid structure, which can promote the expression of the MITF gene in cells through, for example, an RNA activation mechanism.

[0022] In the aforementioned small activating nucleic acid molecules, the lengths of the first and second oligonucleotide chains can be 16-35 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0023] In one embodiment, the first oligonucleotide chain of the small activating nucleic acid molecule of the present invention has at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity, homology, or complementarity with any nucleotide sequence selected from SEQ ID NO: 8-104; or the second oligonucleotide chain of the small activating nucleic acid molecule has at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity, homology, or complementarity with any nucleotide sequence selected from SEQ ID NO: 8-104. In one embodiment, the first oligonucleotide chain of the small activating nucleic acid molecule of the present invention comprises a nucleic acid sequence having at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity, homology, or complementarity with any nucleotide sequence selected from SEQ ID NO:8-104, or is composed of ... Any nucleotide sequence in NO:8-104 comprises at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of identical, homologous, or complementary nucleic acid sequences. In a specific embodiment, the first oligonucleotide chain of the small activating nucleic acid molecule of the present invention may include any nucleotide sequence selected from SEQ ID NO:105-201, and / or its second oligonucleotide chain may include any nucleotide sequence selected from SEQ ID NO:202-298. In one embodiment, the small activating nucleic acid molecule described herein may be synthetic, in vitro transcribed, or vector-expressed.

[0024] All nucleotides in the small activating nucleic acid molecule described herein can be natural, unmodified nucleotides, or may include at least one modification. In one embodiment, the modification in the small activating nucleic acid molecule described herein may include a chemical modification, such as at least one nucleotide having a chemical modification. The chemical modifications used in this invention may include or be selected from one or more of the following modifications, or any combination thereof:

[0025] (1) Modification of the phosphodiester bonds of nucleotides in the nucleotide sequence of the small activated nucleic acid molecule;

[0026] (2) Modification of the 2'-OH of the ribose in the nucleotide sequence of the small activating nucleic acid molecule;

[0027] (3) Modification of the bases in the nucleotide sequence of the small activated nucleic acid molecule;

[0028] (4) At least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.

[0029] The chemical modifications are well known to those skilled in the art. The modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including but not limited to thiophosphate modification and boronyl phosphate modification. Both modifications can stabilize the saRNA structure and maintain high specificity and high affinity of base pairing.

[0030] Ribose modification refers to the modification of the 2'-OH group in the pentose of nucleotides, that is, the introduction of certain substituents at the hydroxyl position of the ribose. For example, including but not limited to 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, 2'-deoxy modification, etc.

[0031] Base modification refers to the modification of the bases of nucleotides, such as, but not limited to, 5′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

[0032] These modifications can increase the bioavailability of small activating nucleic acid molecules, improve their affinity for target sequences, and enhance their resistance to nuclease hydrolysis within cells.

[0033] In addition, to facilitate the entry of small activating nucleic acid molecules into cells, lipophilic groups such as cholesterol can be introduced at the ends of the first and / or second oligonucleotide chains of the small activating nucleic acid molecules, based on the above modifications. This allows them to interact with gene promoter regions in the cell nucleus through the cell membrane and nuclear membrane, which are composed of lipid bilayers.

[0034] The small activating nucleic acid molecule provided by the present invention can effectively activate or upregulate the expression of the MITF gene in cells after contact with or introduction into cells, preferably upregulating the expression by at least 30%.

[0035] Another aspect of the invention relates to nucleic acids encoding the small activating nucleic acid molecules described herein. In one embodiment, the nucleic acid may be a DNA molecule. In one embodiment, the nucleic acid is an expression vector containing a fragment encoding the small activating nucleic acid molecules described herein, which, when introduced into a cell, can express the small activating nucleic acid molecules described herein.

[0036] In another aspect of the invention, a cell comprising the small activating nucleic acid molecule described above or a nucleic acid encoding the small activating nucleic acid molecule described herein is provided. In one embodiment, the small activating nucleic acid molecule of the present invention may be a double-stranded small activating nucleic acid molecule targeting the promoter region of the MITF gene, such as a double-stranded small activating RNA (saRNA) molecule, which includes a first oligonucleotide chain and a second oligonucleotide chain. In another embodiment, the small activating nucleic acid molecule of the present invention may be a single-stranded small activating nucleic acid molecule targeting the promoter region of the MITF gene, such as a double-stranded small activating RNA (saRNA) molecule.

[0037] Another aspect of the invention provides compositions (e.g., pharmaceutical compositions) comprising the small activating nucleic acid molecule described herein or a nucleic acid encoding the small activating nucleic acid molecule described herein and optionally, a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier may comprise or be selected from liposomes, polymers, or peptides.

[0038] In another aspect of the invention, an formulation is provided comprising: the small activating nucleic acid molecule of the invention, a nucleic acid encoding the small activating nucleic acid molecule of the invention, a cell comprising the small activating nucleic acid molecule of the invention or a nucleic acid encoding the small activating nucleic acid molecule of the invention, or a composition comprising the small activating nucleic acid molecule of the invention.

[0039] In another aspect of the invention, a kit is provided comprising: the small activating nucleic acid molecule of the present invention, a nucleic acid encoding the small activating nucleic acid molecule described herein, a cell containing the small activating nucleic acid molecule of the present invention or a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition containing the small activating nucleic acid molecule of the present invention.

[0040] Another aspect of the present invention relates to the use of the small activating nucleic acid molecule described herein, nucleic acid encoding the small activating nucleic acid molecule of the present invention, cell comprising the small activating nucleic acid molecule of the present invention or nucleic acid encoding the small activating nucleic acid molecule of the present invention, or composition comprising the small activating nucleic acid molecule of the present invention in the preparation of a medicament or formulation for activating / upregulating the expression of the MITF gene in cells.

[0041] Another aspect of the present invention relates to a method for activating / upregulating the expression of the MITF gene in cells, the method comprising administering to the cells a small activating nucleic acid molecule of the present invention, a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition or formulation comprising the small activating nucleic acid molecule of the present invention. In one embodiment, the method for activating / upregulating the expression of the MITF gene in cells comprises administering to the cells a small activating nucleic acid molecule of the present invention, a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition comprising the small activating nucleic acid molecule of the present invention. The cells include mammalian cells, such as cells derived from the human body, such as human melanocytes (HEM) and human keratinocytes (NHEK), which may be isolated or present in a mammalian body, such as the human body.

[0042] The small activating nucleic acid molecule of the present invention can be directly introduced into cells, or it can be generated within cells after the nucleic acid sequence encoding the small activating nucleic acid molecule of the present invention is introduced into the cells; the cells are preferably mammalian cells, more preferably human cells. The cells can be in vitro, such as cell lines or cell strains, or they can exist in a mammalian body, such as a human body. The human body can be a patient with a disease or condition associated with insufficient or reduced expression of MITF protein. The small activating nucleic acid molecule of the present invention can be administered in sufficient quantities to treat diseases or conditions associated with a deficiency of MITF protein or insufficient or reduced expression of MITF protein. Specifically, the diseases or conditions associated with a deficiency of MITF protein or insufficient or reduced expression of MITF protein may include, for example, vitiligo.

[0043] In another aspect, the present invention provides an isolated small activating nucleic acid molecule action site for the MITF gene, the site having an arbitrary sequence of 16-35 consecutive nucleotides on the promoter region (SEQ ID NO:1) of the MITF gene. Preferably, the action site comprises or is selected from any sequence of 16-35 consecutive nucleotides on any one of SEQ ID NO:299-305. Specifically, the action site may include or be selected from any nucleotide sequence of SEQ ID NO:8-104.

[0044] Another aspect of the present invention relates to a method for treating a disease or condition in an individual associated with insufficient or reduced expression of the MITF protein, comprising administering to the individual a therapeutically effective amount of the small activating nucleic acid molecule of the present invention, a nucleic acid encoding the small activating nucleic acid molecule of the present invention, a cell containing the small activating nucleic acid molecule of the present invention or a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition containing the small activating nucleic acid molecule of the present invention. In one embodiment, the method of the present invention for treating a disease or condition in an individual associated with insufficient or reduced expression of the MITF protein comprises administering to the individual a therapeutically effective amount of the small activating nucleic acid molecule of the present invention, a nucleic acid encoding the small activating nucleic acid molecule of the present invention, a cell containing the small activating nucleic acid molecule of the present invention or a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition containing the small activating nucleic acid molecule of the present invention and a therapeutically effective amount of other agents, said other agents including, for example, small molecule compounds, antibodies, peptides, proteins, etc. The individual may be a mammal, including, for example, a human. In one embodiment, a disease or condition associated with insufficient or reduced expression of the MITF protein may include, for example, vitiligo. Based on clinical characteristics, it can be divided into vitiligo vulgaris and segmental vitiligo. Vitiligo vulgaris is associated with autoimmune regulation or oxidative stress further damaging melanocytes, while segmental vitiligo is associated with genetic factors. Vitiligo is related to factors such as family history, autoimmune regulation, oxidative stress, and damage to functional melanocytes.

[0045] Another aspect of the present invention relates to a method for treating vitiligo in an individual, comprising administering to the individual a therapeutically effective amount of the small activated nucleic acid molecule of the present invention, a nucleic acid encoding the small activated nucleic acid molecule of the present invention, a cell containing the small activated nucleic acid molecule of the present invention or a nucleic acid encoding the small activated nucleic acid molecule of the present invention, or a composition containing the small activated nucleic acid molecule of the present invention. In one embodiment, the method for treating vitiligo of the present invention comprises administering to an individual a therapeutically effective amount of the small activated nucleic acid molecule of the present invention, a nucleic acid encoding the small activated nucleic acid molecule of the present invention, a cell containing the small activated nucleic acid molecule of the present invention or a nucleic acid encoding the small activated nucleic acid molecule of the present invention, or a composition containing the small activated nucleic acid molecule of the present invention, and a therapeutically effective amount of other agents, said other agents including, for example, small molecule compounds, antibodies, polypeptides, proteins, etc. The individual may be a mammal, including, for example, a human. In the above method, vitiligo includes common vitiligo and segmental vitiligo, etc. In the above method, the individual includes a mammal, for example, a human.

[0046] Another aspect of the present invention relates to the use of the small activating nucleic acid molecule described herein, a nucleic acid encoding the small activating nucleic acid molecule of the present invention, a cell containing the small activating nucleic acid molecule of the present invention or a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition containing the small activating nucleic acid molecule of the present invention in the preparation of a medicament for treating diseases or conditions associated with insufficient or reduced expression of the MITF protein. The individual may be a mammal, such as a human. In one embodiment, the disease associated with insufficient or reduced expression of the MITF protein may include, for example, vitiligo. Preferably, the disease or condition associated with insufficient or reduced expression of the MITF protein is vitiligo; more preferably, the disease or condition associated with insufficient or reduced expression of the MITF protein includes vitiligo vulgaris and segmental vitiligo.

[0047] In one embodiment, the small activating nucleic acid molecule of the present invention, the nucleic acid encoding the small activating nucleic acid molecule of the present invention, the cell containing the small activating nucleic acid molecule of the present invention or the nucleic acid encoding the small activating nucleic acid molecule of the present invention, or the composition containing the small activating nucleic acid molecule of the present invention can be used to prepare a medicament for treating diseases such as vitiligo associated with insufficient or reduced expression of MITF protein. In one embodiment, the diseases associated with insufficient or reduced expression of MITF protein may include, for example, vitiligo. Preferably, the disease or condition associated with insufficient or reduced expression of MITF protein is vitiligo; more preferably, the disease or condition associated with insufficient or reduced expression of MITF protein includes vitiligo vulgaris and segmental vitiligo.

[0048] Another aspect of the present invention relates to the use of the small activating nucleic acid molecule described herein, the nucleic acid encoding the small activating nucleic acid molecule of the present invention, the cell containing the small activating nucleic acid molecule of the present invention or the nucleic acid encoding the small activating nucleic acid molecule of the present invention, or the composition or preparation containing the small activating nucleic acid molecule of the present invention in the preparation of a medicament or combination of medicaments for treating vitiligo. Preferably, vitiligo includes common vitiligo and segmental vitiligo, etc.

[0049] In one embodiment, the use of the small activating nucleic acid molecule of the present invention, a nucleic acid encoding the small activating nucleic acid molecule of the present invention, a cell containing the small activating nucleic acid molecule of the present invention or a nucleic acid encoding the small activating nucleic acid molecule of the present invention, or a composition or formulation containing the small activating nucleic acid molecule of the present invention in the preparation of a medicament or combination of medicaments for treating diseases associated with insufficient or reduced expression of the MITF protein is provided.

[0050] Compared with the prior art, the present invention has the following advantages in one or more aspects:

[0051] The present invention provides a small activating nucleic acid molecule, such as a small activating RNA (saRNA), that can activate / upregulate MITF gene expression. This molecule can persistently activate the MITF gene, thus efficiently and specifically upregulating or restoring the expression of the MITF gene and protein while having low toxicity. It can be used to treat diseases or conditions related to insufficient or reduced MITF protein expression, such as vitiligo, or to prepare drugs or preparations for treating diseases or conditions related to insufficient or reduced MITF protein expression, such as Waardenburg syndrome type 2A and Titzer syndrome. Attached Figure Description

[0052] Figure 1 This study describes the altered human MITF mRNA expression mediated by saRNAs. 239 saRNAs targeting the human MITF promoter were transfected into HEM cells at a final concentration of 25 nM. MITF mRNA expression was analyzed by one-step RT-qPCR 72 hours after transfection. The figure shows the changes in MITF mRNA expression (log2) relative to the control treatment (Mock) from highest to lowest. The ordinate values ​​represent the mean ± SD of the two replicates.

[0053] Figure 2 The diagram shows hotspot regions of saRNAs on the human MITF promoter. 239 saRNAs targeting the human MITF promoter were transfected into HEM cells at a final concentration of 25 nM for 72 hours. MITF mRNA expression was analyzed by one-step RT-qPCR after transfection. The figure shows changes in MITF expression relative to the control treatment (Mock), ordered from -500 to -0 according to the target site location of saRNAs on the MITF promoter. Solid black dots represent functional saRNAs, hollow white dots represent non-functional saRNAs, and dashed lines indicate hotspot regions (H1–H4) and secondary hotspot regions (W1–W3) where functional saRNAs aggregate. The ordinate values ​​represent the mean ± SD of the two replicates.

[0054] Figure 3 To validate high-throughput screening results using a two-step RT-qPCR method. Figure 3 The saRNA shown was transfected into HEM cells at a final concentration of 25 nM for 72 hours. After transfection, RNA was extracted using the Qiagen RNeasy kit, reverse transcribed, and then amplified by qPCR using an ABI 7500 rapid real-time PCR system. The HPRT1 gene was amplified simultaneously as an internal control. The figure shows the relative expression value of MITF to mRNA after single saRNA treatment. Mock, dsCon2, and RAG4-618i represent blank transfection, irrelevant sequence double-stranded RNA transfection, and small interfering RNA control transfection, respectively. The ordinate values ​​represent the mean ± SD of two replicate treatments.

[0055] Figure 4To validate high-throughput screening results using a two-step RT-qPCR method. Figure 4 The saRNA shown was transfected into NHEK cells at a final concentration of 25 nM for 72 hours. After transfection, RNA was extracted using the Qiagen RNeasy kit, reverse transcribed, and then amplified by qPCR using an ABI 7500 rapid real-time PCR system. The HPRT1 gene was amplified simultaneously as an internal control. The figure shows the relative expression value of MITF to mRNA after single saRNA treatment. Mock, dsCon2, and RAG4-618i represent blank transfection, irrelevant sequence double-stranded RNA transfection, and small interfering RNA control transfection, respectively. The ordinate values ​​represent the mean ± SD of two replicate treatments.

[0056] Figure 5 saRNA was used to promote MITF protein expression in human NHEK cells. Figure 5 The saRNA was transfected into NHEK cells at a final concentration of 25 nM for 5 days. Cells were collected, and total cellular protein was extracted. MITF protein expression levels were detected by Western blot, with microtubules (α / β-tubulin) used as an internal control. The figure shows the relative MITF protein expression values ​​in cells treated with a single saRNA. Mock, dsCon2, and RAG4-618i represent blank transfection, irrelevant sequence double-stranded RNA transfection, and small interfering RNA control transfection, respectively.

[0057] Figure 6 saRNA was used to promote MITF protein expression in HEM cells. Figure 6 The saRNA was transfected into HEM cells at a final concentration of 25 nM for 72 hours. Cells were collected, and total cellular protein was extracted. MITF protein expression levels were detected by Western blot, with microtubules (α / β-tubulin) used as an internal control. The figure shows the relative MITF protein expression values ​​in cells treated with a single saRNA. Mock, dsCon2, and RAG4-618i represent blank transfection, irrelevant sequence double-stranded RNA transfection, and small interfering RNA control transfection, respectively. Detailed Implementation

[0058] In this invention, the relevant terms are defined as follows:

[0059] As used herein, the term "complementary" refers to the ability of two oligonucleotide chains to form base pairs with each other. Base pairs are typically formed between nucleotides in antiparallel oligonucleotide chains via hydrogen bonds. Complementary oligonucleotide chains can pair in a Watson-Crick manner (e.g., AT, AU, CG) or in any other manner that allows for the formation of a duplex (e.g., Hoogsteen or anti-Hoogsteen base pairing).

[0060] Complementarity includes both perfect and imperfect complementarity. Perfect complementarity, or 100% complementarity, means that each nucleotide from the first oligonucleotide chain in the double-stranded region of a double-stranded oligonucleotide molecule can form hydrogen bonds with the corresponding nucleotides in the second oligonucleotide chain without any "mismatches." Imperfect complementarity means that not all nucleotide units of the two chains can form hydrogen bonds with each other. For example, for two oligonucleotide chains with a double-stranded region of 20 nucleotides in length, if only two base pairs on each chain can form hydrogen bonds with each other, the oligonucleotide chains exhibit 10% complementarity. In the same example, if 18 base pairs on each chain can form hydrogen bonds with each other, the oligonucleotide chains exhibit 90% complementarity. Substantial complementarity refers to complementarity of at least approximately 75%, approximately 79%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, or approximately 100%.

[0061] As used herein, the term "oligonucleotide" refers to a polymer of nucleotides, including but not limited to single-stranded or double-stranded molecules of DNA, RNA, or DNA / RNA hybrids, comprising oligonucleotide chains with regularly and irregularly alternating deoxyribosyl and ribosyl moieties, as well as modifications of these types of oligonucleotides and naturally occurring or non-natural backbones. The oligonucleotides described in this invention for activating target gene transcription are small activating nucleic acid molecules.

[0062] As used herein, the terms "oligonucleotide chain" and "oligonucleotide sequence" are used interchangeably and refer to a collective term for short-chain nucleotides of 35 bases or less (including nucleotide chains containing deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as mixed oligonucleotide chains formed by one or more deoxynucleotides and one or more ribonucleotides). In this invention, the length of the oligonucleotide chain can be any length from 16 to 35 nucleotides.

[0063] As used in this article, the term "first oligonucleotide chain" can refer to either the sense chain or the antisense chain. The sense chain of a small activating RNA refers to the nucleic acid chain in the small activating RNA duplex that contains the coding chain that is identical to the promoter DNA sequence of the target gene. The antisense chain refers to the nucleic acid chain in the small activating RNA duplex that is complementary to the sense chain.

[0064] As used in this article, the term "second oligonucleotide chain" can also be either the sense chain or the antisense chain. When the first oligonucleotide chain is the sense chain, the second oligonucleotide chain is the antisense chain, and when the first oligonucleotide chain is the antisense chain, the second oligonucleotide chain is the sense chain.

[0065] As used herein, the term "gene" refers to the complete nucleotide sequence required to encode a polypeptide chain or transcribe a functional RNA. A "gene" can be endogenous or wholly or partially recombinant to the host cell (e.g., due to the introduction of exogenous oligonucleotides and coding sequences encoding a promoter or the introduction of a heterologous promoter with a neighboring endogenous coding sequence into the host cell). For example, the term "gene" includes nucleic acid sequences that can consist of exons and introns. A protein-coding sequence is, for example, a sequence contained within an exon in an open reading frame between a start codon and a stop codon. As used herein, "gene" can refer to, for example, gene regulatory sequences such as promoters, enhancers, and all other sequences known in the art that control the transcription, expression, or activity of another gene, whether or not the other gene contains coding or non-coding sequences. In one instance, for example, "gene" can be used to describe a functional nucleic acid containing regulatory sequences such as promoters or enhancers. The expression of a recombinant gene can be controlled by one or more heterologous regulatory sequences.

[0066] As used herein, the term "target gene" can refer to a naturally occurring nucleic acid sequence, transgenic gene, viral or bacterial sequence, chromosomal or extrachromosomal sequence, and / or transient or stable transfection or incorporation into cells and / or their chromatin. Target genes can be protein-coding genes or non-protein-coding genes (e.g., microRNA genes, long non-coding RNA genes). Target genes typically contain a promoter sequence. Designing small activating nucleic acid molecules with identity (homology) with the promoter sequence can achieve positive regulation of the target gene, manifested as upregulation of target gene expression. "Target gene promoter sequence" refers to the non-coding sequence of the target gene. In this invention, the term "complementary to the target gene promoter sequence" refers to the coding strand of that sequence, also known as the non-template strand, i.e., a nucleic acid sequence that is identical to the coding sequence of the gene. "Target site" or "target site sequence" refers to a sequence fragment in the target gene promoter sequence that is homologous or complementary to the positive or negative oligonucleotide chain of the small activating nucleic acid molecule.

[0067] As used herein, the terms "positive chain" and "positive nucleic acid chain" are used interchangeably. A positive oligonucleotide chain of a small activating nucleic acid molecule refers to the first oligonucleotide chain in the duplex of a small activating nucleic acid molecule that contains a coding strand that is identical to the promoter sequence of the target gene.

[0068] As used herein, the terms “antisense strand” and “antisense nucleic acid strand” are used interchangeably. The antisense oligonucleotide strand of a small activating nucleic acid molecule refers to the second oligonucleotide strand in the duplex of the small activating nucleic acid molecule that is complementary to the sense oligonucleotide strand.

[0069] As used herein, the term "coding strand" refers to the DNA strand of the target gene that cannot be transcribed, whose nucleotide sequence is identical to the sequence of the transcribed RNA (in which U replaces T in DNA). The coding strand of the target gene promoter double-stranded DNA sequence described in this invention refers to the promoter sequence located on the same DNA strand as the target gene's coding strand.

[0070] As used herein, the term "template strand" refers to the strand of the double-stranded DNA of the target gene that is complementary to the coding strand, and serves as a template for transcription into RNA. This strand is complementary to the transcribed RNA bases (AU, GC). During transcription, RNA polymerase binds to the template strand and moves along the 3'→5' direction, catalyzing RNA synthesis in the 5'→3' direction. In this invention, the template strand of the target gene promoter double-stranded DNA sequence refers to the promoter sequence located on the same DNA strand as the target gene DNA template strand.

[0071] As used herein, the term "promoter" refers to a sequence that regulates the transcription of protein- or RNA-coding nucleic acid sequences by means of their positional association. Typically, eukaryotic gene promoters contain 100–5,000 base pairs, although this length range is not intended to limit the use of the term "promoter" herein. While promoter sequences are generally located at the 5' end of protein- or RNA-coding sequences, they can also be found in exons and introns.

[0072] As used in this article, the term "transcription start site" refers to a nucleotide on the template strand of a gene that marks the initiation of transcription. Transcription start sites can appear on the template strand of the promoter region. A gene can have more than one transcription start site.

[0073] As used herein, the term "identity" or "homology" refers to the similarity between one oligonucleotide chain (sense or antisense strand) of a small activating RNA and the coding or template strand of a region of the promoter sequence of a target gene. In this document, "identity" or "homology" can be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%.

[0074] As used herein, the terms “protrusion,” “overhang,” and “dangling” are used interchangeably to refer to the non-base-paired nucleotide at the 5' or 3' end of an oligonucleotide chain, which is produced by the extension of one strand beyond the other within a double-stranded oligonucleotide. The single-stranded region extending beyond the 3' and / or 5' ends of the double-stranded oligonucleotide is called a protrusion.

[0075] As used herein, the terms “gene activation” or “activated gene” or “gene upregulation” or “upregulated gene” are used interchangeably to refer to an increase in the transcription, translation, expression, or activity of a nucleic acid, measured by measuring gene transcription levels, mRNA levels, protein levels, enzyme activity, methylation state, chromatin state or conformation, translation level, or their activity or state in a cell or biological system. These activities or states can be measured directly or indirectly. Furthermore, “gene activation,” “activated gene,” “gene upregulation,” and “upregulated gene” refer to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism by which this activation occurs, such as its regulatory role as a regulatory sequence, its transcription into RNA, its translation into protein, and the resulting increase in protein expression.

[0076] As used herein, the terms "small activating RNA," "saRNA," and "small activating nucleic acid molecule" are used interchangeably to refer to nucleic acid molecules capable of promoting gene expression. These molecules can consist of a first nucleic acid fragment (antisense nucleic acid chain, also called an antisense oligonucleotide chain) containing a nucleotide sequence that has sequence identity or homology with a non-coding nucleic acid sequence (e.g., promoter, enhancer, etc.) of a target gene, and a second nucleic acid fragment (sense nucleic acid chain, also called a sense chain or sense oligonucleotide chain) containing a nucleotide sequence complementary to the first nucleic acid fragment, wherein the first and second nucleic acid fragments form a double strand. Small activating nucleic acid molecules can also consist of synthetic or vector-expressed single-stranded RNA molecules capable of forming double-stranded hairpin structures, wherein the first region contains a nucleotide sequence that has sequence identity with the gene's promoter target sequence, and the second region contains a nucleotide sequence complementary to the first region. The length of the double-stranded region of a small activating nucleic acid molecule is typically about 10 to 50 base pairs, about 12 to 48 base pairs, about 14 to 46 base pairs, about 16 to 44 base pairs, about 18 to 42 base pairs, about 20 to 40 base pairs, about 22 to 38 base pairs, about 24 to 36 base pairs, about 26 to 34 base pairs, about 28 to 32 base pairs, and usually about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. Furthermore, the terms "saRNA," "small activating RNA," and "small activating nucleic acid molecule" also include nucleic acids other than the ribonucleotide moiety, including but not limited to modified nucleotides or similar substances.

[0077] As used in this article, the term "hot spot (H region)" refers to a gene promoter region of at least 25 bp in length, in which a cluster of functional small activating nucleic acid (SA) molecules is observed, meaning that at least 30% of SA molecules targeting these hot spots can induce target gene mRNA expression of 1.1-fold or higher. The term "warm spot (W region)" refers to a gene promoter region of at least 25 bp in length, in which a cluster of functional small activating SMILE molecules is observed, meaning that 8-30% of SA molecules targeting these hot spots can induce target gene mRNA expression of 1.1-fold or higher.

[0078] As used in this article, the term "synthesis" refers to the method of synthesizing oligonucleotides, including any method that can synthesize RNA, such as chemical synthesis, in vitro transcription, vector expression, etc.

[0079] This invention upregulates the expression of the MITF gene via RNA activation, thereby increasing the expression level of the MITF protein to treat related diseases, especially vitiligo. In this invention, the MITF gene is sometimes also referred to as the target gene.

[0080] The method for preparing small activating nucleic acid molecules provided by this invention includes sequence design and sequence synthesis.

[0081] The small activating nucleic acid molecule sequence of the present invention can be synthesized by chemical synthesis or by a biotechnology company specializing in nucleic acid synthesis.

[0082] Generally, chemical synthesis methods include the following four processes: (1) synthesis of oligonucleotides; (2) deprotection; (3) purification and separation; and (4) desalting and annealing.

[0083] For example, the specific steps of the chemical synthesis of saRNA described in this invention are as follows:

[0084] (1) Synthesis of oligonucleotides

[0085] On an automated DNA / RNA synthesizer (e.g., Applied Biosystems EXPEDITE 8909), the synthesis of 1 μmol of RNA is set, with the coupling time for each cycle set to 10-15 minutes. The starting material is a solid-phase 5'-O-p-dimethoxytriphenylmethyl-thymidine support. In the first cycle, one base is ligated onto the solid support. Then, in the nth cycle (19 ≥ n ≥ 2), one base is ligated onto the base ligated in the (n-1)th cycle. This cycle is repeated until the synthesis of the entire nucleic acid sequence is completed.

[0086] (2) Deprotection

[0087] Add the solid support containing saRNA to a test tube, and then add 1 mL of an ethanol / ammonia solution (volume ratio 1:3). Seal the tube and incubate it at 25-70°C for 2-30 hours. Filter the solution containing the saRNA solid support and collect the filtrate. Rinse the solid support twice with double-distilled water (1 mL each time) and collect the filtrates. Combine the eluents and dry them under vacuum for 1-12 hours. Then, add 1 mL of tetrabutylammonium fluoride in tetrahydrofuran solution (1M), incubate at room temperature for 4-12 hours, add 2 mL of n-butanol, and centrifuge at high speed to collect the precipitate to obtain the crude saRNA single-stranded product.

[0088] (3) Purification and separation

[0089] The crude saRNA product was dissolved in 2 mL of 1 mol / L triethylamine acetate solution and then separated by high performance liquid chromatography reversed-phase C18 column to obtain the purified saRNA single-stranded product.

[0090] (4) Desalting and annealing

[0091] Salt was removed by size exclusion gel filtration. The sense and antisense oligonucleotide single strands were mixed in 1-2 mL of buffer (10 mM Tris, pH 7.5-8.0, 50 mM NaCl) at the same molar ratio. The solution was heated to 95 °C and then slowly cooled to room temperature to obtain a solution containing saRNA.

[0092] This study found that introducing the above-mentioned saRNA into cells can effectively increase the expression of MITF mRNA and protein.

[0093] The present invention will be further illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0094] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0095] Example 1: Design and Synthesis of saRNA Targeting the Human MITF Gene Promoter

[0096] To screen for functional small activating RNAs capable of activating MITF gene expression, a 19 bp target site was selected starting 500 bp upstream of the TSS, using the 500 bp promoter sequence of MITF as a template. The target sequences were then filtered, retaining sequences based on the following criteria: 1) GC content between 40% and 65%; 2) absence of 5 or more consecutive identical nucleotides; 3) absence of more than 3 dinucleotide repeats; and 4) absence of more than 3 trinucleotide repeats. After filtering, 239 target sequences remained as candidates for further screening. Based on these candidate sequences, corresponding double-stranded small activating RNAs were chemically synthesized.

[0097] In this experiment, the double-stranded small activating RNA used had both sense and antisense strands of 21 nucleotides in length. The 19 nucleotides in the 5' region of the first ribonucleic acid strand (sense strand) of the double-stranded saRNA were 100% identical to the promoter target sequence, and its 3' end contained a TT sequence. The 19 nucleotides in the 5' region of the second ribonucleic acid strand were complementary to the first ribonucleic acid strand sequence, and its 3' end also contained a TT sequence. The two strands of the aforementioned double-stranded saRNA were mixed in equal molar amounts and annealed to form the double-stranded saRNA.

[0098] The human MITF promoter sequence is shown below, corresponding to positions 1 to 500 of SEQ ID NO:1 from 5' to 3' in the sequence listing:

[0099]

[0100]

[0101] Example 2: High-throughput screening of saRNAs targeting the human MITF promoter

[0102] (1) Cell culture and transfection

[0103] Human melanocytes (HEM) (purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd., BNCC350795) and normal human skin keratinocytes (NHEK) (purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd., BNCC340593) were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). Cells were cultured at 37°C with 5% CO2. HEM cells were seeded at 2000 cells per well in 96-well plates and transfected with small activating RNA at a concentration of 25 nM (unless otherwise specified) using RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Transfection time was 72 hours, with two replicates for each treatment.

[0104] (2) One-step RT-qPCR

[0105] After transfection, discard the culture medium, add 150 μl of PBS to each well for washing, discard the PBS, add 50 μl of cell lysis buffer to each well, and incubate at room temperature for 5 minutes. Take 1 μl of cell lysis buffer from each well and use the one-step TB Green™ PrimeScrip™ RT-PCR kit II (Takara, RR086A).

[0106] qPCR analysis was performed using an ABI 7500 Fast Real-time PCR system (Applied Biosystems). Each sample was amplified in triplicate. The PCR reaction conditions are shown in Table 1.

[0107] Table 1 PCR reaction preparation

[0108] 2× One-Step TB Green RT-PCR Buffer 4 2.5μl PrimeScript 1-Step Enzyme Mixture 2 0.2μl Forward and reverse primer mixture (5 μM) 0.4μl <![CDATA[RNase-free dH2O]]> 1.4μl Crude RNA lysate 0.5μl total 5μl

[0109] The reaction conditions were as follows: Stage 1 - Reverse transcription: 42℃ for 5 minutes; 95℃ for 10 seconds; Stage 2 - PCR: 95℃ for 5 seconds, 60℃ for 20 seconds, for 45 cycles. HPRT1 and TBP were used as internal reference genes. The PCR primers used for MITF, HPRT1, and TBP are shown in Table 2, with MITF amplified using the MITF F1 / R1 primer pair.

[0110] Table 2 Primer sequences for RT-qPCR analysis

[0111]

[0112] To calculate the expression value (Erel) of the MITF (target gene) of a certain saRNA transfected sample relative to the control treatment (Mock), the Ct values ​​of the target gene and the two internal reference genes are substituted into Formula 1.

[0113] E re l=2 (CtTm-CtTs) / ((2 (CtR1m-CtR1s) *2 (CtR2m-CtR2s) ) (1 / 2) ) (Formula 1)

[0114] Wherein, CtTm is the Ct value of the target gene from the control (Mock) sample, CtTs is the Ct value of the target gene from the saRNA-treated sample, CtR1m is the Ct value of internal reference gene 1 from the Mock-treated sample, CtR1s is the Ct value of internal reference gene 1 from the saRNA-treated sample, CtR2m is the Ct value of internal reference gene 2 from the control sample, and CtR2s is the Ct value of internal reference gene 2 from the saRNA-treated sample.

[0115] (3) Screening of functional saRNA

[0116] To obtain saRNAs capable of activating MITF transcription, HEM cells were transfected with the aforementioned 239 saRNAs at a concentration of 25 nM. 72 hours after transfection, cells were lysed using the same method as described above, and one-step RT-qPCR analysis was performed to obtain the relative (compared to the control group) expression value of the MITF gene in each saRNA-treated sample. As shown in Table 3, 29 (12.1%) saRNAs showed high activation (≥1.5-fold), 68 (28.5%) saRNAs showed mild activation (≥1.1-fold), and 142 (59.4%) saRNAs did not significantly upregulate MITF expression. The maximum activation magnitude was 2.75-fold, and the maximum inhibition magnitude was 0.38-fold. These saRNAs with activating activity are referred to as functional saRNAs.

[0117] Table 3. Statistics of high-throughput screening results for human MITF saRNA

[0118]

[0119] like Figure 1As shown, the MITF expression changes of human MITF saRNAs are further ranked from highest to lowest. Table 4 shows the active saRNA sequences (functional saRNA sequences), active target sequences, and MITF mRNA expression changes (each active target sequence listed in Table 4 corresponds to SEQ NO: 8-104 in the sequence listing, each sense saRNA sequence corresponds to SEQ ID NO: 105-201 in the sequence listing, and each antisense saRNA sequence corresponds to SEQ ID NO: 202-298 in the sequence listing). Table 5 shows the different hotspot and secondary hotspot regions of the functional saRNAs and their related sequences (SEQ ID NO: 299-305).

[0120] Table 4. Functional saRNA sequences, their active target sequences, and the resulting changes in MITF mRNA expression.

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] Table 5. Hotspot and secondary hotspot regions and sequences of functional saRNAs.

[0127]

[0128] Example 3: saRNA promotes the expression of MITF mRNA in human melanocytes (HEM).

[0129] (1) Cell culture and transfection

[0130] Cell culture was performed as described in Example 2, with human melanocytes (HEM) cultured at 20 × 10⁶ cells per well. 4 Plate the RNA into a six-well plate and transfect small activating RNA at a final concentration of 25 nM. After 72 hours of transfection, use two replicates for each treatment.

[0131] (2) Two-step RT-qPCR

[0132] After transfection, the culture medium was discarded, and 500 μl of cell lysis buffer was added to each well and incubated at room temperature for 5 minutes. RNA was extracted using the Qiagen RNeasy kit, reverse transcribed, and then analyzed by qPCR on an ABI 7500 Fast Real-time PCR system (Applied Biosystems). Each sample was amplified in triplicate. The PCR reaction conditions are shown in Tables 6 and 7.

[0133] Table 6 RT reaction preparation

[0134]

[0135]

[0136]

[0137] Table 7 Preparation of RT-qPCR reactions

[0138] SYBR Premix Ex Taq II (2×) 5μl ROX Reference Dye II (50×) 0.2μl Forward and reverse primer mixture (5 μM) 0.8μl cDNA (RT product) 4μl total 10μl

[0139] The reaction conditions were: 95℃ for 30 seconds, 95℃ for 5 seconds, 60℃ for 30 seconds, for 40 cycles. The HPRT1 gene was also amplified as an internal control, with MITF amplified using the MITF F1 / R1 primer pair. The primers are shown in Table 2.

[0140] To calculate the expression value (Erel) of the MITF (target gene) of a certain saRNA transfected sample relative to the control treatment (Mock), the Ct values ​​of the target gene and one internal reference gene are substituted into Formula 2.

[0141] E rel =2 (CtTm-CtTs) / 2 (CtRm-CtRs) (Formula 2)

[0142] Wherein, CtTm is the Ct value of the target gene from the control (Mock) sample, CtTs is the Ct value of the target gene from the saRNA-treated sample, CtRm is the Ct value of the internal reference gene from the Mock-treated sample, and CtRs is the Ct value of the internal reference gene from the saRNA-treated sample.

[0143] Figure 3The figure shows the relative expression values ​​of MITF mRNA in cells after treatment with different saRNAs. Using the control group (Mock) as a blank transfection control, the relative mRNA expression value in the siRNA (RAG4-618i) group was knocked down by 81.0% compared to the control group, indicating that siRNA transfection as a small interfering RNA control was successful. Compared to the control group, the expression of MITF mRNA increased after treatment with different saRNAs, especially RAG4-175, RAG4-176, and RAG4-290, which upregulated MITF expression by 3.9-fold, 3.1-fold, and 6.1-fold, respectively. Other saRNAs (RAG4-284, RAG4-123, RAG4-396, RAG4-461, RAG4-490, RAG4-316, and RAG4-318) increased the relative expression of MITF mRNA by approximately 2-fold. This demonstrates that the activity of randomly selected functional saRNAs can be verified in HEM cells.

[0144] Example 4: saRNA promotes the expression of MITF mRNA in NHEK cells

[0145] Cell culture was performed as described in Example 2, using human skin keratinocytes (NHEK) at a density of 20 × 10⁶ cells per well. 4 Plate the RNA into six-well plates and transfect with small activating RNA at a final concentration of 25 nM. Leave for 72 hours after transfection, using two replicates for each treatment. Perform two-step RT-qPCR as described in Example 3.

[0146] like Figure 4 The figures show the relative mRNA expression values ​​of MITF in cells after treatment with different saRNAs. Using the control group (Mock) as a blank transfection control, the relative mRNA expression value of the siRNA (RAG4-618i) group decreased by 88.5% compared to the control group, indicating that siRNA transfection as a small interfering RNA control was successful. The activation effect was particularly significant after treatment with RAG4-290 compared to the control group, with the relative mRNA expression value of MITF increasing by 13.1 times. The relative mRNA expression values ​​of the RAG4-175, RAG4-176, and RAG4-316 groups were all higher than the control group, increasing by 5.7 times, 3.7 times, and 3.5 times, respectively, showing significant activation effects. The relative mRNA expression values ​​of the RAG4-284, RAG4-123, RAG4-396, RAG4-461, RAG4-490, and RAG4-318 groups were approximately twice that of the control group, also showing some activation effects. This demonstrates that the activity of randomly selected functional saRNAs can be verified in NHEK cells.

[0147] Example 5: saRNA promotes the expression of MITF protein in NHEK cells.

[0148] Cell culture was performed as described in Example 2, with human melanocytes (NHEK) cultured at 20 × 10⁶ cells per well. 4 Cells were plated into six-well plates and transfected with small activating RNA at a final concentration of 25 nM for 5 days. Lysis was performed using an appropriate amount of cell lysis buffer containing protease inhibitors (1×RIPA buffer, Cell Signaling Technology). Protein quantification was performed using the BCA method, followed by polyacrylamide gel electrophoresis and transfer to a 0.45 μm PVDF membrane. Primary antibodies used were rabbit monoclonal anti-MITF (Cell Signaling Technology, #12590) and α / β-tubulin antibody (Cell Signaling Technology, 2148s) for blot detection; secondary antibodies were anti-rabbit IgG and HRP-linked antibody (Cell Signaling Technology). Signal was detected by scanning the membrane using Image Lab (BIO-RAD, Chemistry Doctm MP Imaging System).

[0149] like Figure 5 The figure shows the relative expression levels of MITF protein in cells after treatment with different saRNAs. Compared with the control group, the relative expression level of MITF protein in the siRNA (RAG4-618i) group decreased by approximately 50%, indicating that siRNA transfection as a small interfering RNA control was successful. Compared with the control group, all saRNAs increased the expression level of MITF protein. Among them, RAG4-416, RAG4-490, and RAG4-318 upregulated MITF protein expression by more than 1.5-fold, with RAG4-318 showing the most significant activation effect, increasing MITF protein expression by 1.71-fold. This demonstrates that the activity of randomly selected functional saRNAs can be verified at the protein level.

[0150] Example 6: saRNA promotes the expression of MITF protein in human melanocytes (HEM).

[0151] Cell culture was performed as described in Example 2, with human melanocytes (HEM) cultured at 20 × 10⁶ cells per well. 4 The protein was plated into six-well plates and transfected with small activating RNA at a final concentration of 25 nM for 72 hours. Protein lysis and detection methods were as described in Example 5.

[0152] like Figure 6The figure shows the relative expression levels of MITF protein in cells after treatment with different saRNAs. Compared with the control group, all saRNAs increased the expression level of MITF protein, with the RAG4-396 and RAG4-490 groups showing a 1.4-fold increase. The MITF protein expression levels in the RAG4-175, RAG4-176, RAG4-290, RAG4-284, and RAG4-123 groups were all higher than the control group, with activation levels exceeding 1.5-fold, indicating a significant activation effect. This demonstrates that the activity of randomly selected functional saRNAs can be verified at the protein level.

[0153] Based on the above results, the applicant, through high-throughput screening of saRNAs targeting the human MITF gene promoter, discovered several human saRNAs that can significantly activate MITF gene expression. These saRNAs can upregulate intracellular MITF gene expression at both the mRNA and protein expression levels, and can be used for diseases or conditions caused by decreased or insufficient MITF protein expression, such as vitiligo, or for the preparation of methods or drugs to treat the aforementioned diseases or conditions.

[0154] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

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[0167] 12. Iannella, G., A. Greco, D. Didona, B. Didona, G. Granata, A. Manno, B. Pasquariello, and G. Magliulo. 2016. 'Vitiligo: Pathogenesis, clinical variants and treatment approaches', Autoimmun Rev, 15: 335-43.

[0168] 13. Lim, HW, PE Grimes, O. Agbai, I. Hamzavi, M. Henderson, M. Haddican, RV Linkner, and M. Lebwohl. 2015. 'Afamelanotide and narrowband UV-Bphototherapy for the treatment of vitiligo: a randomized multicenter trial', JAMA Dermatol, 151: 42-50.

[0169] 14. Grimes, PE, I. Hamzavi, M. Lebwohl, JPO Ortonne, and HW Lim. 2013. 'Theefficacy of afamelanotide and narrowband UV-B phototherapy for repigmentation of vitiligo', JAMA Dermatol, 149: 68-73. sequence list <110> Sino-US Ruikang Nucleic Acid Technology (Nantong) Research Institute Co., Ltd. <120> Nucleic acid molecules targeting the MITF gene and their applications <130> RAC202102 <141> 2021-05-06 <150> CN202010524139.1 <151> 2020-06-10 <160> 305 <170> SIPOSequenceListing 1.0 <210> 1 <211> 500 <212> DNA <213> Homo sapiens <400> 1 gcgaaggaaa gttcttcctc gttgttccaa tccgaggaca agctgatatg tcgcagcagc 60 ccagggaagc atgcgagctg ataggaagtc cttttatttt aagacaggct cgaatgctaa 120 aactttcttg tgccaaaacc cttgactatt ttatttttaa aataagcact tggcgtgccc 180 tcgcagatgt ctgagctgag aggtcggggc gatggtagaa gagcagtcag tgtccattct 240 tattcatatt aagtagccaa gtctgtaccc ttgaagcaag tggggagaga ggagggagag 300 gagctgctga cattgacaat gaatccaaac aggagttgca ctagcggtgt ccaccacgtt 360 gcctctcccc cgcctggcct tctgggagct gtagttttcg tgggagcggc tccccaggcg 420 agctgggaat gccccgcccg ggccgaacta cagatcccag gcggcgctcg gccgccagcc 480 cctcccgccc gggtgcgagt 500 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <400> 2 cgacagaaga aactggagca c 21 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 cccgtggatg gaataaggga aa 22 <210> 4 <211> twenty one <212> DNA <213> Artificial Sequence <400> 4 aaagatggtc aaggtcgcaa g 21 <210> 5 <211> twenty three <212> DNA <213> Artificial Sequence <400> 5 tagtcaaggg catatcctac aac 23 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 tgctcaccca ccaacaattt ag 22 <210> 7 <211> twenty two <212> DNA <213> Artificial Sequence <400> 7 tctgctctga ctttagcacc tg 22 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 gcgaaggaaa gttcttcct 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <400> 9 gaaggaaagt tcttcctcg 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 aaggaaagtt cttcctcgt 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 gaaagttctt cctcgttgt 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 agttcttcct cgttgttcc 19 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <400> 13 gttcttcctc gttgttcca 19 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <400> 14 ttcttcctcg ttgttccaa 19 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <400> 15 cttcctcgtt gttccaatc 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 tcctcgttgt tccaatccg 19 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <400> 17 ctcgttgttc caatccgag 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 tcgttgttcc aatccgagg 19 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <400> 19 cgttgttcca atccgagga 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <400> 20 ttgttccaat ccgaggaca 19 <210> twenty one <211> 19 <212> DNA <213> Artificial Sequence <400> twenty one tgttccaatc cgaggacaa 19 <210> twenty two <211> 19 <212> DNA <213> Artificial Sequence <400> twenty two gttccaatcc gaggacaag 19 <210> twenty three <211> 19 <212> DNA <213> Artificial Sequence <400> twenty three ttccaatccg aggacaagc 19 <210> twenty four <211> 19 <212> DNA <213> Artificial Sequence <400> twenty four tccaatccga ggacaagct 19 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <400> 25 ccaatccgag gacaagctg 19 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <400> 26 caatccgagg acaagctga 19 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <400> 27 aatccgagga caagctgat 19 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <400> 28 ccgaggacaa gctgatatg 19 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <400> 29 gaggacaagc tgatatgtc 19 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <400> 30 aggacaagct gatatgtcg 19 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <400> 31 gacaagctga tatgtcgca 19 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <400> 32 aagctgatat gtcgcagca 19 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <400> 33 agctgatatg tcgcagcag 19 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <400> 34 gctgatatgt cgcagcagc 19 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <400> 35 ctgatatgtc gcagcagcc 19 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <400> 36 gatatgtcgc agcagccca 19 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <400> 37 agcagcccag ggaagcatg 19 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <400> 38 gcatgcgagc tgataggaa 19 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <400> 39 catgcgagct gataggaag 19 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <400> 40 cgagctgata ggaagtcct 19 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <400> 41 gagctgatag gaagtcctt 19 <210> 42 <211> 19 <212> DNA <213> Artificial Sequence <400> 42 tttaagacag gctcgaatg 19 <210> 43 <211> 19 <212> DNA <213> Artificial Sequence <400> 43 ttaagacagg ctcgaatgc 19 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <400> 44 taagacaggc tcgaatgct 19 <210> 45 <211> 19 <212> DNA <213> Artificial Sequence <400> 45 caggctcgaa tgctaaaac 19 <210> 46 <211> 19 <212> DNA <213> Artificial Sequence <400> 46 aggctcgaat gctaaaact 19 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <400> 47 ggctcgaatg ctaaaactt 19 <210> 48 <211> 19 <212> DNA <213> Artificial Sequence <400> 48 gctaaaactt tcttgtgcc 19 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <400> 49 tcttgtgcca aaacccttg 19 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <400> 50 cttgtgccaa aacccttga 19 <210> 51 <211> 19 <212> DNA <213> Artificial Sequence <400> 51 tgtgccaaaa cccttgact 19 <210> 52 <211> 19 <212> DNA <213> Artificial Sequence <400> 52 gtgccaaaac ccttgacta 19 <210> 53 <211> 19 <212> DNA <213> Artificial Sequence <400> 53 gccaaaaccc ttgactatt 19 <210> 54 <211> 19 <212> DNA <213> Artificial Sequence <400> 54 aaaataagca cttggcgtg 19 <210> 55 <211> 19 <212> DNA <213> Artificial Sequence <400> 55 aaataagcac ttggcgtgc 19 <210> 56 <211> 19 <212> DNA <213> Artificial Sequence <400> 56 ccctcgcaga tgtctgagc 19 <210> 57 <211> 19 <212> DNA <213> Artificial Sequence <400> 57 cctcgcagat gtctgagct 19 <210> 58 <211> 19 <212> DNA <213> Artificial Sequence <400> 58 tcgcagatgt ctgagctga 19 <210> 59 <211> 19 <212> DNA <213> Artificial Sequence <400> 59 gcagatgtct gagctgaga 19 <210> 60 <211> 19 <212> DNA <213> Artificial Sequence <400> 60 cagatgtctg agctgagag 19 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <400> 61 agatgtctga gctgagagg 19 <210> 62 <211> 19 <212> DNA <213> Artificial Sequence <400> 62 gtcggggcga tggtagaag 19 <210> 63 <211> 19 <212> DNA <213> Artificial Sequence <400> 63 tcggggcgat ggtagaaga 19 <210> 64 <211> 19 <212> DNA <213> Artificial Sequence <400> 64 cggggcgatg gtagaagag 19 <210> 65 <211> 19 <212> DNA <213> Artificial Sequence <400> 65 ggggcgatgg tagaagagc 19 <210> 66 <211> 19 <212> DNA <213> Artificial Sequence <400> 66 ggcgatggta gaagagcag 19 <210> 67 <211> 19 <212> DNA <213> Artificial Sequence <400> 67 gcgatggtag aagagcagt 19 <210> 68 <211> 19 <212> DNA <213> Artificial Sequence <400> 68 gatggtagaa gagcagtca 19 <210> 69 <211> 19 <212> DNA <213> Artificial Sequence <400> 69 atggtagaag agcagtcag 19 <210> 70 <211> 19 <212> DNA <213> Artificial Sequence <400> 70 tggtagaaga gcagtcagt 19 <210> 71 <211> 19 <212> DNA <213> Artificial Sequence <400> 71 tagaagagca gtcagtgtc 19 <210> 72 <211> 19 <212> DNA <213> Artificial Sequence <400> 72 agaagagcag tcagtgtcc 19 <210> 73 <211> 19 <212> DNA <213> Artificial Sequence <400> 73 gaagagcagt cagtgtcca 19 <210> 74 <211> 19 <212> DNA <213> Artificial Sequence <400> 74 aagagcagtc agtgtccat 19 <210> 75 <211> 19 <212> DNA <213> Artificial Sequence <400> 75 agagcagtca gtgtccatt 19 <210> 76 <211> 19 <212> DNA <213> Artificial Sequence <400> 76 gagcagtcag tgtccattc 19 <210> 77 <211> 19 <212> DNA <213> Artificial Sequence <400> 77 tgaatccaaa caggagttg 19 <210> 78 <211> 19 <212> DNA <213> Artificial Sequence <400> 78 gaatccaaac aggagttgc 19 <210> 79 <211> 19 <212> DNA <213> Artificial Sequence <400> 79 aatccaaaca ggagttgca 19 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <400> 80 ccaaacagga gttgcacta 19 <210> 81 <211> 19 <212> DNA <213> Artificial Sequence <400> 81 caaacaggag ttgcactag 19 <210> 82 <211> 19 <212> DNA <213> Artificial Sequence <400> 82 aaacaggagt tgcactagc 19 <210> 83 <211> 19 <212> DNA <213> Artificial Sequence <400> 83 acaggagttg cactagcgg 19 <210> 84 <211> 19 <212> DNA <213> Artificial Sequence <400> 84 aggagttgca ctagcggtg 19 <210> 85 <211> 19 <212> DNA <213> Artificial Sequence <400> 85 gttgcactag cggtgtcca 19 <210> 86 <211> 19 <212> DNA <213> Artificial Sequence <400> 86 ttgcactagc ggtgtccac 19 <210> 87 <211> 19 <212> DNA <213> Artificial Sequence <400> 87 agcggtgtcc accacgttg 19 <210> 88 <211> 19 <212> DNA <213> Artificial Sequence <400> 88 agtagccaag tctgtaccc 19 <210> 89 <211> 19 <212> DNA <213> Artificial Sequence <400> 89 ctgtaccctt gaagcaagt 19 <210> 90 <211> 19 <212> DNA <213> Artificial Sequence <400> 90 gtacccttga agcaagtgg 19 <210> 91 <211> 19 <212> DNA <213> Artificial Sequence <400> 91 agaggaggga gaggagctg 19 <210> 92 <211> 19 <212> DNA <213> Artificial Sequence <400> 92 aggagggaga ggagctgct 19 <210> 93 <211> 19 <212> DNA <213> Artificial Sequence <400> 93 ccttctggga gctgtagtt 19 <210> 94 <211> 19 <212> DNA <213> Artificial Sequence <400> 94 ggagctgtag ttttcgtgg 19 <210> 95 <211> 19 <212> DNA <213> Artificial Sequence <400> 95 gagctgtagt tttcgtggg 19 <210> 96 <211> 19 <212> DNA <213> Artificial Sequence <400> 96 agctgtagtt ttcgtggga 19 <210> 97 <211> 19 <212> DNA <213> Artificial Sequence <400> 97 gctgtagttt tcgtgggag 19 <210> 98 <211> 19 <212> DNA <213> Artificial Sequence <400> 98 tgtagttttc gtgggagcg 19 <210> 99 <211> 19 <212> DNA <213> Artificial Sequence <400> 99 tagttttcgt gggagcggc 19 <210> 100 <211> 19 <212> DNA <213> Artificial Sequence <400> 100 gttttcgtgg gagcggctc 19 <210> 101 <211> 19 <212> DNA <213> Artificial Sequence <400> 101 cgggccgaac tacagatcc 19 <210> 102 <211> 19 <212> DNA <213> Artificial Sequence <400> 102 gggccgaact acagatccc 19 <210> 103 <211> 19 <212> DNA <213> Artificial Sequence <400> 103 aactacagat cccaggcgg 19 <210> 104 <211> 19 <212> DNA <213> Artificial Sequence <400> 104 actacagatc ccaggcggc 19 <210> 105 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 105 gcgaaggaaa guucuuccut t 21 <210> 106 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 106 gaaggaaagu ucuuccucgt t 21 <210> 107 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 107 aaggaaaguu cuuccucgut t 21 <210> 108 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 108 gaaaguucuu ccucguugut t 21 <210> 109 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 109 aguucuuccu cguuguucct t 21 <210> 110 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 110 guucuuccuc guuguuccat t 21 <210> 111 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 111 uucuuccucg uuguuccaat t 21 <210> 112 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 112 cuuccucguu guuccaauct t 21 <210> 113 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 113 uccucguugu uccaauccgt t 21 <210> 114 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 114 cucguuguuc caauccgagt t 21 <210> 115 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 115 ucguuguucc aauccgaggt t 21 <210> 116 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 116 cguuguucca auccgaggat t 21 <210> 117 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 117 uuguuccaau ccgaggacat t 21 <210> 118 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 118 uguuccaauc cgaggacaat t 21 <210> 119 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 119 guuccaaucc gaggacaagt t 21 <210> 120 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 120 uuccaauccg aggacaagct t 21 <210> 121 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 121 uccaauccga ggacaagcut t 21 <210> 122 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 122 ccaauccgag gacaagcugt t 21 <210> 123 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 123 caauccgagg acaagcugat t 21 <210> 124 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 124 aauccgagga caagcugaut t 21 <210> 125 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 125 ccgaggacaa gcugauaugt t 21 <210> 126 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 126 gaggacaagc ugauauguct t 21 <210> 127 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 127 aggacaagcu gauaugucgt t 21 <210> 128 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 128 gacaagcuga uaugucgcat t 21 <210> 129 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 129 aagcugauau gucgcagcat t 21 <210> 130 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 130 agcugauaug ucgcagcagt t 21 <210> 131 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 131 gcugauaugu cgcagcagct t 21 <210> 132 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 132 cugauauguc gcagcagcct t 21 <210> 133 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 133 gauaugucgc agcagcccat t 21 <210> 134 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 134 agcagcccag ggaagcaugt t 21 <210> 135 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 135 gcaugcgagc ugauaggaat t 21 <210> 136 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 136 caugcgagcu gauaggaagt t 21 <210> 137 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 137 cgagcugaua ggaaguccut t 21 <210> 138 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 138 gagcugauag gaaguccuut t 21 <210> 139 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 139 uuuaagacag gcucgaaugt t 21 <210> 140 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 140 uuaagacagg cucgaaugct t 21 <210> 141 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 141 uaagacaggc ucgaaugcut t 21 <210> 142 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 142 caggcucgaa ugcuaaaact t 21 <210> 143 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 143 aggcucgaau gcuaaaacut t 21 <210> 144 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 144 ggcucgaaug cuaaaacuut t 21 <210> 145 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 145 gcuaaaacuu ucuugugcct t 21 <210> 146 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 146 ucuugugcca aaacccuugt t 21 <210> 147 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 147 cuugugccaa aacccuugat t 21 <210> 148 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 148 ugugccaaaa cccuugacut t 21 <210> 149 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 149 gugccaaaac ccuugacuat t 21 <210> 150 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 150 gccaaaaccc uugacuauut t 21 <210> 151 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 151 aaaauaagca cuuggcgugt t 21 <210> 152 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 152 aaauaagcac uuggcgugct t 21 <210> 153 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 153 cccucgcaga ugucugagct t 21 <210> 154 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 154 ccucgcagau gucugagcut t 21 <210> 155 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 155 ucgcagaugu cugagcugat t 21 <210> 156 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 156 gcagaugucu gagcugagat t 21 <210> 157 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 157 cagaugucug agcugagagt t 21 <210> 158 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 158 agaugucuga gcugagaggt t 21 <210> 159 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 159 gucggggcga ugguagaagt t 21 <210> 160 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 160 ucggggcgau gguagaagat t 21 <210> 161 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 161 cggggcgaug guagaagagt t 21 <210> 162 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 162 ggggcgaugg uagaagagct t 21 <210> 163 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 163 ggcgauggua gaagagcagt t 21 <210> 164 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 164 gcgaugguag aagagcagut t 21 <210> 165 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 165 gaugguagaa gagcagucat t 21 <210> 166 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 166 augguagaag agcagucagt t 21 <210> 167 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 167 ugguagaaga gcagucagut t 21 <210> 168 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 168 uagaagagca gucaguguct t 21 <210> 169 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 169 agaagagcag ucagugucct t 21 <210> 170 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 170 gaagagcagu caguguccat t 21 <210> 171 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 171 aagagcaguc aguguccaut t 21 <210> 172 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 172 agagcaguca guguccauut t 21 <210> 173 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 173 gagcagucag uguccauuct t 21 <210> 174 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 174 ugaauccaaa caggaguugt t 21 <210> 175 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 175 gaauccaaac aggaguugct t 21 <210> 176 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 176 aauccaaaca ggaguugcat t 21 <210> 177 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 177 ccaaacagga guugcacuat t 21 <210> 178 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 178 caaacaggag uugcacuagt t 21 <210> 179 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 179 aaacaggagu ugcacuagct t 21 <210> 180 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 180 acaggaguug cacuagcggt t 21 <210> 181 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 181 aggaguugca cuagcggugt t 21 <210> 182 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 182 guugcacuag cgguguccat t 21 <210> 183 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 183 uugcacuagc gguguccact t 21 <210> 184 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 184 agcggugucc accacguugt t 21 <210> 185 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 185 aguagccaag ucuguaccct t 21 <210> 186 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 186 cuguacccuu gaagcaagut t 21 <210> 187 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 187 guacccuuga agcaaguggt t 21 <210> 188 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 188 agaggaggga gaggagcugt t 21 <210> 189 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 189 aggagggaga ggagcugcut t 21 <210> 190 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 190 ccuucuggga gcuguaguut t 21 <210> 191 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 191 ggagcuguag uuuucguggt t 21 <210> 192 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 192 gagcuguagu uuucgugggt t 21 <210> 193 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 193 agcuguaguu uucgugggat t 21 <210> 194 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 194 gcuguaguuu ucgugggagt t 21 <210> 195 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 195 uguaguuuuc gugggagcgt t 21 <210> 196 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 196 uaguuuucgu gggagcggct t 21 <210> 197 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 197 guuuucgugg gagcggcuct t 21 <210> 198 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 198 cgggccgaac uacagaucct t 21 <210> 199 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 199 gggccgaacu acagauccct t 21 <210> 200 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 200 aacuacagau cccaggcggt t 21 <210> 201 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 201 acuacagauc ccaggcggct t 21 <210> 202 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 202 aggaagaacu uuccuucgct t 21 <210> 203 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 203 cgaggaagaa cuuuccuuct t 21 <210> 204 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 204 acgaggaaga acuuuccuut t 21 <210> 205 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 205 acaacgagga agaacuuuct t 21 <210> 206 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 206 ggaacaacga ggaagaacut t 21 <210> 207 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 207 uggaacaacg aggaagaact t 21 <210> 208 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 208 uuggaacaac gaggaagaat t 21 <210> 209 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 209 gauuggaaca acgaggaagt t 21 <210> 210 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 210 cggauuggaa caacgaggat t 21 <210> 211 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 211 cucggauugg aacaacgagt t 21 <210> 212 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 212 ccucggauug gaacaacgat t 21 <210> 213 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 213 uccucggauu ggaacaacgt t 21 <210> 214 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 214 uguccucgga uuggaacaat t 21 <210> 215 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 215 uuguccucgg auuggaacat t 21 <210> 216 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 216 cuuguccucg gauuggaact t 21 <210> 217 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 217 gcuuguccuc ggauuggaat t 21 <210> 218 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 218 agcuuguccu cggauuggat t 21 <210> 219 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 219 cagcuugucc ucggauuggt t 21 <210> 220 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 220 ucagcuuguc cucggauugt t 21 <210> 221 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 221 aucagcuugu ccucggauut t 21 <210> 222 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 222 cauaucagcu uguccucggt t 21 <210> 223 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 223 gacauaucag cuuguccuct t 21 <210> 224 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 224 cgacauauca gcuuguccut t 21 <210> 225 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 225 ugcgacauau cagcuuguct t 21 <210> 226 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 226 ugcugcgaca uaucagcuut t 21 <210> 227 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 227 cugcugcgac auaucagcut t 21 <210> 228 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 228 gcugcugcga cauaucagct t 21 <210> 229 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 229 ggcugcugcg acauaucagt t 21 <210> 230 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 230 ugggcugcug cgacauauct t 21 <210> 231 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 231 caugcuuccc ugggcugcut t 21 <210> 232 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 232 uuccuaucag cucgcaugct t 21 <210> 233 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 233 cuuccuauca gcucgcaugt t 21 <210> 234 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 234 aggacuuccu aucagcucgt t 21 <210> 235 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 235 aaggacuucc uaucagcuct t 21 <210> 236 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 236 cauucgagcc ugucuuaaat t 21 <210> 237 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 237 gcauucgagc cugucuuaat t 21 <210> 238 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 238 agcauucgag ccugucuuat t 21 <210> 239 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 239 guuuuagcau ucgagccugt t 21 <210> 240 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 240 aguuuuagca uucgagccut t 21 <210> 241 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 241 aaguuuuagc auucgagcct t 21 <210> 242 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 242 ggcacaagaa aguuuuagct t 21 <210> 243 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 243 caaggguuuu ggcacaagat t 21 <210> 244 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 244 ucaaggguuu uggcacaagt t 21 <210> 245 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 245 agucaagggu uuuggcacat t 21 <210> 246 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 246 uagucaaggg uuuuggcact t 21 <210> 247 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 247 aauagucaag gguuuuggct t 21 <210> 248 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 248 cacgccaagu gcuuauuuut t 21 <210> 249 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 249 gcacgccaag ugcuuauuut t 21 <210> 250 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 250 gcucagacau cugcgagggt t 21 <210> 251 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 251 agcucagaca ucugcgaggt t 21 <210> 252 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 252 ucagcucaga caucugcgat t 21 <210> 253 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 253 ucucagcuca gacaucugct t 21 <210> 254 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 254 cucucagcuc agacaucugt t 21 <210> 255 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 255 ccucucagcu cagacaucut t 21 <210> 256 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 256 cuucuaccau cgccccgact t 21 <210> 257 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 257 ucuucuacca ucgccccgat t 21 <210> 258 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 258 cucuucuacc aucgccccgt t 21 <210> 259 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 259 gcucuucuac caucgcccct t 21 <210> 260 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 260 cugcucuucu accaucgcct t 21 <210> 261 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 261 acugcucuuc uaccaucgct t 21 <210> 262 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 262 ugacugcucu ucuaccauct t 21 <210> 263 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 263 cugacugcuc uucuaccaut t 21 <210> 264 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 264 acugacugcu cuucuaccat t 21 <210> 265 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 265 gacacugacu gcucuucuat t 21 <210> 266 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 266 ggacacugac ugcucuucut t 21 <210> 267 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 267 uggacacuga cugcucuuct t 21 <210> 268 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 268 auggacacug acugcucuut t 21 <210> 269 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 269 aauggacacu gacugcucut t 21 <210> 270 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 270 gaauggacac ugacugcuct t 21 <210> 271 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 271 caacuccugu uuggauucat t 21 <210> 272 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 272 gcaacuccug uuuggauuct t 21 <210> 273 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 273 ugcaacuccu guuuggauut t 21 <210> 274 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 274 uagugcaacu ccuguuuggt t 21 <210> 275 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 275 cuagugcaac uccuguuugt t 21 <210> 276 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 276 gcuagugcaa cuccuguuut t 21 <210> 277 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 277 ccgcuagugc aacuccugut t 21 <210> 278 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 278 caccgcuagu gcaacuccut t 21 <210> 279 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 279 uggacaccgc uagugcaact t 21 <210> 280 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 280 guggacaccg cuagugcaat t 21 <210> 281 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 281 caacguggug gacaccgcut t 21 <210> 282 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 282 ggguacagac uuggcuacut t 21 <210> 283 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 283 acuugcuuca aggguacagt t 21 <210> 284 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 284 ccacuugcuu caaggguact t 21 <210> 285 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 285 cagcuccucu cccuccucut t 21 <210> 286 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 286 agcagcuccu cucccuccut t 21 <210> 287 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 287 aacuacagcu cccagaaggt t 21 <210> 288 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 288 ccacgaaaac uacagcucct t 21 <210> 289 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 289 cccacgaaaa cuacagcuct t 21 <210> 290 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 290 ucccacgaaa acuacagcut t 21 <210> 291 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 291 cucccacgaa aacuacagct t 21 <210> 292 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 292 cgcucccacg aaaacuacat t 21 <210> 293 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 293 gccgcuccca cgaaaacuat t 21 <210> 294 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 294 gagccgcucc cacgaaaact t 21 <210> 295 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 295 ggaucuguag uucggcccgt t 21 <210> 296 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 296 gggaucugua guucggccct t 21 <210> 297 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 297 ccgccuggga ucuguaguut t 21 <210> 298 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 298 gccgccuggg aucuguagut t 21 <210> 299 <211> 93 <212> DNA <213> Homo sapiens <400> 299 gcgaaggaaa gttcttcctc gttgttccaa tccgaggaca agctgatatg tcgcagcagc 60 ccagggaagc atgcgagctg ataggaagtc ctt 93 <210> 300 <211> 53 <212> DNA <213> Homo sapiens <400> 300 tttaagacag gctcgaatgc taaaactttc ttgtgccaaa acccttgact att 53 <210> 301 <211> 81 <212> DNA <213> Homo sapiens <400> 301 aaaataagca cttggcgtgc cctcgcagat gtctgagctg agaggtcggg gcgatggtag 60 aagagcagtc agtgtccatt c 81 <210> 302 <211> 42 <212> DNA <213> Homo sapiens <400> 302 tgaatccaaa caggagttgc actagcggtg tccaccacgt tg 42 <210> 303 <211> 57 <212> DNA <213> Homo sapiens <400> 303 agtagccaag tctgtaccct tgaagcaagt ggggagagag gagggagagg agctgct 57 <210> 304 <211> 35 <212> DNA<x <213> Homo sapiens <400> 304 ccttctggga gctgtagttt tcgtgggagc ggctc 35 <210> 305 <211> 27 <212> DNA <213> Homo sapiens <400> 305 [[ID=

Claims

1. A small activating nucleic acid molecule comprising a first oligonucleotide chain and a second oligonucleotide chain, the first oligonucleotide chain and the second oligonucleotide chain forming a double-stranded structure through complete complementarity, the first oligonucleotide chain having 100% homology or complementarity with any nucleotide sequence selected from SEQ ID NO:8-104; and the small activating nucleic acid molecule being able to induce target gene mRNA expression to reach 1.1-fold or more.

2. The small activating nucleic acid molecule according to claim 1, wherein the first oligonucleotide chain comprises any nucleotide sequence selected from SEQ ID NO:105-201, or the second oligonucleotide chain comprises any nucleotide sequence selected from SEQ ID NO:202-298.

3. The small activating nucleic acid molecule according to claim 1, wherein the first oligonucleotide chain is 16-35 nucleotides in length and the second oligonucleotide chain is 16-35 nucleotides in length.

4. The small activating nucleic acid molecule according to claim 1, wherein the small activating nucleic acid molecule is a double-stranded nucleic acid, and the first oligonucleotide chain and the second oligonucleotide chain are respectively located on the two strands of the double-stranded nucleic acid.

5. The small activating nucleic acid molecule according to claim 4, wherein the first oligonucleotide chain and / or the second oligonucleotide chain has protrusions at the 5' end and / or the 3' end.

6. The small activating nucleic acid molecule according to claim 5, wherein the protrusion is a protrusion of 0-6 nucleotides.

7. The small activating nucleic acid molecule according to claim 6, wherein the protrusion is dTdT or dTdTdT.

8. The small activating nucleic acid molecule according to claim 1, wherein the small activating nucleic acid molecule is a single-stranded nucleic acid, wherein the single-stranded nucleic acid has a hairpin structure that can form a double-stranded region, and the first oligonucleotide chain and the second oligonucleotide chain have complementary regions that can form a double-stranded structure.

9. The small activating nucleic acid molecule according to claim 1, wherein the combination of the first nucleotide and the second nucleotide is selected from the group consisting of: SEQ ID NO:105 and SEQ ID NO:202, SEQ ID NO:106 and SEQ ID NO:203, SEQ ID NO:107 and SEQ ID NO:204, SEQ ID NO:108 and SEQ ID NO:205, SEQ ID NO:109 and SEQ ID NO:206, SEQ ID NO:110 and SEQ ID NO:207, SEQ ID NO:111 and SEQ ID NO:208, SEQ ID NO:112 and SEQ ID NO:209, SEQ ID NO:113 and SEQ ID NO:210, SEQ ID NO:114 and SEQ ID NO:211, SEQ ID NO:115 and SEQ ID NO:212, SEQ ID NO:116 and SEQ ID NO:213, SEQ ID NO:117 and SEQ ID NO:214, SEQ ID NO:118 and SEQ ID NO:

219. SEQ ID NO:215, SEQ ID NO:119 and SEQ ID NO:216, SEQ ID NO:120 and SEQ ID NO:217, SEQ ID NO:121 and SEQ ID NO:218, SEQ ID NO:122 and SEQ ID NO:219, SEQ ID NO:123 and SEQ ID NO:220, SEQ ID NO:124 and SEQ ID NO:221, SEQ ID NO:125 and SEQ ID NO:222, SEQ ID NO:126 and SEQ ID NO:223, SEQ ID NO:127 and SEQ ID NO:224, SEQ ID NO:128 and SEQ ID NO:225, SEQ ID NO:129 and SEQ ID NO:226, SEQ ID NO:130 and SEQ ID NO:227, SEQ ID NO:131 and SEQ ID NO:228, SEQ ID NO:132 and SEQ ID NO:229, SEQ ID NO:133 and SEQ ID NO:215, SEQ ID NO:119 and SEQ ID NO:216, SEQ ID NO:120 and SEQ ID NO:217, SEQ ID NO:121 and SEQ ID NO:218, SEQ ID NO:122 and SEQ ID NO:219, SEQ ID NO:123 and SEQ ID NO:220, SEQ ID NO:124 and SEQ ID NO:221, SEQ ID NO:125 and SEQ ID NO:222, SEQ ID NO:126 and SEQ ID NO:223, SEQ ID NO:127 and SEQ ID NO:224, SEQ ID NO:128 and SEQ ID NO:225, SEQ ID NO:129 and SEQ ID NO:226, SEQ ID NO:130 and SEQ ID NO:227, SEQ ID NO:131 and SEQ ID NO:22 NO:230, SEQ ID NO:134 and SEQ ID NO:231, SEQ ID NO:135 and SEQ ID NO:232, SEQ ID NO:136 and SEQ ID NO:233, SEQ ID NO:137 and SEQ ID NO:234, SEQ ID NO:138 and SEQ ID NO:235, SEQ IDSEQ ID NO:139 and SEQ ID NO:236, SEQ ID NO:140 and SEQ ID NO:237, SEQ ID NO:141 and SEQ ID NO:238, SEQ ID NO:142 and SEQ ID NO:239, SEQ ID NO:143 and SEQ ID NO:240, SEQ ID NO:144 and SEQ ID NO:241, SEQ ID NO:145 and SEQ ID NO:242, SEQ ID NO:146 and SEQ ID NO:243, SEQ ID NO:147 and SEQ ID NO:244, SEQ ID NO:148 and SEQ ID NO:245, SEQ ID NO:149 and SEQ ID NO:246, SEQ ID NO:150 and SEQ ID NO:247, SEQ ID NO:151 and SEQ ID NO:248, SEQ ID NO:152 and SEQ ID NO:249, SEQ ID NO:153 and SEQ ID NO:250, SEQ ID SEQ ID NO:154 and SEQ ID NO:251, SEQ ID NO:155 and SEQ ID NO:252, SEQ ID NO:156 and SEQ ID NO:253, SEQ ID NO:157 and SEQ ID NO:254, SEQ ID NO:158 and SEQ ID NO:255, SEQ ID NO:159 and SEQ ID NO:256, SEQ ID NO:160 and SEQ ID NO:257, SEQ ID NO:161 and SEQ ID NO:258, SEQ ID NO:162 and SEQ ID NO:259, SEQ ID NO:163 and SEQ ID NO:260, SEQ ID NO:164 and SEQ ID NO:261, SEQ ID NO:165 and SEQ ID NO:262, SEQ ID NO:166 and SEQ ID NO:263, SEQ ID NO:167 and SEQ ID NO:264, SEQ ID NO:168 and SEQ ID NO:265, SEQ ID NO:169 and SEQ ID NO:266, SEQ ID NO:170 and SEQ ID NO:267, SEQ ID NO:171 and SEQ ID NO:268, SEQ ID NO:172 and SEQ ID NO:269, SEQ ID NO:173 and SEQ ID NO:270, SEQ ID NO:174 and SEQ ID NO:271, SEQSEQ ID NO:175 and SEQ ID NO:272, SEQ ID NO:176 and SEQ ID NO:273, SEQ ID NO:177 and SEQ ID NO:274, SEQ ID NO:178 and SEQ ID NO:275, SEQ ID NO:179 and SEQ ID NO:276, SEQ ID NO:180 and SEQ ID NO:277, SEQ ID NO:181 and SEQ ID NO:278, SEQ ID NO:182 and SEQ ID NO:279, SEQ ID NO:183 and SEQ ID NO:280, SEQ ID NO:184 and SEQ ID NO:281, SEQ ID NO:185 and SEQ ID NO:282, SEQ ID NO:186 and SEQ ID NO:283, SEQ ID NO:187 and SEQ ID NO:284, SEQ ID NO:188 and SEQ ID NO:285, SEQ ID NO:189 and SEQ ID NO:286, SEQ ID SEQ ID NO:190 and SEQ ID NO:287, SEQ ID NO:191 and SEQ ID NO:288, SEQ ID NO:192 and SEQ ID NO:289, SEQ ID NO:193 and SEQ ID NO:290, SEQ ID NO:194 and SEQ ID NO:291, SEQ ID NO:195 and SEQ ID NO:292, SEQ ID NO:196 and SEQ ID NO:293, SEQ ID NO:197 and SEQ ID NO:294, SEQ ID NO:198 and SEQ ID NO:295, SEQ ID NO:199 and SEQ ID NO:296, SEQ ID NO:200 and SEQ ID NO:297, and SEQ ID NO:201 and SEQ ID NO:

298.

10. The small activating nucleic acid molecule according to claim 1, wherein the nucleotides constituting the small activating nucleic acid molecule are natural, unmodified nucleotides.

11. The small activating nucleic acid molecule according to claim 1, wherein one or more nucleotides of the small activating nucleic acid molecule are chemically modified nucleotides.

12. The small activated nucleic acid molecule according to claim 11, wherein the chemical modification is selected from one or more of the following modifications: modification of the phosphodiester bond of the nucleotide, modification of the 2'-OH of the ribose in the nucleotide, and modification of the bases in the nucleotide.

13. The small activated nucleic acid molecule according to claim 11, wherein the chemical modification is selected from one or more of the following modifications: thiophosphate modification, boronized phosphate modification, 2'-fluorine modification, 2'-oxymethyl modification, 2'-oxyethylidene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid modification, 2'-amino modification, 2'-deoxy modification, 5′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

14. The small activating nucleic acid molecule according to claim 11, wherein the first oligonucleotide chain and / or the second oligonucleotide chain are terminally linked to a lipophilic group, wherein the lipophilic group is selected from one or more of liposomes, macromolecules, polypeptides or cholesterol.

15. A nucleic acid molecule comprising a fragment encoding a small activating nucleic acid molecule according to any one of claims 1-14.

16. The nucleic acid molecule according to claim 15, wherein the nucleic acid molecule is an expression vector.

17. A cell comprising a small activating nucleic acid molecule according to any one of claims 1-14 or a nucleic acid molecule according to any one of claims 15-16.

18. A pharmaceutical composition comprising: The small activating nucleic acid molecule according to any one of claims 1-14 or the nucleic acid molecule according to any one of claims 15-16.

19. The pharmaceutical composition of claim 18, further comprising a pharmaceutically acceptable carrier.

20. A formulation comprising a small activating nucleic acid molecule according to any one of claims 1-14, or a nucleic acid molecule according to any one of claims 15-16, or a cell according to claim 17, or a pharmaceutical composition according to any one of claims 18-19.

21. A kit comprising a small activating nucleic acid molecule according to any one of claims 1-14, or a nucleic acid molecule according to any one of claims 15-16, or a cell according to claim 17, or a pharmaceutical composition according to any one of claims 18-19.

22. Use of the small activating nucleic acid molecule according to any one of claims 1-14, or the nucleic acid molecule according to any one of claims 15-16, or the cell according to claim 17, or the pharmaceutical composition according to any one of claims 18-19 in the preparation of a medicament or formulation for treating vitiligo.

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