WO2021249064A1 - 靶向mitf基因的核酸分子及其用途 - Google Patents

靶向mitf基因的核酸分子及其用途 Download PDF

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WO2021249064A1
WO2021249064A1 PCT/CN2021/091834 CN2021091834W WO2021249064A1 WO 2021249064 A1 WO2021249064 A1 WO 2021249064A1 CN 2021091834 W CN2021091834 W CN 2021091834W WO 2021249064 A1 WO2021249064 A1 WO 2021249064A1
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nucleic acid
acid molecule
small activating
small
mitf
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PCT/CN2021/091834
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French (fr)
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李龙承
姜武林
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中美瑞康核酸技术(南通)研究院有限公司
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Priority to JP2022575306A priority Critical patent/JP2023529398A/ja
Priority to CN202180042234.0A priority patent/CN115916975A/zh
Priority to CA3179261A priority patent/CA3179261A1/en
Priority to EP21823138.9A priority patent/EP4166668A1/en
Priority to US18/000,838 priority patent/US20230220389A1/en
Priority to AU2021288166A priority patent/AU2021288166A1/en
Priority to KR1020227041508A priority patent/KR20230022167A/ko
Publication of WO2021249064A1 publication Critical patent/WO2021249064A1/zh

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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

  • the present invention belongs to the field of nucleic acid technology. Specifically, it relates to a nucleic acid molecule having a double-stranded structure related to gene activation, such as a small activating nucleic acid molecule, in particular to a nucleic acid molecule targeting MITF genes, and to a small activating nucleic acid molecule in Activation/up-regulation of microphthalmia-associated transcription factor (MITF) in gene transcription.
  • the present invention generally provides compounds, pharmaceutical compositions and methods of use thereof. More specifically, the present invention will benefit from applications in the treatment of diseases that increase MITF gene expression, such as vitiligo.
  • Vitiligo is an acquired hypopigmented skin disease characterized by the reduction of melanocytes in the local epidermis and hair, and the formation of irregular leukoplakia. According to statistics, patients with vitiligo account for about 0.5%-2% of the global population, with no obvious gender differences (Picardo et al., 2015). According to clinical characteristics, it can be divided into vulgaris vitiligo and segmental vitiligo. Vulgaris is related to autoimmune regulation or oxidative stress further causing damage to melanocytes, and segmental vitiligo is related to genetic factors (Gauthier, Cario Andre, and Taieb 2003; Dell'anna and Picardo 2006).
  • Vitiligo is related to factors such as family inheritance, autoimmune regulation, oxidative stress and functional melanocyte damage.
  • the occurrence of vitiligo has an obvious tendency of family clustering.
  • the positive rate of family history of vitiligo patients is about 6.25% to 40%.
  • the risk of first-degree relatives is 18 times that of the general population. Patients with a positive family history usually get onset earlier ( Sehgal and Srivastava 2007).
  • the cytokines IL-6, TNF- ⁇ , IL-1 ⁇ , IL-8) produced by the immune response in patients with vitiligo are elevated.
  • In vitro experiments have shown that a large number of pro-inflammatory factors can inhibit the synthesis of melanin and the synthesis of melanin.
  • Anti-melanocyte antibodies can be detected in the serum of 50%-93% of patients with vitiligo. These antibodies can activate complement through immune complexes and/or antibody-dependent cell-mediated cytotoxicity leading to the reduction or disappearance of melanocytes (Kemp et al. , 2007).
  • Dell'Anna laboratory proposed that the imbalance of oxidative stress in patients with vitiligo may originate from abnormal cardiolipin in epidermal cells, which can lead to a decrease in the activity of the mitochondrial electron transport chain and induce excessive production of reactive oxygen species (ROS), which in turn leads to Melanocytes die (Dell'Anna et al., 2007).
  • ROS reactive oxygen species
  • melanocytes The loss of melanocytes is a key factor in the pathogenesis of vitiligo.
  • Melanocytes release antigens after physical and chemical damage, which stimulates the body to produce anti-melanocyte antibodies, leading to inactivation of a large number of melanocytes and aggravating epidermal depigmentation.
  • melanin in the skin lesion is damaged or lost when it crosses the epidermis, which may be caused by the adhesion defects of melanocytes, basement membrane and keratinocytes (Kumar, Parsad, and Kanwar 2011).
  • Melanocyte dysfunction is mainly manifested as decreased number of melanocytes, imbalance of melanocyte adhesion and migration, excessive melanocyte apoptosis, and abnormal development and differentiation of melanocytes.
  • 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 DNA in the form of a dimer. It plays a key role in the development of melanocytes, retinal pigment epithelial cells, osteoclasts and mast cells.
  • MITF and related factors transcription factor EB transcription factor EB, TFEB
  • TFE3 and TFEC together constitute the MiT family (Hemesath et al., 1994).
  • MITF is the only factor in the MiT family that plays an important role in the development of normal melanocytes (Levy, Khaled, and Fisher 2006).
  • MITF is not only a necessary regulator for the development, proliferation and survival of melanocytes, but also plays a vital role in regulating the expression of related enzymes and melanosome proteins.
  • psoralen drugs are commonly used as photosensitizers and long-wave ultraviolet rays to perform psoralen photochemotherapy (PUVA) in patients with vitiligo to improve the synthesis of melanocytes in the skin lesions and anti-apoptosis (Iannella et al., 2016) .
  • Alfamelanotide (Afamelanotide) is the only drug approved by the U.S. Food and Drug Administration (FDA) for preclinical testing, but Alfamelanotide needs to be tested in combination with narrow-band ultraviolet B phototherapy (NB-UVB).
  • NB-UVB narrow-band ultraviolet B phototherapy
  • MCIR melanocorticosteroid receptor 1
  • alfamelatide can combine with MCIR to produce melanin (Lim et al., 2015; Grimes et al., 2013), but it is specific The duration of action of NB-UVB and the dose of the drug need to be further tested.
  • autologous scar epidermal transplantation, autologous micro-transplantation, autologous melanocyte transplantation, mixed epidermal transplantation and micro-staining method also have good curative effects.
  • this type of surgery has strict contraindications and treatment methods are limited.
  • An objective of the present invention is to provide small activating nucleic acid molecules based on the RNA activation process, which increase the expression of MITF protein by activating/up-regulating the transcription of MITF genes, or using small activating nucleic acid molecules to prepare treatments related to MITF protein deficiency or deficiency Medicine for the disease or condition.
  • Another object of the present invention is to provide a composition or preparation containing small activating nucleic acid molecules.
  • Another object of the present invention is to provide a method for activating/up-regulating the expression of MITF gene in cells.
  • Another object of the present invention is to provide the use of small activating nucleic acid molecules or compositions or preparations containing them in preparation for treating diseases or conditions related to MITF protein deficiency or deficiency, such as treatment of vitiligo.
  • Another objective of the present invention is to provide an isolated MITF gene small activation nucleic acid molecule target site, wherein the target site includes any consecutive 16-sequence selected from SEQ ID NO: 299-SEQ ID NO: 305
  • the 35-nucleotide sequence or the sequence consisting of any of the above-mentioned consecutive 16-35 nucleotides has at least 75%, such as at least about 79%, about 80%, about 85%, about 90%, about 95%, about Sequence with 99% or 100% homology.
  • a small activating nucleic acid molecule such as a small activating RNA (saRNA) molecule, the small activating nucleic acid molecule at least comprising a first oligonucleotide strand that is identical to human MITF Any contiguous segment with a length of 16-35 nucleotides in the promoter region of a gene has at least 75% homology or complementarity.
  • saRNA small activating RNA
  • the human MITF gene promoter region is preferably a region (SEQ ID NO: 1) from the transcription start site (TSS) of the MITF gene to 500 nucleotides upstream thereof, and the first oligonucleotide
  • TSS transcription start site
  • SEQ ID NO: 1 The nucleotide chain and any continuous segment of 16-35 nucleotides in length from the transcription start site (TSS) of the MITF gene to the 500 nucleotides upstream of it (SEQ ID NO: 1) Have at least 75% homology or complementarity.
  • one strand of the small activation nucleic acid molecule includes a region selected from -500 to -408 (region H1, SEQ ID NO: 299) and -403 to -351 from the transcription start site in the MITF gene promoter.
  • region H1, SEQ ID NO: 299 region H1, SEQ ID NO: 299
  • -403 to -351 from the transcription start site in the MITF gene promoter.
  • consecutive 16- 35 nucleotides have at least 75%, for example, at least about 79%, about 80%, about 85%, about
  • one strand of the small activating nucleic acid molecule of the present invention has at least 75%, such as at least about 79%, about 80, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity.
  • one strand of the small activation nucleic acid molecule of the present invention includes at least 75%, such as at least about 79%, about 80%, and any nucleotide sequence selected from SEQ ID NO: 8-104. , About 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity of nucleic acid sequences.
  • the small activating nucleic acid molecule of the present invention includes a double-stranded small activating nucleic acid molecule targeted to the promoter region of the MITF gene, such as a small activating RNA (saRNA) molecule, which includes a first oligonucleotide strand and a second oligonucleotide strand.
  • saRNA small activating RNA
  • the first oligonucleotide chain and the second oligonucleotide chain of the small activation nucleic acid molecule of the present invention may exist on two different nucleic acid chains, or may exist on the same nucleic acid chain.
  • at least one strand of the small activation nucleic acid molecule can have a protrusion (or called a pendant) at the 5'end and/or 3'end. ), for example, there may be an overhang of 0-6 nucleotides at the 3'end, such as an overhang of 0, 1, 2, 3, 4, 5, or 6 nucleotides.
  • both chains of the small activation nucleic acid molecule of the present invention have protrusions.
  • the 3'ends of the two chains of the small activation nucleic acid molecule may have protrusions of 0-6 nucleotides, for example, Overhangs of 0, 1, 2, 3, 4, 5, or 6 nucleotides, most preferably, overhangs of 2 or 3 nucleotides.
  • the nucleotide type of the overhang can be dT (thymidine deoxynucleotide, or written as T).
  • the protrusion at the 5'end and/or the 3'end is dTdT or dTdTdT.
  • the small activation nucleic acid molecule of the present invention may also include a small activation nucleic acid molecule that can form a double-stranded region hairpin structure, such as a single-stranded small activation RNA molecule.
  • the small activating nucleic acid molecule of the present invention includes a single-stranded small activating RNA molecule targeted to the promoter region of the MITF gene, wherein the single-stranded small activating nucleic acid molecule can form a double-stranded hairpin structure.
  • the small activating nucleic acid molecule of the present invention may be a hairpin type single-stranded nucleic acid molecule, wherein the first oligonucleotide chain
  • the nucleotide 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 a cell through, for example, an RNA activation mechanism.
  • the length of the first oligonucleotide strand and the second oligonucleotide strand may 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.
  • the first oligonucleotide strand of the small activation nucleic acid molecule of the present invention has at least 75%, such as at least about 79%, and any nucleotide sequence selected from SEQ ID NO: 8-104, About 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity or homology or complementarity, or the second oligonucleotide strand of the small activating nucleic acid molecule and the selected Any nucleotide sequence from SEQ ID NO: 8-104 has at least 75%, such as at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100 % Identity or homology or complementarity.
  • At least one nucleotide in the nucleotide sequence of the small activation nucleic acid molecule is a locked nucleic acid.
  • the chemical modification is well known to those skilled in the art, and the modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including but not limited to phosphorothioate modification and borating phosphate modification. Both modifications can stabilize the structure of saRNA and maintain the high specificity and affinity of base pairing.
  • Ribose modification refers to the modification of the 2'-OH in the nucleotide pentose, that is, the introduction of certain substituents at the hydroxyl position of the ribose sugar, for example, including but not limited to, 2'-fluoro modification, 2'-oxymethyl Group modification, 2'-oxyethylene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, 2'-deoxy modification, etc.
  • Base modification refers to the modification of nucleotide bases, for example, including but not limited to, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, 2,6- Diaminopurine modification and so on.
  • the small activating nucleic acid molecule provided by the present invention can effectively activate or up-regulate the expression of the MITF gene in the cell after being in contact with or introduced into the cell, and preferably the expression is up-regulated by at least 30%.
  • nucleic acids encoding the small activation nucleic acid molecules described herein.
  • the nucleic acid may be a DNA molecule.
  • nucleic acid is an expression vector that contains a fragment encoding the small activation nucleic acid molecule described herein. After the expression vector is introduced into a cell, the small activation nucleic acid molecule described herein can be expressed.
  • the small activating nucleic acid molecule of the present invention may be a double-stranded small activating nucleic acid molecule targeted to the promoter region of the MITF gene, such as a double-stranded small activating RNA (saRNA) molecule, which includes a first oligonucleotide strand and The second oligonucleotide chain.
  • saRNA double-stranded small activating RNA
  • the small activating nucleic acid molecule of the present invention may be a single-stranded small activating nucleic acid molecule that targets the promoter region of the MITF gene, such as a double-stranded small activating RNA (saRNA) molecule.
  • saRNA small activating RNA
  • compositions for example, a pharmaceutical composition
  • the composition comprising the small activation nucleic acid molecule of the present invention or the nucleic acid encoding the small activation nucleic acid molecule described herein and optionally, a pharmaceutically acceptable Accepted carrier.
  • the pharmaceutically acceptable carrier may include or be selected from liposomes, high molecular polymers or polypeptides.
  • kits comprising: the small activating nucleic acid molecule of the present invention, the nucleic acid encoding the small activating nucleic acid molecule described herein, the small activating nucleic acid molecule of the present invention, or the coding book
  • Another aspect of the present invention relates to 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 application of the composition containing the small activating nucleic acid molecule of the present invention in the preparation of a medicine or preparation for activating/up-regulating the expression of the MITF gene in a cell.
  • the method for activating/up-regulating the expression of the MITF gene in a cell comprises administering to the cell the small activating nucleic acid molecule of the present invention, the nucleic acid encoding the small activating nucleic acid molecule of the present invention, or the nucleic acid containing the small activating nucleic acid molecule of the present invention.
  • the above-mentioned cells include mammalian cells, such as cells from the human body, such as human melanocytes (HEM) and human skin keratinocytes (NHEK).
  • the above-mentioned cells may be in vitro or in a mammalian body, such as a human body. .
  • the small activating nucleic acid molecule of the present invention can be administered in a sufficient amount to achieve the treatment of diseases or conditions related to the lack of MITF protein or the lack or reduction of MITF protein expression.
  • the disease or condition related to the lack of MITF protein amount or the insufficient or decreased expression of MITF may include, for example, vitiligo.
  • the method for treating diseases or conditions associated with insufficient or decreased MITF protein expression in an individual of the present invention includes administering to the individual a therapeutically effective amount of the small activation nucleic acid molecule of the present invention, encoding the small activation of the present invention.
  • diseases or conditions associated with insufficient or reduced MITF protein expression may include, for example, vitiligo. According to clinical characteristics, it can be divided into vulgaris vitiligo and segmental vitiligo. Vulgaris vitiligo is related to autoimmune regulation or oxidative stress further causing damage to melanocytes, and segmental vitiligo is related to genetic factors. Vitiligo is related to factors such as family inheritance, autoimmune regulation, oxidative stress and functional melanocyte damage.
  • Another aspect of the present invention relates to a method of treating vitiligo in an individual, 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, and comprising the small activating nucleic acid of the present invention
  • the disease or condition associated with insufficient or decreased MITF protein expression is vitiligo, and more preferably, the disease or condition associated with insufficient or decreased MITF protein expression includes vitiligo vulgaris, segmental vitiligo and the like.
  • 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 are provided ,
  • the composition containing the small activating nucleic acid molecule of the present invention can be used to prepare drugs for treating diseases related to the underexpression or reduction of MITF protein, such as vitiligo.
  • the diseases associated with insufficient or decreased MITF protein expression may include, for example, vitiligo.
  • the disease or condition associated with insufficient or decreased MITF protein expression is vitiligo, and more preferably, the disease or condition associated with insufficient or decreased MITF protein expression includes vitiligo vulgaris, segmental vitiligo, and the like.
  • Another aspect of the present invention relates to 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 application of the composition or preparation containing the small activating nucleic acid molecule of the present invention in the preparation of a medicine or a combination of medicines for the treatment of vitiligo.
  • vitiligo includes vitiligo vulgaris, segmental vitiligo, and the like.
  • 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 are provided , Or application of a composition or preparation containing the small activating nucleic acid molecule of the present invention in the preparation of a medicine or a combination of medicines for treating diseases related to insufficient or reduced MITF protein expression.
  • the present invention has the following beneficial effects in one or more aspects:
  • the small activating nucleic acid molecules such as small activating RNA (saRNA) that can activate/upregulate the expression of MITF genes provided by the present invention can permanently activate MITF genes, thereby efficiently and specifically upregulating or restoring the expression of MITF genes and proteins with low It can be used to treat diseases or disorders related to the underexpression or reduction of MITF protein, such as vitiligo, or to prepare drugs or preparations for the treatment of diseases or disorders related to underexpression or reduction of MITF protein, such as Wadenberg's synthesis Sign type 2A and Titzer syndrome.
  • saRNA small activating RNA
  • Figure 1 shows the changes in human MITF mRNA expression mediated by saRNA.
  • 239 saRNAs targeted to the human MITF promoter were transfected into HEM cells at a final concentration of 25nM. 72 hours after transfection, MITF mRNA expression was analyzed by one-step RT-qPCR.
  • the graph shows the mRNA expression changes (log2) of MITF relative to the control treatment (Mock), sorted from highest to lowest.
  • the ordinate value represents the mean ⁇ SD of 2 repeated treatments.
  • Figure 2 shows the hot spots of saRNA 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. After the transfection, the expression of MITF mRNA was analyzed by one-step RT-qPCR. The figure shows the change in MITF expression relative to the control treatment (Mock), sorted according to the target position of saRNA on the MITF promoter from -500 to -0.
  • the black solid dots indicate functional saRNA
  • the white hollow dots indicate non-functional saRNA
  • the dashed bars indicate the hotspots (H1 ⁇ H4) and sub-hotspots (W1 ⁇ W3) where functional saRNAs gather.
  • the ordinate value represents the mean ⁇ SD of 2 repeated treatments.
  • Figure 3 shows the two-step RT-qPCR verification high-throughput screening results.
  • the saRNA shown in Figure 3 was used to transfect HEM cells at a final concentration of 25nM for 72 hours. After transfection, RNA was extracted with Qiagen RNeasy kit. After reverse transcription, qPCR amplification was performed with ABI 7500 fast real-time PCR system, and HPRT1 gene was amplified at the same time as Internal reference.
  • the graph shows the expression value of MITF relative to mRNA after a single saRNA-treated cell. Mock, dsCon2 and RAG4-618i are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively. The ordinate value represents the mean ⁇ SD of 2 repeated treatments.
  • Figure 4 shows the two-step RT-qPCR verification high-throughput screening results.
  • NHEK cells were transfected with the saRNA shown in Figure 4 at a final concentration of 25nM for 72 hours. After the transfection, RNA was extracted with Qiagen RNeasy kit, and after reverse transcription, the ABI 7500 fast real-time PCR system was used for qPCR amplification, and HPRT1 gene was amplified at the same time As an internal reference.
  • the graph shows the expression value of MITF relative to mRNA after a single saRNA-treated cell. Mock, dsCon2 and RAG4-618i are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively. The ordinate value represents the mean ⁇ SD of 2 repeated treatments.
  • Figure 5 shows how saRNA promotes MITF protein expression in human NHEK cells.
  • NHEK cells were transfected with the saRNA shown in Figure 5 at a final concentration of 25nM for 5 days. The cells were collected and the total cell protein was extracted. Western blot was used to detect the expression level of MITF protein, and tubulin ( ⁇ / ⁇ -Tubulin) was used as an internal control.
  • the graph shows the relative protein expression of MITF for a single saRNA-treated cell.
  • Mock, dsCon2 and RAG4-618i are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • Complementary refers to the ability of two oligonucleotide strands to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains.
  • Complementary oligonucleotide strands can be base paired in a Watson-Crick manner (e.g., AT, AU, CG), or in any other manner that allows duplex formation (e.g., Hoogsteen type or reverse Hoogsteen type base pairing) for base pairing.
  • oligonucleotide chain exhibits 10% Complementarity.
  • the oligonucleotide strand exhibits 90% complementarity.
  • Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% complementarity.
  • oligonucleotide chain and “oligonucleotide sequence” are used interchangeably and refer to the general term for short-chain nucleotides of less than 35 bases (including deoxyribonucleic acid DNA, ribonucleic acid Nucleotide chains including RNA also include mixed oligonucleotide chains formed by one or more deoxynucleotides and one or more ribonucleotides).
  • the length of the oligonucleotide chain can be any length from 16 to 35 nucleotides.
  • first oligonucleotide strand can be either the sense strand or the antisense strand.
  • the sense strand of the small activating RNA refers to the small activating RNA duplex containing the promoter DNA sequence of the target gene.
  • the coding strand has the same nucleic acid strand, and the antisense strand refers to the nucleic acid strand complementary to the sense strand in the small activating RNA duplex.
  • the term "second oligonucleotide strand" can also be a sense strand or an antisense strand.
  • the second oligonucleotide strand is the antisense strand
  • the second oligonucleotide strand is the sense strand.
  • gene refers to the entire nucleotide sequence required to encode a polypeptide chain or transcribe a functional RNA.
  • a “gene” may be a gene endogenous to the host cell or fully or partially recombined (e.g., due to the introduction of an exogenous oligonucleotide and coding sequence encoding a promoter or a heterologous promoter adjacent to an endogenous coding sequence Into host cells).
  • the term “gene” includes nucleic acid sequences that can be composed of exons and introns.
  • a sequence encoding a protein is, for example, a sequence contained within an exon in an open reading frame between a start codon and a stop codon.
  • gene may refer to including, 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, regardless of whether the other gene contains coding or non-coding sequences.
  • 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, regardless of whether the other gene contains coding or non-coding sequences.
  • “gene” can be used to describe a functional nucleic acid containing regulatory sequences such as promoters or enhancers. The expression of recombinant genes can be controlled by one or more heterologous regulatory sequences.
  • target gene promoter sequence refers to the non-coding sequence of the target gene.
  • the target gene promoter sequence refers to the coding strand of the sequence, also known as the non-template strand. It is a nucleic acid sequence that is the same sequence as the gene coding sequence.
  • Target or target sequence refers to a sequence fragment homologous or complementary to the sense oligonucleotide strand or antisense oligonucleotide of the small activating nucleic acid molecule in the promoter sequence of the target gene.
  • sense strand and “sense nucleic acid strand” are used interchangeably.
  • the sense oligonucleotide strand of a small activation nucleic acid molecule refers to the small activation nucleic acid molecule duplex containing the promoter sequence of the target gene.
  • the coding strand has the same identity as the first oligonucleotide strand.
  • coding strand refers to the DNA strand that cannot be transcribed in the target gene.
  • the nucleotide sequence of the strand is consistent with the sequence of the RNA generated by transcription (in RNA, U replaces the DNA in the DNA). T).
  • the coding strand of the double-stranded DNA sequence of the target gene promoter in the present invention refers to the promoter sequence on the same DNA strand as the target gene DNA coding strand.
  • template strand refers to another strand in the double-stranded DNA of the target gene that is complementary to the coding strand, and that strand that can be used as a template to be transcribed into RNA, which strand is complementary to the base of the transcribed RNA (AU, GC).
  • RNA polymerase binds to the template strand and moves along the 3' ⁇ 5' direction of the template strand, catalyzing the synthesis of RNA in the 5' ⁇ 3' direction.
  • the template strand of the double-stranded DNA sequence of the target gene promoter in the present invention refers to the promoter sequence on the same DNA strand as the target gene DNA template strand.
  • promoter refers to a sequence that regulates the transcription of protein-encoding or RNA-encoding nucleic acid sequences by being positionally associated with them.
  • a eukaryotic gene promoter contains 100 to 5,000 base pairs, although this length range is not meant to limit the term “promoter” as used herein.
  • the promoter sequence is generally located at the 5'end of the protein coding or RNA coding sequence, the promoter sequence can also be present in exon and intron sequences.
  • transcription initiation site refers to a nucleotide that marks the initiation of transcription on the template strand of a gene.
  • the transcription initiation site can appear on the template strand in the promoter region.
  • a gene can have more than one transcription start site.
  • identity refers to the coding strand between one of the oligonucleotide strands (sense strand or antisense strand) of the small activating RNA and a region of the promoter sequence of the target gene Or the similarity in the template chain.
  • the “identity” or “homology” may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% .
  • overhang As used herein, the terms “overhang”, “overhang”, and “overhang” are used interchangeably and refer to non-base paired nucleotides at the end (5' or 3') of an oligonucleotide chain, which are extended beyond One of the strands in the double-stranded oligonucleotide is produced by the other strand.
  • the single-stranded region that extends beyond the 3'and/or 5'end of the duplex is called an overhang.
  • gene activation or “activation of genes” or “gene up-regulation” or “up-regulation of genes” are used interchangeably and refer to the measurement of gene transcription level, mRNA level, protein level, enzyme activity, methylation State, chromatin state or configuration, translation level, or its activity or state in a cell or biological system are used to determine the increase in transcription, translation, or expression or activity of a certain nucleic acid. These activities or states can be measured directly or indirectly.
  • gene activation refers to the increase in activity associated with nucleic acid sequences, regardless of the mechanism by which such activation occurs, for example, it acts as a regulatory sequence, It is transcribed into RNA, is translated into protein and increases protein expression.
  • the small activation nucleic acid molecule can also be composed of a synthetic or vector-expressed single-stranded RNA molecule that can form a double-stranded region hairpin structure, wherein the first region contains a nucleotide sequence that has sequence identity with the target sequence of the promoter of the gene, The nucleotide sequence contained in the second region is complementary to the first region.
  • the length of the duplex region of a small activation nucleic acid molecule is generally about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, and about 16 to about 44 base pairs.
  • Base pairs about 18 to about 42 base pairs, about 20 to about 40 base pairs, about 22 to about 38 base pairs, about 24 to about 36 base pairs, about 26 to about 34 base pairs, about 28 to about 32 base pairs, usually about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 Base pairs.
  • the terms "saRNA”', "small activating RNA” and “small activating nucleic acid molecule” also contain nucleic acids other than ribonucleotide moieties, including but not limited to modified nucleotides or the like.
  • hot spot refers to a gene promoter region with a length of at least 25 bp. In these regions, there is an aggregation of functional small activating nucleic acid molecule targets, that is, these hot spots are targeted. At least 30% of the small activating nucleic acid molecules can induce the target gene mRNA expression to reach 1.1 times or more; the term “warm spot (W region)” refers to the gene promoter region with a length of at least 25bp, in these regions, The aggregation of functional small activating nucleic acid molecule targets, that is, 8-30% of small activating nucleic acid molecules targeting these hot spots can induce the mRNA expression of target genes by 1.1 times or more.
  • the preparation method of the small activation nucleic acid molecule provided by the present invention includes sequence design and sequence synthesis.
  • the synthesis of the small activating nucleic acid molecule sequence of the present invention can be synthesized by a chemical synthesis method or by entrusting a biotechnology company specializing in nucleic acid synthesis.
  • the method of chemical synthesis includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting and annealing.
  • the first cycle connects a base to the solid support, and then in the nth cycle (19 ⁇ n ⁇ 2), in the first cycle One base is connected to the connected base in n-1 cycles, and this cycle is repeated until the synthesis of all nucleic acid sequences is completed.
  • the obtained crude saRNA product was dissolved in 2 ml of triethylamine acetate solution with a concentration of 1 mol/L, and then separated by a high-pressure liquid chromatography reverse phase C18 column to obtain a purified saRNA single-stranded product.
  • a target with a size of 19bp was selected starting from -500bp upstream of TSS. Then filter the target sequence.
  • the criteria for retaining the target sequence are: 1) The GC content is between 40% and 65%; 2) Does not contain 5 or more than 5 consecutive identical nucleotides; 3) Does not contain more than 3 dinucleotide repeats; 4) Does not contain more than 3 trinucleotide repeats. After filtering, the remaining 239 target sequences will enter the screening process as candidates. Based on these candidate sequences, the corresponding double-stranded small activating RNA was chemically synthesized.
  • the length of the sense and antisense strands of the double-stranded small activating RNA used in this experiment are both 21 nucleotides, and 19 of the 5'regions of the first ribonucleic acid strand (sense strand) of the double-stranded saRNA
  • the nucleotide has 100% identity with the promoter target sequence, and its 3'end contains the TT sequence; the 19 nucleotides in the 5'region of the second ribonucleic acid strand are complementary to the first ribonucleic acid strand sequence.
  • The'end contains the TT sequence.
  • the two strands of the aforementioned double-stranded saRNA are mixed in the same amount of moles and annealed to form a double-stranded saRNA.
  • RNAiMax (Invitrogen, Carlsbad, CA) was used to transfect small activation RNA at a concentration of 25 nM (unless otherwise specified) according to the manufacturer’s instructions. The transfection time was 72. Hours, use 2 replicates for each treatment.
  • the qPCR analysis was performed on the ABI 7500 FastReal-time PCR system (Applied Biosystems), and each sample was amplified in 3 multiple wells.
  • the PCR reaction conditions are shown in Table 1.
  • stage 1-reverse transcription reaction 42°C for 5 minutes; 95°C for 10 seconds; stage 2-PCR reaction: 95°C for 5 seconds, 60°C for 20 seconds, and 45 cycles of amplification.
  • stage 2-PCR reaction 95°C for 5 seconds, 60°C for 20 seconds, and 45 cycles of amplification.
  • HPRT1 and TBP as internal reference genes.
  • the PCR primers used for MITF, HPRT1 and TBP are shown in Table 2.
  • MITF uses MITF F1/R1 primer pair for amplification.
  • 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 the internal reference gene 1 from the Mock-treated sample
  • CtR1s is from the saRNA treatment
  • CtR2m is the Ct value of the internal reference gene 2 from the control treatment sample
  • CtR2s is the Ct value of the internal reference gene 2 from the saRNA treatment sample.
  • saRNA that can activate the transcription of MITF
  • HEM cells were transfected with the above 239 saRNAs at a transfection concentration of 25nM. 72 hours after transfection, the cells were lysed and analyzed by one-step RT-qPCR using the same method as described above. Obtain the relative (compared with the control treatment group) expression value of the MITF gene of each saRNA treatment sample.
  • 29 (12.1%) saRNAs showed high activation ( ⁇ 1.5 times), 68 (28.5%) saRNAs showed mild activation ( ⁇ 1.1 times), and 142 (59.4%)
  • a saRNA did not significantly up-regulate the expression of MITF.
  • the maximum amplitude of activation is 2.75 times, and the maximum inhibition amplitude is 0.38 times.
  • the MITF expression changes of human MITF saRNA are ranked from highest to lowest.
  • the MITF active saRNA sequence (functional saRNA sequence), active target sequence and MITF mRNA expression changes are shown in Table 4 (each active target sequence listed in Table 4 corresponds to SEQ NO: 8-104 in the sequence table , Each saRNA sense sequence corresponds to SEQ ID NO: 105-201 in the sequence list, and each saRNA antisense sequence corresponds to SEQ ID NO: 202-298 in the sequence list).
  • Table 5 shows the different hotspots and sub-hotspot regions of functional saRNA and related sequences (SEQ ID NO: 299-305).
  • Example 3 saRNA promotes the expression of MITF mRNA in human melanocytes (HEM)
  • HEM Human melanocytes
  • Reagent volume SYBR Premix Ex Taq II(2 ⁇ ) 5 ⁇ l ROX Reference Dye II (50 ⁇ ) 0.2 ⁇ l Forward and reverse primer mix (5 ⁇ M) 0.8 ⁇ l cDNA (RT product) 4 ⁇ l total 10 ⁇ l
  • saRNAs (RAG4-284, RAG4-123, RAG4-396, RAG4-461, RAG4-490, RAG4-316 and RAG4-318) increased the relative expression of MITFmRNA by about 2 times. This shows that the activity of randomly selected functional saRNA can be verified in HEM cells.
  • the cell culture was as described in Example 2.
  • Human skin keratinocytes (NHEK) were plated at 20 ⁇ 104 per well into a six-well plate, and small activating RNA was transfected at a final concentration of 25nM. The transfection was used for 72 hours. Each treatment was used 2 multiple holes.
  • the two-step RT-qPCR is as described in Example 3.
  • Figure 4 shows the relative mRNA expression of MITF in cells after different saRNA treatments. Taking the control group (Mock) as a blank transfection control, compared with the control group, the relative expression value of mRNA in the siRNA (RAG4-618i) group decreased by 88.5%, indicating that siRNA was successfully transfected as a small interfering RNA control. Compared with the control group, the activation effect after treatment with RAG4-290 was particularly obvious, and the expression value of MITF relative to mRNA increased by 13.1 times.
  • the relative mRNA expression values of RAG4-175, RAG4-176 and RAG4-316 groups were higher than those of the control group, and their relative mRNA expression values increased by 5.7 times, 3.7 times, and 3.5 times, respectively, which had obvious activation effects.
  • the relative mRNA expression values of RAG4-284, RAG4-123, RAG4-396, RAG4-461, RAG4-490 and RAG4-318 groups were about two times higher than those of the control group, and they also had a certain activation effect. This shows that the activity of randomly selected functional saRNA can be verified in NHEK cells.
  • the cell culture was as described in Example 2.
  • Human melanocytes (NHEK) were plated at 20 ⁇ 104 per well into a six-well plate, and small activating RNA was transfected at a final concentration of 25 nM for 5 days.
  • cell lysis buffer (1 ⁇ RIPA buffer, Cell Signaling Technology) containing protease inhibitors for lysis.
  • the protein was quantified by BCA method, and then separated by polyacrylamide gel electrophoresis and transferred to a 0.45 ⁇ m PVDF membrane.
  • Figure 5 shows the relative expression of MITF protein in cells after different saRNA treatments.
  • the relative expression of MITF protein in the siRNA (RAG4-618i) group decreased by about 50%, indicating that siRNA was successfully transfected as a small interfering RNA control.
  • each saRNA can increase the expression of MITF protein.
  • RAG4-416, RAG4-490 and RAG4-318 up-regulate the expression of MITF protein by more than 1.5 times, especially RAG4-318 has the most obvious activation effect. , Which increased the expression of MITF protein by 1.71 times. This shows that the activity of randomly selected functional saRNA can be verified at the protein level.
  • Example 6 saRNA promotes the expression of MITF protein in human melanocytes (HEM)
  • the cell culture was as described in Example 2.
  • Human melanocytes (HEM) were plated into a six-well plate at 20 ⁇ 104 per well, and small activation RNA was transfected at a final concentration of 25 nM for 72 hours.
  • the protein cleavage and detection methods are as described in Example 5.
  • Figure 6 shows the relative expression of MITF protein in cells after different saRNA treatments. Compared with the control group, each saRNA can increase the expression of MITF protein, and the protein expression of RAG4-396 and RAG4-490 groups increased by 1.4 times.
  • the expression levels of MITF protein in RAG4-175, RAG4-176, RAG4-290, RAG4-284 and RAG4-123 groups were all higher than those in the control group, and the activation level was more than 1.5 times, and the activation effect was obvious. This shows that the activity of randomly selected functional saRNA can be verified at the protein level.
  • saRNAs targeting human MITF gene promoters can up-regulate the expression of MITF genes in cells at the level of mRNA and protein expression, and can be used for diseases or disorders caused by decreased or insufficient MITF protein expression, such as vitiligo, or for the preparation of methods or drugs for the treatment of the aforementioned diseases or disorders.
  • Vitiligo compendium of clinico-epidemiological features, Indian J Dermatol Venereol Leprol, 73: 149-56.

Abstract

提供了一种小激活核酸分子，该小激活核酸分子是靶向MITF基因的核酸分子，其至少包含第一寡核苷酸链。还提供了包含该小激活核酸分子的组合物或制剂及其用途。使用该小激活核酸分子或含有该小激活核酸分子的组合物或制剂可激活或上调MITF基因在细胞中的表达，治疗与MITF蛋白表达不足或减少相关的疾病或状况。

Description

靶向MITF基因的核酸分子及其用途 技术领域
本发明属于核酸技术领域，具体来讲，涉及一种与基因激活相关的具有双链结构的核酸分子，例如小激活核酸分子，尤其涉及靶向MITF基因的核酸分子，还涉及小激活核酸分子在激活/上调小眼畸形转录因子(microphthalmia-associated transcription factor，MITF)在基因转录中的应用。本发明一般地提供化合物，药物组合物及其使用方法，更具体地，本发明将受益于增加MITF基因表达的疾病，如白癜风治疗中的应用。
背景技术
白癜风(vitiligo)是一种以局部表皮及毛发黑色素细胞减少，进而形成不规则白斑为特征的后天色素减退性皮肤病。据统计，白癜风患者占全球人口的比例约0.5％-2％，无明显的性别差异(Picardo等，2015)。根据临床特征可分为寻常型白癜风和节段型白癜风，寻常型白癜风与自身免疫调节或氧化应激进一步造成黑色素细胞受损有关，节段型白癜风与遗传因素有关(Gauthier，Cario Andre，and Taieb 2003；Dell′anna and Picardo 2006)。
白癜风与家族遗传、自身免疫调节、氧化应激和功能性黑色素细胞受损等因素有关。白癜风的发生具有明显的家族聚集倾向，白癜风患者的家族史阳性率约为6.25％～40％，一级亲属的患病风险是一般人群的18倍，有阳性家族史的患者通常发病较早(Sehgal and Srivastava2007)。白癜风患者由于免疫反应产生的细胞因子(IL-6、TNF-α、IL-1β、IL-8)升高，体外实验表明大量的促炎因子会抑制黑素的合成以及与黑素合成有关的转录因子的表达(Kotobuki等，2012)。有50％-93％的白癜风患者血清中可检测到抗黑色素细胞抗体，这些抗体可通过免疫复合物激活补体和(或)抗体依赖细胞介导的细胞毒作用导致黑色素细胞减少或消失(Kemp等，2007)。Dell′Anna实验室提出白癜风患者体内氧化应激的失衡可能来源于表皮细胞中异常的心磷脂，它能导致线粒体电子传递链活性下降，诱导活性氧族(reactive oxygenspecies，ROS)过度产生，进而导致黑色素细胞死亡(Dell′Anna等，2007)。
黑色素细胞的丢失是白癜风发病的关键因素，黑色素细胞受到理化损伤后释放抗原，刺激机体产生抗黑色素细胞抗体，导致大量的黑色素细胞失活，加重表皮色素脱失。实验 表明皮损中的黑色素在其穿越表皮时损伤或丢失，这可能是由于黑色素细胞与基底膜、角质形成细胞的粘附缺陷所致(Kumar，Parsad，and Kanwar 2011)。黑色素细胞功能失调主要表现为黑色素细胞数量减少、黑色素细胞黏附和迁移失调、黑色素细胞过度凋亡和黑色素细胞发育、分化失常等。
由MITF基因编码的小眼畸形转录因子是一种具有碱性-螺旋-环-螺旋-亮氨酸拉链(bHLH-Zip)结构的转录因子，以二聚体形式结合DNA。在黑色素细胞、视网膜色素上皮细胞、破骨细胞和肥大细胞的发育过程中起关键作用。MITF与相关因子转录因子EB(transcription factor EB，TFEB)、TFE3和TFEC一起构成MiT家族(Hemesath等，1994)。MITF是MiT家族中唯一对正常黑色素细胞发育有重要作用的因子(Levy，Khaled，and Fisher 2006)。MITF不仅是黑色素细胞发育、增殖和存活的必要调节因子，而且对调节相关酶和黑素体蛋白表达具有至关重要的作用。酪氨酸酶、TYRP1和DCT三种主要色素沉着相关酶的启动子中均含有MITF的通用结合位点(TCATGTG)，为MITF转录调控的靶基因(Bentley，Eisen，and Goding 1994)。
已发现MITF突变可导致多种人类疾病，包括瓦登伯格综合征2A型(Waardenburg syndrome type 2A，WS2A)、蒂策综合征(Tietz syndrome)以及COMMAD综合征，都表现出严重的耳聋和色素沉着障碍，其中前两者表现为常染色体显性遗传。
现阶段虽治疗白癜风的方法多样，但仍没有彻底治愈白癜风的方法。单纯改善外观可以用化妆品掩饰白斑，其他治疗方法有类固醇激素、紫外线照射、手术等。紫外线-B照射可以使白斑的边界模糊化，但光疗法有致癌的风险。早期Kwinter等利用高效糖皮质激素局部外涂治疗70例儿童白癜风患者，其有效率达64％(Kwinter等，2007)，但复发率高。临床上常用补骨脂类药物作为光敏剂配合长波紫外线对白癜风患者进行补骨脂素光化学疗法(PUVA)，用以改善皮损处黑色素细胞的合成和抗凋亡等作用(Iannella等，2016)。阿法美拉肽(Afamelanotide)是唯一被美国食品药品监督管理局(FDA)批准进行临床前试验的药物，但阿法美拉肽需与窄波段紫外线B光疗(NB-UVB)进行联合试验。因为需要窄波段紫外线B光疗(NB-UVB)激活黑素皮质激素受体1(MCIR)，阿法美拉肽才能和MCIR结合进而产生黑色素(Lim等，2015；Grimes等，2013)，但是具体的NB-UVB作用时间以及药物的剂量还需要进一步的试验。外科疗法中的自体吸疤表皮移植、自体微移植、自体黑 色素细胞移植、混合表皮移植和微着色法等也有很好的疗效，但此类手术有严格的禁忌症，治疗方法局限。
这些治疗方法虽各有一定效果，但容易复发，副作用大，药物的有效剂量还有待进一步确定，给临床治疗带来很大的局限性。
发明内容
为解决上述一方面或几方面的问题，本发明提供了一种基于RNA激活过程的小激活核酸分子(small activating RNA，saRNA)，通过激活/上调MITF基因表达来治疗由MITF蛋白质缺乏或不足或者由MITF单等位基因突变导致的疾病或状况，如白癜风、瓦登伯格综合征2A型或蒂策综合征。
本发明的一个目标是提供基于RNA激活过程的小激活核酸分子，其通过激活/上调MITF基因转录，从而增加MITF蛋白的表达量，或者小激活核酸分子用于制备治疗与MITF蛋白质缺乏或不足相关的疾病或状况的药物。
本发明的另一目标是提供包含小激活核酸分子的组合物或制剂。
本发明的又一目标为提供小激活核酸分子或包含其的组合物或制剂在制备用于激活/上调MITF基因在细胞中的表达的药物中的应用。
本发明的又一目标为提供激活/上调MITF基因在细胞中的表达的方法。
本发明的又一目标为提供激活/上调MITF基因在细胞中的表达的方法，可用于治疗因MITF单等位基因突变所致的疾病，如瓦登伯格综合征2A型和蒂策综合征。
本发明的再一目标为提供小激活核酸分子或包含其的组合物或制剂在制备用于治疗与MITF蛋白质缺乏或不足相关的疾病或状况如白癜风治疗中的应用。
本发明的另一目标为提供分离的MITF基因小激活核酸分子靶位点，其中所述靶位点包括选自SEQ ID NO：299-SEQ ID NO：305的任一条序列上的任意连续16-35个核苷酸的序列或与上述任意连续16-35个核苷酸组成的序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％或100％的同源性的序列。
在本发明的一个方面，提供了小激活核酸分子例如小激活RNA(saRNA)分子，所述小激活核酸分子至少包含第一寡核苷酸链，所述第一寡核苷酸链与人MITF基因启动子区 的任一长度为16-35个核苷酸的连续片段具有至少75％的同源性或互补性。进一步地，所述人MITF基因启动子区优选为从MITF基因的转录起始位点(TSS)至其上游的500个核苷酸的区域(SEQ ID NO：1)，所述第一寡核苷酸链与从MITF基因的转录起始位点(TSS)至其上游的500个核苷酸的区域(SEQ ID NO：1)中的任一长度为16-35个核苷酸的连续片段具有至少75％的同源性或互补性。
在本发明的一个方面，提供了小激活核酸分子例如小激活RNA(saRNA)分子，其激活或者上调细胞中MITF基因的表达，所述小激活核酸分子的一条链与MITF基因启动子区的任一长度为16-35个核苷酸的核酸序列具有至少75％的同源性或互补性，启动子区是指包括转录起始位点上游的500个核苷酸(其序列为SEQ ID NO：1)，从而实现MITF基因表达的激活或者上调。具体地，小激活核酸分子的一条链包括与选自与MITF基因启动子中距转录起始位点的-500至-408区域(区域H1，SEQ ID NO：299)、-403至-351区域(区域H2，SEQ ID NO：300)、-342至-262区域(区域H3，SEQ ID NO：301)、-181至-140区域(区域H4，SEQ ID NO：302)、-250至-193区域(区域W1，SEQ ID NO：303)、-123至-89区域(区域W2，SEQ ID NO：304)、-62至-36区域(区域W3，SEQ ID NO：305)中的连续16-35个核苷酸具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％或100％的同源性或互补性的核酸序列，热点和次热点区域序列如表5所示。更具体地，本发明的小激活核酸分子的一条链与选自SEQ ID NO：8-104的任一核苷酸序列具有至少75％、例如至少约79％、约80、约85％、约90％、约95％、约99％、或约100％的同源性或互补性。在一个具体的实施方式中，本发明的小激活核酸分子的一条链包括与选自SEQ ID NO：8-104的任一核苷酸序列具有至少75％、例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同源性或互补性的核酸序列。在另一实施方式中，本发明的小激活核酸分子的一条链由与选自SEQ ID NO：8-104的任一核苷酸序列具有至少75％、例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同源性或互补性的核酸序列组成。在还一实施方式中，本发明的小激活核酸分子的一条链是与选自SEQ ID NO：8-104的任一核苷酸序列具有至少75％、例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同源性或互补性的核酸序列。
本发明的小激活核酸分子包括靶向MITF基因启动子区的双链小激活核酸分子例如小 激活RNA(saRNA)分子，其包含第一寡核苷酸链和第二寡核苷酸链，第一寡核苷酸链与MITF基因启动子中距转录起始位点的-500至-408区域(区域H1，SEQ ID NO：299)、-403至-351区域(区域H2，SEQ ID NO：300)、-342至-262区域(区域H3，SEQ ID NO：301)、-181至-140区域(区域H4，SEQ ID NO：302)、-250至-193区域(区域W1，SEQ ID NO：303)、-123至-89区域(区域W2，SEQ ID NO：304)、-62至-36区域(区域W3，SEQ ID NO：305)中的连续16-35个核苷酸具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同源性或互补性，第一寡核苷酸链和第二寡核苷酸链能通过互补形成双链核酸结构，双链核酸结构能够激活MITF基因在细胞中的表达。
本发明的小激活核酸分子的第一寡核苷酸链和第二寡核苷酸链可以存在于两条不同的核酸链上，也可以存在于同一条核酸链上。当第一寡核苷酸链和第二寡核苷酸链分别位于两条链上时，小激活核酸分子的至少一条链可以在5’端和/或3’端具有突出(或称为悬垂)，例如在3’端可以具有0-6个核苷酸的突出，如，0、1、2、3、4、5或6个核苷酸的突出。优选地，本发明的小激活核酸分子的两条链都具有突出，更优选地，小激活核酸分子的两条链的3’端均可以具有0-6个核苷酸的突出，例如，具有0、1、2、3、4、5或6个核苷酸的突出，最优选地，具有2个或3个核苷酸的突出。优选地，突出端的核苷酸类型可以是dT(胸腺嘧啶脱氧核苷酸，或写作T)。优选地，5’端和/或3’端的突出为dTdT或dTdTdT。
本发明的小激活核酸分子也可以包括可形成双链区发夹结构的小激活核酸分子，例如单链小激活RNA分子。在一个实施方式中，本发明的小激活核酸分子包括靶向MITF基因启动子区的单链小激活RNA分子，其中所述单链小激活核酸分子可形成双链区发夹结构。当第一寡核苷酸链和第二寡核苷酸链存在于同一条核酸链上时，优选地，本发明的小激活核酸分子可以为发夹型单链核酸分子，其中第一寡核苷酸链和第二寡核苷酸链具有能形成双链核酸结构的互补区域，所述双链核酸结构可以通过例如RNA激活机制促进MITF基因在细胞中的表达。
上述小激活核酸分子中，第一寡核苷酸链和第二寡核苷酸链的长度可以分别为16-35个核苷酸，例如，可以为16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35个核苷酸。
在一个实施方式中，本发明的小激活核酸分子的第一寡核苷酸链与选自SEQ ID NO： 8-104中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性或互补性，或者小激活核酸分子的第二寡核苷酸链与选自SEQ ID NO：8-104中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性或互补性。在一个实施方式中，本发明的小激活核酸分子的第一寡核苷酸链包括与选自SEQ ID NO：8-104中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性或互补性的核酸序列，或者由与选自SEQ ID NO：8-104中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性或互补性的核酸序列组成，或本发明的小激活核酸分子的第二寡核苷酸链包括与选自SEQ ID NO：8-104中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性或互补性的核酸序列，或者由与选自SEQ ID NO：8-104中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性或互补性的核酸序列组成。在具体的实施方式中，本发明的小激活核酸分子的第一寡核苷酸链可以包括选自SEQ ID NO：105-201的任一核苷酸序列，和/或其第二寡核苷酸链可以包括选自SEQ ID NO：202-298的任一核苷酸序列。在一个实施方式中，本文所述的小激活核酸分子可以是合成的、体外转录的或者载体表达的。
本文所述小激活核酸分子中所有的核苷酸都可以为天然的未经化学修饰的核苷酸，也可以包括至少一种修饰。在一个实施方式中，本文所述的小激活核酸分子中的修饰可以包括化学修饰，如在至少一个核苷酸可以具有化学修饰，本发明使用的化学修饰可以包括或选自如下修饰中的一种或多种，或其任意组合：
(1)对所述小激活核酸分子的核苷酸序列中核苷酸的磷酸二酯键的修饰；
(2)对所述小激活核酸分子的核苷酸序列中的核糖的2’-OH的修饰；
(3)对所述小激活核酸分子的核苷酸序列中的碱基的修饰；
(4)所述小激活核酸分子的核苷酸序列中的至少一个核苷酸为锁核酸。
所述化学修饰为本领域技术人员所公知，所述磷酸二酯键的修饰是指对磷酸二酯键中的氧进行修饰，包括但不限于，硫代磷酸修饰和硼烷化磷酸盐修饰。两种修饰都能稳定 saRNA结构，保持碱基配对的高特异性和高亲和力。
核糖修饰是指对核苷酸戊糖中2’-OH的修饰，即，在核糖的羟基位置引入某些取代基，例如，包括但不限于，2’-氟代修饰、2’-氧甲基修饰、2’-氧亚乙基甲氧基修饰、2，4’-二硝基苯酚修饰、锁核酸(LNA)、2’-氨基修饰、2’-脱氧修饰等。
碱基修饰是指对核苷酸的碱基进行修饰，例如，包括但不限于，5′-溴尿嘧啶修饰、5′-碘尿嘧啶修饰、N-甲基脲嘧啶修饰、2，6-二氨基嘌呤修饰等。
这些修饰可以增加小激活核酸分子的生物可利用性，提高与靶序列的亲和性，增强在细胞内抵抗核酸酶水解的能力。
此外，为了促进小激活核酸分子进入细胞，可以在以上修饰的基础上，在小激活核酸分子的第一寡核苷酸链和/或第二寡核苷酸链的末端引入例如胆固醇等亲脂性基团，以利于通过由脂质双分子层构成的细胞膜及核膜与细胞核内的基因启动子区发生作用。
本发明提供的小激活核酸分子在与细胞接触或引入细胞后可有效激活或上调细胞中MITF基因的表达，优选情况下表达至少上调30％。
本发明的另一方面还涉及编码本文所述的小激活核酸分子的核酸。在一个实施方式中，所述核酸可以是DNA分子。在一个实施方式中，所述核酸为表达载体，该表达载体包含编码本文所述的小激活核酸分子的片段，将该表达载体引入细胞后，可表达本文所述的小激活核酸分子。
在本发明的另一方面，提供了包含上文所述的小激活核酸分子或编码本文所述的小激活核酸分子的核酸的细胞。在一个实施方式中，本发明的小激活核酸分子可以是靶向MITF基因启动子区的双链小激活核酸分子例如双链小激活RNA(saRNA)分子，其包括第一寡核苷酸链和第二寡核苷酸链。在另一实施方式中，本发明的小激活核酸分子可以是靶向MITF基因启动子区的单链小激活核酸分子例如双链小激活RNA(saRNA)分子。
本发明的另一方面提供了组合物(例如药物组合物)，该组合物包含本发明所述的小激活核酸分子或编码本文所述的小激活核酸分子的核酸和任选地，药学上可接受的载体。在一个实施方式中，所述药学上可接受的载体可以包括或选自脂质体、高分子聚合物或多肽。
在本发明的另一方面，提供了制剂，该制剂包括：本发明所述的小激活核酸分子、编 码本发明所述的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物。
在本发明的另一方面，提供了试剂盒，所述试剂盒包括：本发明的小激活核酸分子、编码本文所述的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物。
本发明的另一方面涉及本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物在制备用于激活/上调MITF基因在细胞中的表达的药物或制剂中的应用。
本发明的另一方面还涉及激活/上调MITF基因在细胞中的表达的方法，该方法包括给所述细胞施用本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、或包含本发明的小激活核酸分子的组合物或制剂。在一个实施方式中，激活/上调MITF基因在细胞中的表达的方法包括给所述细胞施用本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、或包含本发明的小激活核酸分子的组合物。上述细胞包括哺乳动物细胞，例如来自人体的细胞，如人黑色素细胞(HEM)和人皮肤角质形成细胞(NHEK)，上述细胞可以是离体的，也可以存在于哺乳动物体中，如人体中。
本发明的小激活核酸分子可以被直接导入细胞中，也可以是将编码本发明的小激活核酸分子的核酸序列导入细胞后在细胞内产生；所述细胞优选为哺乳动物细胞，更优选为人类细胞。上述细胞可以是离体的，如细胞系或细胞株等，也可以存在于哺乳动物体中，如人体中。该人体可以是具有与MITF蛋白表达不足或减少相关的疾病或状况的患者。本发明所述的小激活核酸分子可以被施以足够的量以实现对与MITF蛋白量的缺乏或与MITF蛋白表达不足或减少相关的疾病或状况的治疗。具体情况下，所述与MITF蛋白量的缺乏或与MITF表达不足或减少相关的疾病或状况可以包括例如白癜风等。
本发明另一方面提供了分离的MITF基因小激活核酸分子作用位点，该位点具有MITF基因的启动子区(SEQ ID NO：1)上任意连续的16-35个核苷酸的序列，优选情况下，所述作用位点包括或选自SEQ ID NO：299-305的任一条序列上的任意连续16-35个核苷酸的序列。具体地，所述作用位点可以包括或选自SEQ ID NO：8-104中的任一核苷酸序列。
本发明的另一方面涉及治疗个体中与MITF蛋白表达不足或减少相关的疾病或状况的方法，包括给所述个体施用治疗有效量的本发明的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物。在一个实施方式中，本发明的治疗个体中与MITF蛋白表达不足或减少相关的疾病或状况的方法包括给个体施用治疗有效量的本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物和治疗有效量的其它剂，所述其它剂包括例如小分子化合物、抗体、多肽、蛋白等。所述个体可以是哺乳动物，包括例如人。在一个实施方案中，与MITF蛋白表达不足或减少相关的疾病或状况可以包括例如白癜风。根据临床特征可分为寻常型白癜风和节段型白癜风，寻常型白癜风与自身免疫调节或氧化应激进一步造成黑色素细胞受损有关，节段型白癜风与遗传因素有关。白癜风与家族遗传、自身免疫调节、氧化应激和功能性黑色素细胞受损等因素有关。
本发明的另一方面涉及治疗个体中白癜风的方法，包括给所述个体施用治疗有效量的本发明的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物。在一个实施方式中，本发明的治疗白癜风的方法包括给个体施用治疗有效量的本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物和治疗有效量的其它剂，所述其它剂包括例如小分子化合物、抗体、多肽、蛋白等。所述个体可以是哺乳动物，包括例如人。在上述方法中，白癜风包括寻常型白癜风和节段型白癜风等。在上述方法中，个体包括哺乳动物，例如人。
本发明的另一方面涉及本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物在制备用于治疗与MITF蛋白表达不足或减少相关的疾病或状况的药物中的应用。所述个体可以是哺乳动物，例如人。在一个实施方案中，所述与MITF蛋白表达不足或减少相关的疾病可以包括例如白癜风等。优选地，与MITF蛋 白表达不足或减少相关的疾病或状况是白癜风，更优选地，与MITF蛋白表达不足或减少相关的疾病或状况包括寻常型白癜风和节段型白癜风等。
在一个实施方式中，提供了本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物可以用于制备治疗与MITF蛋白表达不足或减少相关的疾病如白癜风的药物。在一个实施方案中，所述与MITF蛋白表达不足或减少相关的疾病可以包括例如白癜风等。优选地，与MITF蛋白表达不足或减少相关的疾病或状况是白癜风，更优选地，与MITF蛋白表达不足或减少相关的疾病或状况包括寻常型白癜风和节段型白癜风等。
本发明的另一方面涉及本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物或制剂在制备用于治疗白癜风的药物或药物组合中的应用。优选地，白癜风包括寻常型白癜风和节段型白癜风等。
在一个实施方式中，提供了本发明所述的小激活核酸分子、编码本发明的小激活核酸分子的核酸、包含本发明的小激活核酸分子或编码本发明的小激活核酸分子的核酸的细胞、或包含本发明的小激活核酸分子的组合物或制剂在制备用于治疗与MITF蛋白表达不足或减少相关的疾病的药物或药物组合中的应用。
与现有技术相比，本发明具有以下一方面或几方面的有益效果：
本发明提供的能够激活/上调MITF基因表达的小激活核酸分子例如小激活RNA(saRNA)，能够持久地激活MITF基因，因而高效、特异地上调或恢复MITF基因和蛋白的表达并同时具有较低的毒副作用，可用于治疗与MITF蛋白表达不足或减少相关的疾病或病症例如白癜风，或者制备用于治疗与MITF蛋白表达不足或减少相关的疾病或病症的药物或制剂，例如瓦登伯格综合征2A型和蒂策综合征等。
附图说明
图1为saRNA介导的人MITF mRNA表达改变。靶向人MITF启动子的239个saRNA以25nM终浓度转染HEM细胞，转染72小时后用一步法RT-qPCR分析MITF mRNA表达。图示为 相对于对照处理(Mock)的MITF的mRNA表达改变(log2)从最高到最低排序。纵坐标值代表2个重复处理的平均值±SD。
图2为saRNA在人MITF启动子上的热点区域。靶向人MITF启动子的239个saRNA以25nM终浓度转染HEM细胞72小时，转染结束后用一步法RT-qPCR分析MITF mRNA表达。图示为相对于对照处理(Mock)的MITF表达改变，按照saRNA在MITF启动子上的靶点位置从-500到-0排序。黑色实心点表示功能性saRNA，白色空心点表示非功能性saRNA，虚线条框示功能性saRNA聚集的热点区(H1～H4)和次热点区(W1～W3)。纵坐标值代表2个重复处理的平均值±SD。
图3为二步法RT-qPCR验证高通量筛选结果。用图3所示saRNA以25nM终浓度转染HEM细胞72小时，转染结束后用QiagenRNeasy试剂盒提取RNA，反转录后用ABI 7500快速实时PCR系统进行qPCR扩增，同时扩增HPRT1基因作为内参。图示为单个saRNA处理细胞后MITF相对mRNA的表达值。Mock、dsCon2和RAG4-618i分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。纵坐标值代表2个重复处理的平均值±SD。
图4为二步法RT-qPCR验证高通量筛选结果。用图4所示saRNA以25nM终浓度转染NHEK细胞72小时，转染结束后用Qiagen RNeasy试剂盒提取RNA，反转录后用ABI 7500快速实时PCR系统进行qPCR扩增，同时扩增HPRT1基因作为内参。图示为单个saRNA处理细胞后MITF相对mRNA的表达值。Mock、dsCon2和RAG4-618i分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。纵坐标值代表2个重复处理的平均值±SD。
图5为saRNA促进人NHEK细胞中MITF蛋白表达。用图5所示saRNA以25nM终浓度转染NHEK细胞5天，收集细胞并提取细胞总蛋白后用western blot检测MITF蛋白表达水平，同时检测微管蛋白(α/β-Tubulin)作为内参。图示为单个saRNA处理的细胞的MITF相对蛋白表达值。Mock、dsCon2和RAG4-618i分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。
图6为saRNA促进HEM细胞中MITF蛋白表达。用图6所示saRNA以25nM终浓度转染HEM细胞72小时，收集细胞并提取细胞总蛋白后用western blot检测MITF蛋白表达水平，同时检测微管蛋白(α/β-Tubulin)作为内参。图示为单个saRNA处理的细胞的MITF相对蛋白表达值。Mock、dsCon2和RAG4-618i分别为空白转染、无关序列双链RNA转染及小干 扰RNA对照转染。
具体实施方式
在本发明中，相关术语采用如下定义：
如本文所用的术语“互补”是指两条寡核苷酸链彼此形成碱基对的能力。碱基对通常由反向平行的寡核苷酸链中的核苷酸之间通过氢键形成。互补寡核苷酸链可以沃森-克里克(Watson-Crick)方式碱基配对(例如，A-T，A-U，C-G)，或以允许形成双链体的任何其他方式(例如Hoogsteen型或者反向Hoogsteen型碱基配对)进行碱基配对。
互补包括完全互补和不完全互补两种情况。完全互补或100％互补是指双链寡核苷酸分子的双链区中来自第一条寡核苷酸链的每个核苷酸可以与第二条寡核苷酸链相应位置的核苷酸形成氢键而没有“错配”的情况。不完全互补是指两条链的核苷酸单元不能全部互相氢键结合的情况。例如，对于两条双链区为20个核苷酸长度的寡核苷酸链，如果每条链上只有两个碱基对可以彼此氢键结合，则寡核苷酸链展现出10％的互补性。在同一实例中，如果每条链上的18个碱基对可以彼此氢键结合，则寡核苷酸链展现出90％的互补性。实质互补是指至少约75％、约79％、约80％、约85％、约90％、约95％、约99％、或约100％的互补。
如本文所用的术语“寡核苷酸”是指核苷酸的聚合物，包括但不限于DNA，RNA或DNA/RNA杂交体的单链或双链分子，包括规则地和不规则地交替的脱氧核糖基部分和核糖基部分的寡核苷酸链，以及这些种类的寡核苷酸的修饰和以及天然存在的或非天然存在的骨架。本发明中所述的用于激活靶基因转录的寡核苷酸为小激活核酸分子。
如本文所用的术语“寡核苷酸链”和“寡核苷酸序列”可互换地使用，是指35个以下碱基的短链核苷酸的总称(包括脱氧核糖核酸DNA、核糖核酸RNA在内的核苷酸链，也包括由一个或多个脱氧核苷酸以及一个或多个核糖核苷酸共同形成的混合寡核苷酸链)。在本发明中，寡核苷酸链的长度可以是16至35个核苷酸的任一长度。
如本文所用，术语“第一寡核苷酸链”可以是正义链也可以是反义链，小激活RNA的正义链是指小激活RNA双链体中含与靶基因的启动子DNA序列的编码链具有同一性的核酸链，反义链是指小激活RNA双链体中与正义链互补的核酸链。
如本文所用，术语“第二寡核苷酸链”也可以是正义链或者反义链。当第一寡核酸链为正义链时，第二寡核酸链为反义链，当第一寡核酸链为反义链时，第二寡核酸链为正义链。
如本文所用的术语“基因”是指编码一条多肽链或转录一条功能RNA所需的全部核苷酸序列。“基因”可以是对于宿主细胞而言内源的或完全或部分重组的基因(例如，由于引入编码启动子的外源寡核苷酸和编码序列或将邻近内源编码序列的异源启动子导入宿主细胞)。例如，术语“基因”包括可以由外显子和内含子组成的核酸序列。编码蛋白质的序列是，例如，包含在起始密码子和终止密码子之间的开放阅读框中的外显子内的序列，如本文所用，“基因”可以指包括例如基因调控序列例如启动子，增强子和本领域已知的控制另一基因的转录，表达或活性的所有其他序列，无论另一基因是否包含编码序列或非编码序列。在一种情况下，例如，“基因”可以用于描述包含调控序列例如启动子或增强子的功能性核酸。重组基因的表达可以通过一种或多种异源调节序列来控制。
如本文所用的术语“靶基因”可以是天然存在于生物体中的核酸序列、转基因、病毒或细菌序列、染色体或染色体外和/或瞬时或稳定转染或掺入细胞和/或其染色质。靶基因可以为蛋白质编码基因，也可为非蛋白质编码基因(例如微小RNA基因、长链非编码RNA基因)。靶基因通常含有启动子序列，设计与启动子序列具有同一性(也称同源性)的小激活核酸分子可以实现对靶基因的正向调控，表现为靶基因表达的上调。“靶基因启动子序列”是指靶基因的非编码序列，在本发明中涉及“与靶基因启动子序列互补”中靶基因启动子序列是指该序列的编码链，亦称非模板链，即为与该基因编码序列为同一序列的核酸序列。“靶点”或“靶点序列”是指靶基因启动子序列中小激活核酸分子的正义寡核苷酸链或反义寡核苷酸与之同源或互补的序列片段。
如本文所用，术语“正义链”、“正义核酸链”可互换地使用，小激活核酸分子的正义寡核苷酸链是指小激活核酸分子双链体中含与靶基因的启动子序列的编码链具有同一性的第一寡核苷酸链。
如本文所用，术语“反义链”、“反义核酸链”可互换地使用，小激活核酸分子的反义寡核苷酸链是指小激活核酸分子双链体中与正义寡核苷酸链互补的第二寡核苷酸链。
如本文所用的术语“编码链”是指靶基因中不能进行转录的那一条DNA链，该链的核 苷酸序列与转录生成的RNA的序列一致(在RNA中是以U取代了DNA中的T)。本发明中所述的靶基因启动子双链DNA序列的编码链是指与靶基因DNA编码链在同一条DNA链上的启动子序列。
如本文所用的术语“模板链”是指靶基因的双链DNA中与编码链互补的另一条链，可作为模板转录为RNA的那条链，该链与转录的RNA碱基互补(A-U，G-C)。在转录过程中，RNA聚合酶与模板链结合，并沿着模板链的3′→5′方向移动，按照5′→3′方向催化RNA的合成。本发明中所述的靶基因启动子双链DNA序列的模板链是指与靶基因DNA模板链在同一条DNA链上的启动子序列。
如本文所用的术语“启动子”是指通过与蛋白质编码或RNA编码核酸序列在位置上关联而对它们的转录发挥调控作用的序列。通常，真核基因启动子包含100～5,000个碱基对，尽管此长度范围并不意味着限制本文所用的术语“启动子”。虽然启动子序列一般位于蛋白质编码或者RNA编码序列的5′端，但启动子序列也可存在于外显子及内含子序列中。
如本文所用的术语“转录起始位点”是指在基因的模板链上标志转录起始的核苷酸。转录起始位点可出现于启动子区的模板链上。一个基因可以有多于一个的转录起始位点。
如本文所用的术语“同一性”或“同源性”是指小激活RNA的其中一条寡核苷酸链(正义链或者反义链)与靶基因的启动子序列的某一区域的编码链或者模板链存在的相似性。在本文中，所述“同一性”或“同源性”可以是至少约75％、约79％、约80％、约85％、约90％、约95％、约99％或约100％。
如本文所用的术语“突出”、“overhang”、“悬垂”可互换地使用，是指寡核苷酸链末端(5′或3′)非碱基配对核苷酸，其是由延伸超出双链寡核苷酸内的其中一条链的另一条链产生的。延伸超出双链体3′和/或5′端的单链区域被称为突出。
如本文所用，术语“基因激活”或“激活基因”或“基因上调”或“上调基因”可互换地使用，是指通过测量基因转录水平、mRNA水平、蛋白水平、酶活性、甲基化状态、染色质状态或构型、翻译水平、或其在细胞或生物系统中的活性或状态来测定某一核酸转录、翻译或表达或活性的增加。这些活动或状态可以直接或间接的测定。此外，“基因激活”、“激活基因”、“基因上调”、“上调基因”是指与核酸序列相关的活性增加，而不管发生这种激活的机制如何，例如其作为调节序列发挥调控作用、被转录成RNA，被翻译为蛋白质并增 加蛋白质的表达。
如本文所用，术语“小激活RNA”、“saRNA”、“小激活核酸分子”可互换地使用，是指能够促进基因表达的核酸分子，并且可以由包含与靶基因的非编码核酸序列(例如启动子、增强子等)具有序列同一性或同源性的核苷酸序列的第一核酸片段(反义核酸链，也称反义寡核苷酸链)和包含与第一核酸片段互补的核苷酸序列的第二核酸片段(正义核酸链，也称有义链或正义寡核苷酸链)组成，其中所述第一核酸片段和第二核酸片段形成双链体。小激活核酸分子也可以由合成的或者载体表达的可形成双链区发夹结构的单链RNA分子组成，其中第一区域包含与基因的启动子靶序列具有序列同一性的核苷酸序列，第二区域包含的核苷酸序列与第一区域互补。小激活核酸分子的双链体区域长度通常为约10至约50个碱基对、约12个至约48个碱基对、约14个至约46个碱基对、约16个至约44个碱基对、约18个至约42个碱基对、约20个至约40个碱基对、约22个至约38个碱基对、约24个至约36个碱基对、约26个至约34个碱基对、约28个至约32个碱基对、通常约10个、约15个、约20、约25、约30、约35、约40、约45、约50个碱基对。此外，术语“saRNA”’、“小激活RNA”和“小激活核酸分子”还含有除核糖核苷酸部分之外的核酸，包括但不限于修饰的核苷酸或类似物。
如本文所用，术语“热点(hot spot，H区)”是指长度至少为25bp的基因启动子区域，在这些区域，呈现出功能性小激活核酸分子靶点的聚集，即靶向这些热点区域的小激活核酸分子至少30％能够诱导靶基因mRNA表达达到1.1倍及以上；术语“次热点(warm spot，W区)”是指长度至少为25bp的基因启动子区域，在这些区域，呈现出功能性小激活核酸分子靶点的聚集，即靶向这些热点区域的小激活核酸分子8-30％能够诱导靶基因mRNA表达达到1.1倍及以上。
如本文所用，术语“合成”是指寡核苷酸的合成方式，包括任何能够合成RNA的方式，例如化学合成、体外转录、载体表达等。
本发明通过RNA激活方式上调MITF基因的表达，通过增加MITF蛋白的表达量来治疗相关疾病，尤其是白癜风。本发明中MITF基因有时也称为靶基因。
本发明提供的小激活核酸分子的制备方法包括序列设计和序列合成。
本发明的小激活核酸分子序列的合成可以采用化学合成的方法，或者委托专门从事 核酸合成的生物技术公司合成。
一般来说，化学合成的方法包括以下四个过程：(1)寡聚核糖核苷酸的合成；(2)脱保护；(3)纯化分离；(4)脱盐及退火。
例如，本发明所述saRNA化学合成的具体步骤如下：
(1)寡聚核糖核苷酸的合成
在自动DNA/RNA合成仪(例如，Applied Biosystems EXPEDITE8909)上设定合成1微摩尔的RNA，同时设定每个循环的偶联时间为10-15分钟，起始物为固相连接的5’-O-对二甲氧基三苯甲基-胸苷支持物，第一个循环在固相支持物上连接一个碱基，然后在第n次(19≥n≥2)循环中，在第n-1次循环所连接的碱基上连接一个碱基，重复此循环直至完成全部核酸序列的合成。
(2)脱保护
将连接有saRNA的固相支持物加入到试管中，并在此试管中加入1毫升的乙醇/氨水溶液(体积比为1∶3)，然后密封，置于25-70℃温箱中，孵育2-30小时，过滤含有saRNA的固相支持物的溶液并收集滤液，用双蒸水淋洗固相支持物2次(每次1毫升)并收集滤液，合并收集洗脱液，在真空条件下干燥1-12小时。然后，加入1毫升四丁基氟化铵的四氢呋喃溶液(1M)，室温放置4-12小时，再加入2毫升正丁醇，高速离心收集沉淀即得到saRNA单链的粗产物。
(3)纯化分离
将得到的saRNA的粗产物溶解于2毫升浓度为1摩尔/升的三乙胺乙酸盐溶液中，然后通过高压液相色谱反相C18柱进行分离，得到纯化的saRNA单链产物。
(4)脱盐及退火
用体积排阻凝胶过滤法去除盐份，将正义链和反义链的寡聚核糖核酸单链按相同摩尔比混合在1-2毫升的缓冲液中(10mM Tris，pH＝7.5-8.0，50mM NaCl)，将此溶液加热至95℃，然后缓缓将此溶液冷却至室温，得到含有saRNA的溶液。
本研究发现，将上述saRNA导入细胞后，能够有效增加MITF mRNA和蛋白质的表达。
下面结合具体实施例及附图，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常 规条件，例如Sambrook等，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为对本公开的范围的限定。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1靶向人MITF基因启动子的saRMA的设计与合成
为了筛选能够激活MITF基因表达的功能性小激活RNA，以MITF 500bp的启动子序列为模板，从TSS上游-500bp处开始选定大小为19bp的靶点。然后对靶点序列进行过滤处理，保留靶点序列的标准为：1)GC含量在40％～65％之间；2)不含有5个或者多于5个的连续同一核苷酸；3)不含多于3个的二核苷酸重复序列；4)不含多于3个的三核苷酸重复序列。过滤后，剩余239个靶点序列作为候选进入筛选过程。基于这些候选序列，化学合成相应的双链小激活RNA。
其中，该实验中使用的双链小激活RNA的正义和反义链的长度均为21个核苷酸，所述双链saRNA的第一核糖核酸链(正义链)的5’区域的19个核苷酸与启动子靶点序列具有100％的同一性，其3’末端含有TT序列；第二核糖核酸链的5’区域的19个核苷酸与第一核糖核酸链序列互补，其3’末端含有TT序列。将前述双链saRNA的两条链以同等量的摩尔数混合，退火后形成双链saRNA。
人MITF启动子序列如下所示，其对应于序列表中SEQ ID NO：1从5’至3’的位置1至位置500：
Figure PCTCN2021091834-appb-000001
Figure PCTCN2021091834-appb-000002
实施例2靶向人MITF启动子的saRNA的高通量筛选
(1)细胞培养和转染
人黑色素细胞(HEM)(购自北京北纳创联生物科技有限公司，BNCC350795)和正常人皮肤角质形成细胞(NHEK)(购自北京北纳创联生物科技有限公司，BNCC340593)培养在DMEM培养基(Gibco)中，培养基含有10％小牛血清(Sigma-Aldrich)和1％青霉素/链霉素(Gibco)。细胞在5％的CO 2，37℃条件下培养。HEM细胞以每孔2000个细胞铺板到96孔板中，依照制造商的说明，使用RNAiMax(Invitrogen，Carlsbad，CA)以25nM(除非另有说明)浓度转染小激活RNA，转染时间为72小时，每种处理使用2个复孔。
(2)一步法RT-qPCR
转染结束后，弃掉培养基，每孔加入150μl PBS清洗一次，弃掉PBS，每孔加入50μl细胞裂解液，室温孵育5分钟。每孔取1μl细胞裂解液使用一步法TB GreenTM PrimeScripTM RT-PCR试剂盒II(Takara，RR086A)。
在ABI 7500快速实时(FastReal-time)PCR系统(AppliedBiosystems)进行qPCR分析，每个样本重复3个复孔扩增，PCR反应条件见表1。
表1 PCR反应制备
试剂 体积
2×一步法TB Green RT-PCR缓冲液4 2.5μl
PrimeScript 1step酶混合物2 0.2μl
正向和反向引物混合物(5μM) 0.4μl
无RNase dH 2O 1.4μl
粗制裂解物(RNA) 0.5μl
总计 5μl
反应条件为：阶段1-反转录反应：42℃，5分钟；95℃10秒；阶段2-PCR反应：95℃ 5秒，60℃ 20秒，扩增45个循环。以HPRT1及TBP为内参基因。MITF、HPRT1及TBP所用PCR引物见表2，其中MITF用MITF F1/R1引物对扩增。
表2 RT-qPCR分析的引物序列
Figure PCTCN2021091834-appb-000003
为了计算某个saRNA转染样本的MITF(目的基因)的相对于对照处理(Mock)的表达值(Erel)，用公式1代入目的基因及2个内参基因的Ct值计算。
E rel＝2( CtTm-CtTs)/((2 (CtR1m-CtR1s)*2 (CtR2m-CtR2s)) (1/2))   (从式1)
其中，CtTm为来自对照处理(Mock)样本的目的基因的Ct值，CtTs为来自saRNA处理样本的目的基因的Ct值，CtR1m为来自Mock处理样本的内参基因1的Ct值，CtR1s为来自saRNA处理样本的内参基因1的Ct值，CtR2m为来自对照处理样本的内参基因2的Ct值，CtR2s为来自saRNA处理样本的内参基因2的Ct值。
(3)功能性saRNA筛选
为了获得能够激活MITF转录的saRNA，用上述239个saRNA分别转染HEM细胞，转染浓度为25nM，转染72小时后以如上所述相同的方法，裂解细胞并进行一步法RT-qPCR分析，得到每个saRNA处理样本的MITF基因的相对(与对照处理组比较)表达值。如表3所示，有29(12.1％)个saRNA显示出高度激活作用(≥1.5倍)，68(28.5％)个saRNA显示出轻度激活作用(≥1.1倍)，有142(59.4％)个saRNA对MITF的表达不产生显著的上调影响。激活的最大幅度为2.75倍，最大抑制幅度为0.38倍，这些具有激活活性的saRNA被称为功能性saRNA。
表3人MITF saRNA高通量筛选结果统计
Figure PCTCN2021091834-appb-000004
如图1所示，进一步显示了人MITF saRNA的MITF表达改变从最高到最低排序。其中MITF活性saRNA序列(功能性saRNA序列)、活性靶点序列以及MITF mRNA表达改变，如表4所示(表4所列出的各活性靶点序列对应序列表中的SEQ NO：8-104，各saRNA正义序列对应序列表中的SEQ ID NO：105-201，各saRNA反义序列对应序列表中的SEQ ID NO：202-298)。表5所示为功能性saRNA所处的不同热点和次热点区域及相关序列(SEQ ID NO：299-305)。
表4功能性saRNA序列、其活性靶点序列以及其导致的MITF mRNA表达改变
Figure PCTCN2021091834-appb-000005
Figure PCTCN2021091834-appb-000006
Figure PCTCN2021091834-appb-000007
Figure PCTCN2021091834-appb-000008
Figure PCTCN2021091834-appb-000009
表5功能性saRNA的热点和次热点区域及序列
Figure PCTCN2021091834-appb-000010
实施例3 saRNA促进人黑色素细胞(HEM)中MITF mRNA的表达
(1)细胞培养和转染
细胞培养如实施例2所述，人黑色素细胞(HEM)以每孔20×104铺板到六孔板中，以25nM终浓度转染小激活RNA，转染72小时，每种处理使用2个复孔。
(2)二步法RT-qPCR
转染结束后，弃掉培养基，每孔加入500μl细胞裂解液，室温孵育5分钟。用Qiagen RNeasy试剂盒提取RNA，反转录后在ABI 7500快速实时(Fast Real-time)PCR系统(Applied Biosystems)进行qPCR分析，每个样本重复3个复孔扩增，PCR反应条件见表6和表7。
表6 RT反应制备
Figure PCTCN2021091834-appb-000011
Figure PCTCN2021091834-appb-000012
表7 RT-qPCR反应制备
试剂 体积
SYBR Premix Ex Taq II(2×) 5μl
ROX参考染料II(50×) 0.2μl
正向和反向引物混合物(5μM) 0.8μl
cDNA(RT产物) 4μl
总计 10μl
反应条件为：95℃ 30秒，95℃ 5秒，60℃ 30秒，扩增40个循环。同时扩增HPRT1基因作为内参，其中MITF用MITF F1/R1引物对扩增，扩增引物见表2。
为了计算某个saRNA转染样本的MITF(目的基因)的相对于对照处理(Mock)的表达值(Erel)，用公式2代入目的基因及1个内参基因的Ct值计算。
E rel＝2 (CtTm-CtTs)/2 (CtRm-CtRs)  (公式2)
其中，CtTm为来自对照处理(Mock)样本的目的基因的Ct值，CtTs为来自saRNA处理样本的目的基因的Ct值，CtRm为来自Mock处理样本的内参基因的Ct值，CtRs为来自saRNA处理样本的内参基因的Ct值。
图3所示为不同saRNA处理后细胞中MITF相对mRNA的表达值。以对照组(Mock)作为空白转染对照，与对照组相比，siRNA(RAG4-618i)组的mRNA相对表达值敲降了81.0％，这说明siRNA作为小干扰RNA对照转染是成功的。与对照组相比，不同saRNA处理细胞后其MITF mRNA的表达均有升高，尤其是RAG4-175，RAG4-176和RAG4-290分别上调了MITF表达达3.9倍、3.1倍和6.1倍。其它的saRNA(RAG4-284，RAG4-123， RAG4-396，RAG4-461，RAG4-490，RAG4-316和RAG4-318)增加了MITFmRNA相对表达达到2倍左右。这说明随机选择的功能性saRNA其活性在HEM细胞能够被验证。
实施例4 saRNA促进NHEK细胞中MITF mRNA的表达
细胞培养如实施例2所述，人皮肤角质化形成细胞(NHEK)以每孔20×104铺板到六孔板中，以25nM终浓度转染小激活RNA，转染72小时，每种处理使用2个复孔。二步法RT-qPCR如实施例3所述。
如图4所示为不同saRNA处理后细胞中MITF相对mRNA的表达值。以对照组(Mock)作为空白转染对照，与对照组相比，siRNA(RAG4-618i)组的mRNA相对表达值下降了88.5％，这说明siRNA作为小干扰RNA对照转染是成功的。与对照组相比，经RAG4-290处理后的激活效果尤为明显，MITF相对mRNA的表达值上升了13.1倍。RAG4-175，RAG4-176和RAG4-316组的mRNA相对表达值均高于对照组，其mRNA相对表达值分别上升了5.7倍、3.7倍和3.5倍，有明显的激活效果。RAG4-284，RAG4-123，RAG4-396，RAG4-461，RAG4-490和RAG4-318组的mRNA相对表达值大约高于对照组2倍，也有一定的激活效果。在这说明随机选择的功能性saRNA的活性在NHEK细胞能够被验证。
实施例5 saRNA促进NHEK细胞中MITF蛋白的表达
细胞培养如实施例2所述，人黑色素细胞(NHEK)以每孔20×104铺板到六孔板中，以25nM终浓度转染小激活RNA，转染5天。使用适量含有蛋白酶抑制剂的细胞裂解液(1×RIPA缓冲液，Cell Signaling Technology)裂解。BCA法进行蛋白质定量，随后进行聚丙烯酰胺凝胶电泳分离并转至0.45μm的PVDF膜。所用一抗为：兔单克隆抗MITF(Cell Signaling Technology，#12590)，α/β-微管蛋白抗体(Cell Signaling Technology，2148s)对印迹进行检测；二抗用抗兔IgG，HRP-连接的抗体(Cell Signaling Technology)。用Image Lab(BIO-RAD，Chemistry Doctm MP Imaging System)扫膜检测信号。
如图5所示为不同saRNA处理后细胞中MITF蛋白的相对表达量。与对照组相比，siRNA(RAG4-618i)组的MITF蛋白相对表达量下降了约50％，这说明siRNA作为小干扰RNA对照转染是成功的。与对照组相比，各saRNA均能提高MITF蛋白的表达量，其中，RAG4-416，RAG4-490和RAG4-318上调MITF蛋白表达量均在1.5倍以上，尤以 RAG4-318激活效果最为明显，其增加MITF蛋白表达量达1.71倍。这说明随机选择的功能性saRNA的活性可以在蛋白水平被验证。
实施例6 saRNA促进人黑色素细胞(HEM)中MITF蛋白的表达
细胞培养如实施例2所述，人黑色素细胞(HEM)以每孔20×104铺板到六孔板中，以25nM终浓度转染小激活RNA，转染72小时。蛋白裂解及检测方法如实施例5所述。
如图6所示为不同saRNA处理后细胞中MITF蛋白的相对表达量。与对照组相比，各saRNA均能提高MITF蛋白的表达量，RAG4-396和RAG4-490组的蛋白表达量上升了1.4倍。RAG4-175，RAG4-176，RAG4-290，RAG4-284和RAG4-123各组MITF蛋白表达量均高于对照组，其激活水平均在1.5倍以上，激活效果明显。这说明随机选择的功能性saRNA的活性可以在蛋白水平被验证。
综合上述结果，申请人通过高通量筛选靶向人MITF基因启动子的saRNA，发现了多个能够显著激活MITF基因表达的人saRNA。这些saRNA能够在mRNA和蛋白表达层面上调细胞内MITF基因的表达，可用于MITF蛋白表达下降或者不足引发的疾病或病症，如白癜风，或用于制备治疗上述疾病或病症的方法或药物。
以上已经描述了本公开的各实施例，上述说明是示例性的，并非穷尽性的，并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下，对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。本文中所用术语的选择，旨在最好地解释各实施例的原理、实际应用或对市场中的技术改进，或者使本技术领域的其它普通技术人员能理解本文披露的各实施例。
参考文献：
1.Picardo，M.，M.L.Dell′Anna，K.Ezzedine，I.Hamzavi，J.E.Harris，D.Parsad，and A.Taieb.2015.′Vitiligo′，Nat Rev Dis Primers，1：15011.
2.Gauthier，Y.，M.Cario Andre，and A.Taieb.2003.′A critical appraisal of vitiligo etiologic theories.Is melanocyte loss a melanocytorrhagy？′，Pigment Cell Res，16：322-32.
3.Dell′anna，M.L.，and M.Picardo.2006.′A review and a new hypothesis for non-immunological pathogenetic mechanisms in vitiligo′，Pigment Cell Res，19：406-11.
4.Sehgal，V.N.，and G.Srivastava.2007.′Vitiligo：compendium of clinico-epidemiological  features′，Indian J Dermatol Venereol Leprol，73：149-56.
5.Kotobuki，Y.，A.Tanemura，L.Yang，S.Itoi，M.Wataya-Kaneda，H.Murota，M.Fujimoto，S.Serada，T.Naka，and I.Katayama.2012.′Dysregulation of melanocyte function by Th17-related cytokines：significance of Th17 cell infiltration in autoimmune vitiligo vulgaris′，Pigment Cell Melanoma Res，25：219-30.
6.Kemp，E.H.，N.G.Gavalas，D.J.Gawkrodger，and A.P.Weetman.2007.′Autoantibody responses to melanocytes in the depigmenting skin disease vitiligo′，Autoimmun Rev，6：138-42.7.Dell′Anna，M.L.，M.Ottaviani，V.Albanesi，A.P.Vidolin，G.Leone，C.Ferraro，A.Cossarizza，L.Rossi，and M.Picardo.2007.′Membrane lipid alterations as a possible basis for melanocyte degeneration in vitiligo′，J Invest Dermatol，127：1226-33.
8.Kumar，R.，D.Parsad，and A.J.Kanwar.2011.′Role of apoptosis and melanocytorrhagy：a comparative study of melanocyte adhesion in stable and unstable vitiligo′，Br J Dermatol，164：187-91.
9.Hemesath，T.J.，E.Steingrimsson，G.McGill，M.J.Hansen，J.Vaught，C.A.Hodgkinson，H.Arnheiter，N.G.Copeland，N.A.Jenkins，and D.E.Fisher.1994.′microphthalmia，a critical factor in melanocyte development，defines a discrete transcription factor family′，Genes Dev，8：2770-80.
10.Levy，C.，M.Khaled，and D.E.Fisher.2006.′MITF：master regulator of melanocyte development and melanoma oncogene′，Trends Mol Med，12：406-14.
11.Kwinter，J.，J.Pelletier，A.Khambalia，and E.Pope.2007.′High-potency steroid use in children with vitiligo：a retrospective study′，J Am Acad Dermatol，56：236-41.
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.
13.Lim，H.W.，P.E.Grimes，O.Agbai，I.Hamzavi，M.Henderson，M.Haddican，R.V.Linkner，and M.Lebwohl.2015.′Afamelanotide and narrowband UV-B phototherapy for the treatment of vitiligo：a randomized multicenter trial′，JAMA Dermatol，151：42-50.
14.Grimes，P.E.，I.Hamzavi，M.Lebwohl，J.P.Ortonne，and H.W.Lim.2013.′The efficacy of afamelanotide and narrowband UV-B phototherapy for repigmentation of vitiligo′，JAMA Dermatol，149：68-73.

Claims (28)

  1. 一种小激活核酸分子，所述小激活核酸分子至少包含第一寡核苷酸链，所述第一寡核苷酸链与人MITF基因启动子区的任一长度为16-35个核苷酸的连续片段具有至少75％的同源性或互补性。
  2. 根据权利要求1所述的小激活核酸分子，其中所述小激活核酸分子包含第一寡核苷酸链和第二寡核苷酸链，所述第一寡核苷酸链和第二寡核苷酸链通过完全互补或不完全互补形成双链结构。
  3. 根据权利要求2所述的小激活核酸分子，其中所述第一寡核苷酸链与SEQ ID NO：299、SEQ ID NO：300、SEQ ID NO：301、SEQ ID NO：302、SEQ ID NO：303、SEQ ID NO：304或SEQ ID NO：305中的任一长度为16-35个核苷酸的连续片段具有至少75％的同源性或互补性。
  4. 根据权利要求2所述的小激活核酸分子，其中所述第一寡核苷酸链与选自SEQ ID NO：8-104的任一核苷酸序列具有至少75％的同源性或互补性。
  5. 根据权利要求4所述的小激活核酸分子，其中所述第一寡核苷酸链包括选自SEQ ID NO：105-201的任一核苷酸序列，或所述第二寡核苷酸链包括选自SEQ ID NO：202-298的任一核苷酸序列。
  6. 根据权利要求2-5任一项所述的小激活核酸分子，其中所述第一寡核苷酸链的长度为16-35个核苷酸，所述第二寡核苷酸链的长度为16-35个核苷酸。
  7. 根据权利要求2-6任一项所述的小激活核酸分子，其中所述小激活核酸分子为双链核酸，所述第一寡核苷酸链和所述第二寡核苷酸链分别位于所述双链核酸的两条链上。
  8. 根据权利要求7所述的小激活核酸分子，其中所述第一寡核苷酸链和/或所述第二寡核苷酸链在5’端和/或3’端具有突出。
  9. 根据权利要求8所述的小激活核酸分子，其中所述突出为0-6个核苷酸的突出。
  10. 根据权利要求9所述的小激活核酸分子，其中所述突出为dTdT或dTdTdT。
  11. 根据权利要求2-6任一项所述的小激活核酸分子，其中所述小激活核酸分子为单链核酸，所述单链核酸中具有可形成双链区的发夹结构，所述第一寡核苷酸链和所述第二寡核苷酸链具有可形成双链结构的互补区域。
  12. 根据权利要求2-11任一项所述的小激活核酸分子，其中所述第一寡核苷酸链和所述第二寡核苷酸链具有至少75％的互补性。
  13. 根据权利要求1-12任一项所述的小激活核酸分子，其中构成所述小激活核酸分子的核苷酸为天然的未经化学修饰的核苷酸。
  14. 根据权利要求1-12任一项所述的小激活核酸分子，其中所述小激活核酸分子的一个或多个核苷酸为具有化学修饰的核苷酸。
  15. 根据权利要求14所述的小激活核酸分子，其中所述化学修饰为选自以下修饰的一种或多种：对核苷酸的磷酸二酯键的修饰、对核苷酸中核糖的2’-OH的修饰、对核苷酸中碱基的修饰。
  16. 根据权利要求14所述的小激活核酸分子，其中所述化学修饰为选自以下修饰的一种或多种：硫代磷酸修饰、硼烷化磷酸盐修饰、2’-氟代修饰、2’-氧甲基修饰、2’-氧亚乙基甲氧基修饰、2，4’-二硝基苯酚修饰、锁核酸修饰、2’-氨基修饰、2’-脱氧修饰、5′-溴尿嘧啶修饰、5′-碘尿嘧啶修饰、N-甲基脲嘧啶修饰、2，6-二氨基嘌呤修饰。
  17. 根据权利要求14-16任一项所述的小激活核酸分子，其中所述第一寡核苷酸链和/或所述第二寡核苷酸链的末端连接亲脂性基团，所述亲脂性基团为选自脂质体、高分子化合物、多肽或胆固醇的一种或多种。
  18. 一种核酸分子，其包含编码根据权利要求1-13任一项所述的小激活核酸分子的片段。
  19. 根据权利要求18所述的核酸分子，其中所述核酸分子为表达载体。
  20. 一种细胞，其包含根据权利要求1-17任一项所述的小激活核酸分子或根据权利要求18-19任一项所述的核酸分子。
  21. 一种药物组合物，包含：
    根据权利要求1-17任一项所述的小激活核酸分子或根据权利要求18-19任一项所述的核酸分子，和
    任选地，药学上可接受的载体。
  22. 一种制剂，其包含根据权利要求1-17任一项所述的小激活核酸分子，或根据权利要求18-19任一项所述的核酸分子，或根据权利要求20所述的细胞，或根据权利要求21所 述的药物组合物。
  23. 一种试剂盒，其包含根据权利要求1-17任一项所述的小激活核酸分子，或根据权利要求18-19任一项所述的核酸分子，或根据权利要求20所述的细胞，或根据权利要求21所述的药物组合物。
  24. 根据权利要求1-17任一项所述的小激活核酸分子，或根据权利要求18-19任一项所述的核酸分子，或根据权利要求20所述的细胞，或根据权利要求21所述的药物组合物在制备用于激活或上调MITF基因在细胞中的表达的药物或制剂中的用途。
  25. 一种激活或上调MITF基因在细胞中的表达的方法，所述方法包括向所述细胞使用根据权利要求1-17任一项所述的小激活核酸分子，或根据权利要求18-19任一项所述的核酸分子，或根据权利要求21所述的组合物，或根据权利要求22所述的试剂。
  26. 一种治疗与MITF蛋白表达不足或减少相关的疾病或状况的方法，所述方法包括向有需要的个体施用根据权利要求1-17任一项所述的小激活核酸分子，或根据权利要求18-19任一项所述的核酸分子，或根据权利要求21所述的组合物，或根据权利要求22所述的制剂。
  27. 根据权利要求1-17任一项所述的小激活核酸分子，或根据权利要求18-19任一项所述的核酸分子，或根据权利要求20所述的细胞，或根据权利要求21所述的药物组合物在制备用于治疗与MITF蛋白表达不足或减少相关的疾病或状况的药物中的用途。
  28. 根据权利要求27所述的用途，其中，所述与MITF蛋白表达不足或减少相关的疾病或状况为白癜风，或瓦登伯格综合征2A型，或蒂策综合征。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102429894A (zh) * 2011-12-07 2012-05-02 中国药科大学 氟西汀治疗色素脱色疾病的用途
CN106032532A (zh) * 2015-03-17 2016-10-19 中国医学科学院北京协和医院 一种小激活rna及其制备方法和应用
CN109071603A (zh) * 2016-02-18 2018-12-21 凯尔格恩有限公司 呈现黑色素生成促进活性的肽及其的用途
CN110959042A (zh) * 2018-04-10 2020-04-03 中美瑞康核酸技术(南通)研究院有限公司 一种新型小激活rna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102429894A (zh) * 2011-12-07 2012-05-02 中国药科大学 氟西汀治疗色素脱色疾病的用途
CN106032532A (zh) * 2015-03-17 2016-10-19 中国医学科学院北京协和医院 一种小激活rna及其制备方法和应用
CN109071603A (zh) * 2016-02-18 2018-12-21 凯尔格恩有限公司 呈现黑色素生成促进活性的肽及其的用途
CN110959042A (zh) * 2018-04-10 2020-04-03 中美瑞康核酸技术(南通)研究院有限公司 一种新型小激活rna

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
DELL'ANNA, M. L.M. OTTAVIANIV. ALBANESIA. P. VIDOLING. LEONEC. FERRAROA. COSSARIZZAL. ROSSIM. PICARDO: "Membrane lipid alterations as a possible basis for melanocyte degeneration in vitiligo", J INVEST DERMATOL, vol. 127, 2007, pages 1226 - 33
DELL'ANNA, M. L.M. PICARDO: "A review and a new hypothesis for non-immunological pathogenetic mechanisms in vitiligo", PIGMENT CELL RES, vol. 19, 2006, pages 406 - 11
GAUTHIER, YM. CARIO ANDREA. TAIEB: "A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy?", PIGMENT CELL RES, vol. 16, 2003, pages 322 - 32
GRIMES, P. E.I. HAMZAVIM. LEBWOHLJ. P. ORTONNEH. W. LIM: "The efficacy of afamelanotide and narrowband UV-B phototherapy for repigmentation of vitiligo", JAMA DERMATOL, vol. 149, 2013, pages 68 - 73, XP008162255, DOI: 10.1001/2013.jamadermatol.386
HEMESATH, T. J.E. STEINGRIMSSONG. MCGILLM. J. HANSENJ. VAUGHTC. A. HODGKINSONH. ARNHEITERN. G. COPELANDN. A. JENKINSD. E. FISHER: "microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family", GENES DEV, vol. 8, 1994, pages 2770 - 80, XP000909088
IANNELLA, G.A. GRECOD. DIDONAB. DIDONAG. GRANATAA. MANNOB. PASQUARIELLOG. MAGLIULO: "Vitiligo: Pathogenesis, clinical variants and treatment approaches", AUTOIMMUN REV, vol. 15, 2016, pages 335 - 43, XP029432853, DOI: 10.1016/j.autrev.2015.12.006
KEMP, E. H.N. G. GAVALASD. J. GAWKRODGERA. P. WEETMAN: "Autoantibody responses to melanocytes in the depigmenting skin disease vitiligo", AUTOIMMUN REV, vol. 6, 2007, pages 138 - 42, XP005876573, DOI: 10.1016/j.autrev.2006.09.010
KOTOBUKI, YA. TANEMURAL. YANGS. ITOIM. WATAYA-KANEDAH. MUROTAM. FUJIMOTOS. SERADAT. NAKAI. KATAYAMA: "Dysregulation of melanocyte function by Th17-related cytokines: significance of Thl7 cell infiltration in autoimmune vitiligo vulgaris", PIGMENT CELL MELANOMA RES, vol. 25, 2012, pages 219 - 30
KUMAR, R.D. PARSADA. J. KANWAR: "Role of apoptosis and melanocytorrhagy: a comparative study of melanocyte adhesion in stable and unstable vitiligo", BR J DERMATOL, vol. 164, 2011, pages 187 - 91
KWINTER, J.J. PELLETIERA. KHAMBALIAE. POPE: "High-potency steroid use in children with vitiligo: a retrospective study", JAM ACAD DERMATOL, vol. 56, 2007, pages 236 - 41, XP005860328, DOI: 10.1016/j.jaad.2006.08.017
LEVY, C.M. KHALEDD. E. FISHER: "MITF: master regulator of melanocyte development and melanoma oncogene", TRENDS MOL MED, vol. 12, 2006, pages 406 - 14, XP028058641, DOI: 10.1016/j.molmed.2006.07.008
LIM, H. W., P. E. GRIMES, O. AGBAI, I. HAMZAVI, M. HENDERSON, M. HADDICAN, R. V. LINKNER, M. LEBWOHL: "Afamelanotide and narrowband UV-B phototherapy for the treatment of vitiligo: a randomized multicenter trial", JAMA DERMATOL, vol. 151, 2015, pages 42 - 50, XP009186730, DOI: 10.1001/jamadermatol.2014.1875
PICARDO, M., M. L. DELL'ANNA, K. EZZEDINE, I. HAMZAVI, J. E. HARRIS, D. PARSAD, A. TAIEB.: "Vitiligo", NAT REV DIS PRIMERS, vol. 1, 2015, pages 15011
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SEHGAL, V N.G. SRIVASTAVA: "Vitiligo: compendium of clinico-epidemiological features", INDIAN J DERMATOL VENEREOL LEPROL, vol. 73, 2007, pages 149 - 56

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