Double-stranded siRNA molecules and uses thereof

Abstract

The application discloses a double-stranded siRNA molecule and application thereof. The double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence as shown in SEQ ID NO. 39 or a nucleotide sequence with at least 90% sequence identity with SEQ ID NO. 39, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 40 or a nucleotide sequence with at least 90% sequence identity with SEQ ID NO. 40. The double-stranded siRNA molecule of the application considers sequence specificity, nucleic acid stability, local tissue action efficiency and application safety, and can effectively overcome problems in existing whitening or anti-pigmentation technologies, such as relatively indirect target points, insufficient curative effect maintenance, easy recurrence and long-term use limitation and the like. The conjugate of the application can effectively down-regulate MITF expression, inhibit melanin synthesis, and is used for whitening, improving or relieving yellow-brown spots and other pigmentation.

Description

Technical Field

[0001] This invention relates to a double-stranded siRNA molecule and its applications, belonging to the field of biomedicine. Background Technology

[0002] Skin color is primarily determined by the synthesis, maturation, and transport of melanin within melanocytes, as well as the distribution of melanin granules within keratinocytes. Factors such as ultraviolet radiation, inflammation, endocrine changes, genetic factors, and abnormal skin barrier function can all affect the melanin production process, leading to problems such as dull skin tone, pigmentation, age spots, and melasma. Melasma, in particular, is a common acquired hyperpigmentation disorder, clinically manifesting as symmetrical brown or dark brown patches on sun-exposed areas of the face. It is characterized by complex etiology, a prolonged course, a high recurrence rate, and the difficulty in maintaining treatment.

[0003] Melanin production is influenced by multiple signaling pathways and regulatory factors. Tyrosinase (TYR), tyrosinase-associated protein 1 (TYRP1), and dopachrome tautomerase (DCT / TYRP2) are important functional proteins in melanin synthesis, while microphthalmia-associated transcription factor (MITF) is a crucial transcription factor regulating melanocyte differentiation, survival, and the expression of genes related to melanin synthesis. MITF can regulate the expression of multiple melanin synthesis-related genes upstream; therefore, regulating MITF expression levels is one of the important technical approaches for intervening in melanin production.

[0004] Existing technologies for whitening, fading dark spots, or improving pigmentation mainly include inhibiting tyrosinase activity, anti-oxidation, anti-inflammation, promoting keratin renewal, inhibiting melanin transport, chemical peels, laser or phototherapy, and topical application of active substances such as hydroquinone, retinoic acid, azelaic acid, kojic acid, arbutin, tranexamic acid, and their derivatives. While these technologies can improve melanin deposition or uneven skin tone to some extent, they still have limitations, including relatively indirect target targeting, significant individual variability, unstable onset or maintenance effects, easy recurrence after discontinuation, potential irritation or intolerance with long-term use, and the need for professional medical procedures in some treatments, which may induce post-inflammatory hyperpigmentation. Especially for chronic, recurrent pigmentation problems such as melasma, relying solely on existing small-molecule topical preparations or physical therapy methods still cannot simultaneously meet the requirements of clear targeting, long-term maintenance, safety, and ease of use.

[0005] RNA interference (RNAi) technology can specifically silence target gene expression at the post-transcriptional level using small interfering RNAs (siRNAs), providing a new technical approach for targeted regulation of the melanin synthesis pathway. Compared with traditional small molecule active substances, siRNAs typically have advantages such as high sequence specificity, the ability to directly reduce target mRNA levels, and the ability to target gene targets that are difficult for traditional small molecules to act on directly. Therefore, designing siRNA molecules targeting key regulatory factors of melanin synthesis, such as MITF, holds promise for reducing the expression of related proteins upstream in the melanin production pathway, thereby inhibiting melanin synthesis and improving pigmentation.

[0006] However, as oligonucleotide molecules, siRNA still faces several technical obstacles in practical applications. First, unmodified naked siRNA is easily degraded by nucleases, resulting in insufficient in vitro and in vivo stability. Second, siRNA molecules typically carry a strong negative charge and have a relatively large molecular weight, making it difficult for them to autonomously cross cell membranes and skin barriers, limiting their delivery efficiency to local skin tissues. Third, the silencing efficiency varies significantly between different target regions and different siRNA sequences, and candidate sequences obtained through conventional sequence design or prediction do not necessarily possess ideal cellular or in vivo activity. Furthermore, inappropriate sequence or modification designs may lead to off-target effects, immune stimulation, reduced activity, or safety risks. Therefore, simply proposing the use of siRNA to inhibit melanin production cannot fully address the comprehensive balance between targeted sequence selection, structural stability, cellular uptake, in vivo activity, and safety in local skin applications.

[0007] To improve the performance of siRNA molecules, current technologies typically employ chemical modifications such as 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, and thiophosphate, or attach ligands such as cholesterol, fatty acids, sugars, and peptides to the ends of oligonucleotides, aiming to enhance nuclease stability, tissue distribution, cellular uptake, or drug efficacy persistence. However, there are complex interactions between the siRNA's target sequence, sense and antisense strand lengths, modification sites, modification ratios, terminal modification groups, and target ligands. Modification schemes cannot be simply superimposed to obtain molecules with both stability and high silencing activity. Over-modification or inappropriate modification site selection can also affect the loading of the RNA-induced silencing complex (RISC), antisense strand recognition, and target mRNA cleavage efficiency, thereby reducing gene silencing effectiveness.

[0008] Therefore, there is still a need in this field for a double-stranded siRNA molecule that can effectively downregulate MITF expression, inhibit melanin synthesis, and is suitable for whitening, improving or alleviating pigmentation-related scenarios such as melasma. At the same time, this type of siRNA molecule should also take into account sequence specificity, nucleic acid stability, local tissue action efficiency, and application safety to overcome the problems of relatively indirect target targeting, insufficient efficacy maintenance, easy recurrence, and limited long-term use in existing whitening or anti-pigmentation technologies. Summary of the Invention

[0009] The purpose of this invention is to provide a double-stranded siRNA molecule and its applications.

[0010] In a first aspect, the present invention provides a naked sequence of a double-stranded siRNA molecule.

[0011] Specifically, the present invention provides a double-stranded siRNA molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.39 or a nucleotide sequence containing at least 90% sequence identity with SEQ ID NO.39, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.40 or a nucleotide sequence containing at least 90% sequence identity with SEQ ID NO.40.

[0012] In some implementations, the length of the positive chain does not exceed 35, 33, 31, 29, 28, 27, 26, 25, 24, 23, 22, or 21 nucleotides.

[0013] In some embodiments, the length of the antisense strand does not exceed 37, 35, 33, 31, 29, 28, 27, 26, 25, 24, or 23 nucleotides.

[0014] Preferably, the length of the sense strand is 19-27 nucleotides, and / or the length of the antisense strand is 19-29 nucleotides.

[0015] Preferably, the length of the sense strand is 21-27 nucleotides, and / or the length of the antisense strand is 23-29 nucleotides.

[0016] More preferably, the length of the sense strand is 21 to 23 nucleotides, and / or the length of the antisense strand is 23 to 25 nucleotides.

[0017] In some embodiments, the nucleotide sequence of the positive strand contains at least 90% sequence identity with SEQ ID NO. 39, and may be further defined as at least 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0018] In some embodiments, the nucleotide sequence of the antisense strand contains at least 90% sequence identity with SEQ ID NO. 40, and may be further defined as at least 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0019] In some implementations, one or more nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0020] In some embodiments, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO. 39 or comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO. 39, and / or the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO. 40 or comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO. 40.

[0021] Preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO.39 or contains a sequence that is at least 90% identical to the sequence of SEQ ID NO.39, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO.40 or contains a sequence that is at least 90% identical to the sequence of SEQ ID NO.40.

[0022] In some embodiments, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO. 39 or contains at least 90% sequence identity with SEQ ID NO. 39, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO. 40 or contains at least 90% sequence identity with SEQ ID NO. 40, wherein the length of the sense strand is 21 to 27 nucleotides, and the length of the antisense strand is 23 to 29 nucleotides.

[0023] Preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO.39 or contains at least 90% sequence identity with SEQ ID NO.39, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO.40 or contains at least 90% sequence identity with SEQ ID NO.40, wherein the length of the sense strand is 21-23 nucleotides, and the length of the antisense strand is 23-25 ​​nucleotides.

[0024] Preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO.39, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO.40, wherein the length of the sense strand is 21-27 nucleotides, and the length of the antisense strand is 23-29 nucleotides.

[0025] More preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO.39, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO.40, wherein the length of the sense strand is 21-23 nucleotides, and the length of the antisense strand is 23-25 ​​nucleotides.

[0026] A second aspect of the present invention provides a modification scheme for double-stranded siRNA.

[0027] In some implementations, one or more nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

[0028] Preferably, the modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or any combination thereof, and the sense strand and / or the antisense strand contains one or more phosphate thioester modified nucleotide inter-bonds.

[0029] In some embodiments, the positive strand comprises the following modified nucleotides: one or more nucleotides in positions 4-16 from the 5' end that are 2'-fluorinated.

[0030] In some embodiments, the antisense strand comprises nucleotides modified such that one or more nucleotides with a 2'-fluorination modification are contained in positions 2-20 from the 5' end.

[0031] In some implementations, the justice chain and antisense chain include modifications selected from any of the following groups: (1) The sense strand contains the following modifications: nucleotides with 2'-fluorination at positions 4, 6, 8, 10 and 14 from the 5' end; and / or the antisense strand contains the following modifications: nucleotides with 2'-fluorination at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 20 from the 5' end.

[0032] (2) The sense strand contains the following modifications: nucleotides with 2'-fluorination at positions 6, 8, 9, and 10 starting from the 5' end; and / or the antisense strand contains the following modifications: nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14, and 16 starting from the 5' end.

[0033] (3) The sense strand contains the following modifications: nucleotides with 2'-fluorination at positions 7, 9, 10, and 11 starting from the 5' end; and / or the antisense strand contains the following modifications: nucleotides with 2'-fluorination at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 starting from the 5' end.

[0034] (4) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 4, 6, 8, 10, and 14 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 6, 14, and 16 starting from the 5' end.

[0035] (5) The sense strand contains the following modification: nucleotides with 2'-fluorination at positions 4, 6, 8, 10, and 14 starting from the 5' end; and / or the antisense strand contains the following modification: nucleotides with 2'-fluorination at positions 2, 6, 8, 14, and 16 starting from the 5' end.

[0036] Preferably, the sense strand comprises a nucleotide with a 2'-fluorinated modification at positions 4, 6, 8, 10 and 14 starting from the 5' end; and / or the antisense strand comprises a nucleotide with a 2'-fluorinated modification at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 20 starting from the 5' end.

[0037] Preferably, the sense strand comprises the following modification: nucleotides with 2'-fluorination at positions 6, 8, 9, and 10 starting from the 5' end; and / or the antisense strand comprises the following modification: nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14, and 16 starting from the 5' end.

[0038] In some embodiments, the positive strand further comprises the following modifications: the 5' end of the positive strand contains two phosphate-thioester-modified nucleotide inter-bonds, the 3' end of the positive strand contains two phosphate-thioester-modified nucleotide inter-bonds, and the remaining nucleotides of the positive strand are nucleotides modified with 2'-O-methyl.

[0039] In some implementations, the chain of justice further includes the following modifications: a) The first and second nucleotide bonds starting from the 5' end, and the second and third nucleotide bonds starting from the 3' end, are phosphate thioester bonds; and / or b) The remaining sites on the positive strand are nucleotides modified with 2'-methoxy groups.

[0040] In some embodiments, the antisense strand further comprises modifications selected from the following: the 5' end of the antisense strand contains two phosphate-thioester-modified nucleotide inter-bonds, the 3' end of the antisense strand contains two phosphate-thioester-modified nucleotide inter-bonds, and the remaining nucleotides of the antisense strand are nucleotides modified with 2'-O-methyl.

[0041] In some embodiments, the antisense chain further includes modifications selected from: a) The first and second nucleotide bonds starting from the 5' end, and the second and third nucleotide bonds starting from the 3' end, are phosphate thioester bonds; and / or b) The remaining sites of the antisense strand are nucleotides modified with 2'-O-methyl.

[0042] In some implementations, the chain of justice includes the following modifications: a) Nucleotides with 2'-fluorinated modification at positions 4, 6, 8, 10, and 14 starting from the 5' end; b) Phosphothiophosphate bonds at the first and second nucleotide internucleotide bonds starting from the 5' end, and at the second and third nucleotide internucleotide bonds starting from the 3' end; and c) Nucleotides with 2'-O-methyl modification at the remaining positions; and The antisense strand comprises the following modifications: b) nucleotides with 2'-fluorination at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5' end; b) nucleotides with 1st and 2nd internucleotide bonds from the 5' end, and 2nd and 3rd internucleotide bonds from the 3' end, being phosphate thioester bonds; and c) nucleotides with 2'-O-methyl modifications at the remaining positions.

[0043] In some implementations, the chain of justice includes the following modifications: a) Nucleotides with 2'-fluorination at positions 6, 8, 9, and 10 starting from the 5' end; b) Phosphothiophosphate bonds at the first and second nucleotide internucleotide bonds starting from the 5' end, and at the second and third nucleotide internucleotide bonds starting from the 3' end; and c) Nucleotides with 2'-O-methylation at the remaining positions; and The antisense strand comprises the following modifications: b) nucleotides with 2'-fluorination at positions 2, 3, 5, 7, 10, 14, and 16 starting from the 5' end; c) nucleotides with 2'-O-methyl modification at the remaining positions.

[0044] In some implementations, the chain of justice includes the following modifications: a) Nucleotides with 2'-fluorination at positions 7, 9, 10, and 11 starting from the 5' end; b) Phosphothiophosphate bonds at the first and second nucleotide internucleotide bonds starting from the 5' end, and at the second and third nucleotide internucleotide bonds starting from the 3' end; and c) Nucleotides with 2'-O-methylation at the remaining positions; and The antisense strand comprises the following modifications: b) nucleotides with 2'-fluorination at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5' end; b) nucleotides with 1st and 2nd internucleotide bonds from the 5' end, and 2nd and 3rd internucleotide bonds from the 3' end, being phosphate thioester bonds; and c) nucleotides with 2'-O-methyl modifications at the remaining positions.

[0045] In some implementations, the chain of justice includes the following modifications: a) nucleotides with 2'-fluorination at positions 4, 6, 8, 10, and 14 starting from the 5' end; b) nucleotides with the first and second internucleotide bonds starting from the 5' end, and the second and third internucleotide bonds starting from the 3' end, being phosphate thioester bonds; and c) nucleotides with 2'-O-methylation at the remaining positions; and the antisense strand comprising the following modifications: nucleotides with 2'-fluorination at positions 2, 6, 14, and 16 starting from the 5' end; b) nucleotides with the first and second internucleotide bonds starting from the 5' end, and the second and third internucleotide bonds starting from the 3' end, being phosphate thioester bonds; and c) nucleotides with 2'-O-methylation at the remaining positions.

[0046] In some implementations, the chain of justice includes the following modifications: a) Nucleotides with 2'-fluorinated modification at positions 4, 6, 8, 10, and 14 starting from the 5' end; b) Phosphothiophosphate bonds at the first and second nucleotide internucleotide bonds starting from the 5' end, and at the second and third nucleotide internucleotide bonds starting from the 3' end; and c) Nucleotides with 2'-O-methyl modification at the remaining positions; and The antisense strand contains the following modifications: b) nucleotides with 2'-fluorination at positions 2, 6, 8, 14, and 16 starting from the 5' end; c) phosphate thioester bonds at the first and second nucleotide internucleotide bonds starting from the 5' end, and at the second and third nucleotide internucleotide bonds starting from the 3' end; and nucleotides with 2'-O-methyl modification at the remaining positions.

[0047] A third aspect of the present invention provides a modified double-stranded siRNA.

[0048] Specifically, the present invention provides a modified double-stranded siRNA, the double-stranded siRNA molecule comprising a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the nucleotide sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61 or comprises a nucleotide sequence that is at least 90% sequence identical to SEQ ID NO. 53, 55, 57, 59 or 61, and the nucleotide sequence of the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62 or comprises a nucleotide sequence that is at least 90% sequence identical to SEQ ID NO. 54, 56, 58, 60 or 62.

[0049] In some implementations, the length of the positive chain does not exceed 35, 33, 31, 29, 28, 27, 26, 25, 24, 23, 22, or 21 nucleotides.

[0050] In some embodiments, the length of the antisense strand does not exceed 37, 35, 33, 31, 29, 28, 27, 26, 25, 24, or 23 nucleotides.

[0051] Preferably, the length of the sense strand is 19-27 nucleotides, and / or the length of the antisense strand is 19-29 nucleotides.

[0052] Preferably, the length of the sense strand is 21-27 nucleotides, and / or the length of the antisense strand is 23-29 nucleotides.

[0053] More preferably, the length of the sense strand is 21-23 nucleotides, and / or the length of the antisense strand is 23-25 ​​nucleotides.

[0054] In some embodiments, the nucleotide sequence of the positive strand contains at least 90% sequence identity with SEQ ID NO. 53, 55, 57, 59 or 61, and may be further defined as at least 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0055] In some embodiments, the nucleotide sequence of the antisense strand contains at least 90% sequence identity with SEQ ID NO. 53, 55, 57, 59 or 61, and may be further defined as at least 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0056] In some embodiments, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61, and / or the nucleotide sequence of the antisense strand comprises or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62, and / or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62.

[0057] Preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61, and the nucleotide sequence of the antisense strand comprises or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62, or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62.

[0058] In some embodiments, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61, and the nucleotide sequence of the antisense strand comprises or contains a sequence that is at least 90% identical to the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62, and the sequence contains at least 90% identical to the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62, wherein the length of the sense strand is 21 to 27 nucleotides, and the length of the antisense strand is 23 to 29 nucleotides.

[0059] Preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises or contains a sequence identical to at least 90% of the sequence of SEQ ID NO. 53, 55, 57, 59 or 61, and the nucleotide sequence of the antisense strand comprises or contains a sequence identical to at least 90% of the sequence of SEQ ID NO. 54, 56, 58, 60 or 62, wherein the sense strand is 21 to 23 nucleotides in length, and the antisense strand is 23 to 25 nucleotides in length.

[0060] Preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62, wherein the length of the sense strand is 21 to 27 nucleotides, and the length of the antisense strand is 23 to 29 nucleotides.

[0061] More preferably, a double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises the sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61, and the nucleotide sequence of the antisense strand comprises the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62, wherein the length of the sense strand is 21 to 23 nucleotides, and the length of the antisense strand is 23 to 25 nucleotides.

[0062] In some embodiments, the 3' end of the sense chain is modified with a targeting ligand, and / or the 5' end of the antisense chain is modified with a 5'-vinylphosphonate.

[0063] The targeting ligand is selected from one of the following: cholesterol, fatty acids, cyclic RGD peptides, N-acetylgalactosamine, folic acid, and more preferably, the targeting ligand is behenic acid.

[0064] In some embodiments, the 5' end of the antisense chain contains a 5'-vinylphosphonate (VP) modification.

[0065] A fourth aspect of the present invention provides a coupling of formula (I): R1—B—R2 Formula (I), Wherein R1 is a targeting ligand, delivery enhancer, or hydrophobic modifier, B is an optional linker, and R2 is a double-stranded siRNA molecule selected from those described in this invention.

[0066] In some embodiments, the R1 is selected from: cholesterol, fatty acids, cyclic RGD peptides, N-acetylgalactosamine (GalNAc), folic acid (FA), steroids, lithocholic acid (LCA), open-ring steroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosuccinic acid (DCA).

[0067] R1 is docosuccinic acid (DCA), which can also be called docosuccinic acid, docosanoic acid, behenic acid, oleic acid, succinic acid, etc. It is a carboxylic acid and saturated fatty acid with the molecular formula C. 21 H43 COOH, structural formula as shown below: .

[0068] Preferably, wherein R1 is .

[0069] In some embodiments, the connector is selected from one of the following groups: , , , , , , , , or , where * represents the linker to the target ligand and ** represents the linker to the double-stranded siRNA molecule; Where n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0070] In some implementations, ** indicates coupling with the positive strand of the double-stranded siRNA molecule.

[0071] Preferably, ** indicates coupling with the 3' end of the positive strand of the double-stranded siRNA molecule.

[0072] In some embodiments, the 5' end of the antisense chain contains a 5'-vinylphosphonate (VP) modification.

[0073] In some embodiments, the coupling of the present invention has a structure as shown in the following formula: .

[0074] In a fifth aspect, the present invention also provides a delivery formulation or cell containing the double-stranded siRNA molecule or the conjugate described herein.

[0075] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the siRNA, the conjugate, the delivery formulation, or the cell of the present invention.

[0076] The pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0077] In some implementations, "pharmaceutical composition" means a mixture of substances suitable for administration to a subject.

[0078] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0079] Preferably, "pharmaceutically acceptable carrier" means a medium or diluent that does not interfere with the oligonucleotide structure. Some of these carriers enable the pharmaceutical composition to be formulated, for example, into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, pastes, suspensions, and lozenges for oral administration by a subject.

[0080] Preferably, the carrier or excipient comprises at least one functional excipient selected from the group consisting of: skeleton material, thickener, gelling agent, humectant, emulsifier, crosslinking agent, crosslinking regulator, filler, transdermal facilitator, film-forming material, solvent, cosolvent, preservative, antioxidant, pH adjuster, buffer, chelating agent, plasticizer, wetting agent, dispersant, masking agent, and flavoring agent.

[0081] The above-mentioned functional excipients can be used alone or in any combination as needed for the dosage form, as long as there is no adverse interaction between them or with the double-stranded siRNA molecule.

[0082] In a seventh aspect, the present invention provides the use of the double-stranded siRNA molecule, the conjugate, the delivery formulation or cell, and the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment and / or improvement of MITF gene-mediated diseases or conditions.

[0083] This invention provides the use of the double-stranded siRNA molecule, the conjugate, the delivery formulation or cell, and the pharmaceutical composition in the preparation of cosmetics for improving MITF gene-mediated diseases or conditions.

[0084] In some implementations, the MITF gene-mediated diseases or conditions include MITF gene-mediated pigmentary skin diseases.

[0085] Preferably, the pigmentary skin condition includes melasma.

[0086] In some embodiments, the present invention provides the use of the double-stranded siRNA molecule, the conjugate, the delivery formulation or cell, or the pharmaceutical composition in the preparation of skin whitening products.

[0087] Preferably, the whitening product includes, but is not limited to, drugs, pharmaceutical compositions, and cosmetics.

[0088] In some implementations, the skin whitening is achieved by inhibiting melanin synthesis.

[0089] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The double-stranded siRNA molecule of the present invention takes into account sequence specificity, nucleic acid stability, local tissue action efficiency and application safety, and can effectively overcome the problems of relatively indirect target, insufficient efficacy maintenance, easy recurrence and long-term use in existing whitening or anti-pigmentation technologies.

[0090] 2. The conjugates of the present invention can effectively downregulate MITF expression and inhibit melanin synthesis, and are suitable for whitening, improving or alleviating pigmentation-related scenarios such as melasma. Attached Figure Description

[0091] Figure 1 The effect of 1 nM transfection concentration of siRNA sequence on MITF expression in SK-MEL-5 cells; Figure 2 The effect of 0.1 nM transfection concentration of siRNA sequence on MITF expression in SK-MEL-5 cells; Figure 3 The effect of 1 nM transfection concentration of siRNA sequence on MITF expression in B16 cells; Figure 4 The graph shows the expression levels of MITF in SK-MEL-5 cells after different chemical modifications of MITFSil-20. Figure 5 The expression level of MITF in mouse skin after intradermal injection of chemically modified MITFSil-20 coupled with the same delivery system; Figure 6 Effects of MITFSil-20-1 on pigmentation in chloasma mice: representative images of mouse ears; Figure 7 Masso-Fontana melanin staining results: A: Normal group (Ctrl.); B: Model control group (Mel.); Figure 8 Results of Masso-Fontana melanin staining: A: Hydroquinone cream group (HQ); B: MITFSil-20 group; Figure 9 Effects of MITFSil-20-1 on pigmentation in chloasma mice: A: Effect of MITFSil-R70-1 on melanin area; B: Relative expression level of TYR protein in mouse ear skin. Detailed Implementation

[0092] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0093] Example 1: Listing of raw sequences and modified sequences Tables 1 to 3 show the raw sequence information and modified (modification schemes MITFSil-20-M1 to M5) sequence information designed in this invention.

[0094] Table 1. siRNA naked sequence information

[0095] Table 2. siRNA naked sequence information (continued)

[0096] Table 3 Modification schemes and modified sequence information

[0098] In this context, uppercase letters C, G, U, A, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with 2'-O-methyl; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with 2'-fluorine; and lowercase letter s indicates that the nucleotide adjacent to the left of the letter s is a nucleotide modified with phosphate thioester.

[0099] In this invention, the modified sequences are coupled with the same delivery system, specifically, DCA is coupled at the 3' end of the SS chain and 5VP is coupled at the 5' end of the AS chain; where the uppercase letter DCA represents docosuccinic acid (DCA) modification; and the uppercase letter 5VP represents 5'-vinylphosphonate modification. The specific structure is shown below: .

[0100] Example 2: Screening candidate naked sequences for MITF gene knockout activity using human cell lines Experimental methods: (1) siRNA transfection Cell seeding: SK-MEL-5 cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0707) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were seeded into plates. The cell density was adjusted to 6 × 10⁶ cells per well. 4 The cells were seeded into a 24-well plate.

[0101] Preparation of transfection complex: 50 μL of Opti-MEM medium was mixed with 5 μL of siRNA as described in Table 1 (to achieve a final transfection concentration of 0.1 nM or 1 nM in the 24-well plate). 50 μL of Opti-MEM medium was mixed with 1 μL of RNAimax transfection reagent (Invitrogen, catalog number 13778150). The two mixtures were then combined and allowed to stand for 15 min to obtain the transfection complex. This transfection complex was added to 24-well plates containing cells and incubated at 37°C with 5% CO2 for 48 h. A total of 52 experimental groups were obtained, with three replicates per group. In addition to the experimental groups, an NC group was set up as a negative control for each cell transfection.

[0102] (2) Real-time quantitative PCR analysis In step (1), cells were lysed 48 h after transfection in each group. Total RNA was extracted from the cells using a column extraction kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC112-01-50rxns, hereinafter the same) and reverse transcribed to obtain cDNA samples. Using the GAPDH gene as an internal reference, real-time quantitative PCR was performed using a dye method and a QuantStudio 5 fluorescence quantitative PCR instrument. The reaction detection system was as follows: 4 μL of reverse transcribed cDNA sample, 5 μL of qPCR mix, 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 0.6 μL of water, totaling 10 μL; the reaction program was as follows: ① 95℃, 30s; ② 40 cycles: 95℃, 5s; 60℃, 30s; ③ 95℃, 15s; 60℃, 60s; 95℃, 15s. The primers used are shown in Table 4. Table 4 Primer sequence information

[0103] Experimental results: After the PCR reaction was completed, use 2 -ΔΔCt Methods: Relative quantitative analysis was performed using a reference gene as a standard. Results are shown in Tables 5-6 and 6. Figures 1-2 As shown. In this embodiment, all siRNAs used are naked nucleic acid molecules without chemical modification.

[0104] Table 5. Inhibitory activity of 1 nM transfection concentration siRNA sequence in SK-MEL-5 cells.

[0105] Table 6. Inhibitory activity of 0.1 nM transfection concentration siRNA sequence in SK-MEL-5 cells

[0106] Experimental Conclusions: The screening results of human SK-MEL-5 cells show that different MITF siRNA candidate sequences exhibit significant differences in their inhibitory activity against MITF mRNA, indicating that different siRNA sequences designed for the same MITF target gene do not necessarily have the same or similar gene silencing effects. At a siRNA transfection concentration of 1 nM, MITFSil-20 can reduce the relative expression level of MITF mRNA in SK-MEL-5 cells to 11.93%, demonstrating strong MITF gene silencing activity; at a siRNA transfection concentration of 0.1 nM, MITFSil-20 can still reduce the relative expression level of MITF mRNA to 53.7%, indicating that it still has significant inhibitory activity at lower concentrations.

[0107] Example 3: Screening candidate naked sequences for MITF gene knockout activity using mouse cell lines Experimental methods: (1) siRNA transfection Cell seeding: B16 cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number ZQ0186) were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C with 5% CO2. Cells were seeded into plates when they were in the logarithmic growth phase and in good condition (70% confluence). The cell density was adjusted to 6 × 10⁶ cells per well. 4 The cells were seeded into a 24-well plate.

[0108] Preparation of transfection complexes: 50 μL of Opti-MEM medium was mixed with 5 μL of the 7 siRNAs screened in Example 1 (to achieve a final transfection concentration of 1 nM in the 24-well plate), and 50 μL of Opti-MEM medium was mixed with 1 μL of RNAiMax transfection reagent. These two mixtures were then combined and allowed to stand for 15 min to obtain the transfection complexes. The transfection complexes were added to 24-well plates containing cells and incubated at 37°C with 5% CO2 for 48 h, resulting in 7 experimental groups, with 3 replicates per group. In addition to the experimental groups, an NC group was set up as a negative control for each cell transfection.

[0109] (2) Real-time quantitative PCR analysis Step (1) After 48 hours of transfection, cells in each group were lysed, and total RNA was extracted from the cells using a column extraction kit and reverse transcribed to obtain cDNA samples. Using the GAPDH gene as an internal reference, real-time quantitative PCR was performed using a dye method and a QuantStudio 5 fluorescence quantitative PCR instrument. The reaction detection system was as follows: 4 μL of reverse transcribed cDNA sample, 5 μL of qPCRmix, 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), and 0.6 μL of water, totaling 10 μL; the reaction program was as follows: ① 95℃, 30s; ② 40 cycles: 95℃, 5s; 60℃, 30s; ③ 95℃, 15s; 60℃, 60s; 95℃, 15s. The primers used are shown in Table 7. Table 7 Primer sequence information

[0111] Experimental results: After the PCR reaction was completed, use 2 -ΔΔCt The method involved relative quantitative analysis using a reference gene as a standard, and the results were as follows: Figure 3 As shown in Table 8. All siRNAs used in this embodiment are naked nucleic acid molecules without chemical modification.

[0112] Table 8. Inhibitory activity of 1 nM transfection concentration siRNA sequence in B16 cells

[0113] Experimental conclusion: Furthermore, in murine B16 cells, MITFSil-20 reduced the relative expression of MITF mRNA to 53%, demonstrating superior murine MITF inhibitory activity among the tested candidate sequences.

[0114] Based on the screening results of human SK-MEL-5 cells and mouse B16 cells, MITFSil-20 exhibits both strong inhibitory activity against human MITF and superior inhibitory activity against mouse MITF, demonstrating good consistency in activity across cell lines. Therefore, it is a suitable candidate sequence for subsequent chemical modification optimization and in vivo efficacy validation. Thus, MITFSil-20 was selected for further research.

[0115] Example 4: Effects of different modification schemes on the in vitro activity of candidate naked sequences Experimental methods: (1) siRNA transfection Cell seeding: SK-MEL-5 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% CO2. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were seeded into plates. The cell density was adjusted to 6 × 10⁶ cells per well. 4 The cells were seeded into a 24-well plate.

[0116] Preparation of transfection complexes: Mix 50 μL of Opti-MEM medium with 5 μL of the modified siRNA (MITFSil-20-M1~M5) described in Table 2 (to make the final transfection concentrations in the 24-well plates 1 nM, 0.5 nM and 0.1 nM, respectively), and mix 50 μL of Opti-MEM medium with 1 μL of RNAimax transfection reagent; then mix the two mixtures and let stand for 15 min to obtain the transfection complexes. Add the transfection complexes to the 24-well plates containing cells and incubate in a 5% CO2, 37℃ incubator for 48 h. A total of 15 experimental groups were obtained, with 3 replicates for each group.

[0117] (2) Real-time quantitative PCR analysis Step (1) Cells in each group were lysed 48 h after transfection, and total RNA was extracted using a column extraction kit and reverse transcribed. Using the GAPDH gene as an internal reference, real-time quantitative PCR was performed using a dye method and a QuantStudio 5 fluorescence quantitative PCR instrument. The reaction detection system was as follows: 4 μL of reverse transcribed sample cDNA, 5 μL of qPCR mix, 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), and 0.6 μL of water, totaling 10 μL. The reaction program was as follows: ① 95℃, 30s; ② 40 cycles: 95℃, 5s; 60℃, 30s; ③ 95℃, 15s; 60℃, 60s; 95℃, 15s. The primers used are shown in Table 3.

[0118] Experimental results: The results are as follows Figure 4 As shown, for candidate naked sequences, modification scheme 1 (MITFSil-20-M1) and modification scheme 3 (MITFSil-20-M3) showed significantly better in vitro activity than other modification schemes (p < 0.001).

[0119] Experimental Conclusions: In vitro data showed that at 0.1 nM, modification schemes 1 (MITFSil-20-M1) and 3 (MITFSil-20-M3) exhibited significantly superior activity compared to modification scheme 4 (MITFSil-20-M4, p < 0.05). At a siRNA transfection concentration of 0.5 nM, modification schemes 1 and 3 showed significantly superior activity compared to the other three modification schemes (p < 0.05 compared to modification schemes 2 and 5; p < 0.001 compared to modification scheme 4). At a siRNA transfection concentration of 1 nM, modification schemes 1 and 3 showed significantly superior activity compared to the other three modification schemes (p < 0.01 compared to modification scheme 2; p < 0.001 compared to modification schemes 4 and 5). Based on these results, modification schemes 1 and 3 are the preferred modification schemes for this sequence. Further comparisons of in vivo activity will be conducted using these two chemical modification schemes coupled with the same delivery system.

[0120] Example 5: Effects of different modification schemes on the in vivo activity of candidate naked sequences Experimental methods: Female C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks were used in the experiment. Mice were randomly divided into groups of five. Each mouse received a single subcutaneous injection of 0.15 mg of the test sample (MITFSil-20-M1, MITFSil-20-M3, and negative control NC) into the back. The sample was dissolved in PBS solution, and skin tissue was collected from the injection site after hair removal on day 3. All small nucleic acid molecules used in this experimental system were pre-modified chemically and conjugated with functional delivery molecules. RNA was extracted from tissues using the Trizol method, reverse transcribed, and detected by qPCR. The GAPDH gene was used as an internal reference gene, and real-time quantitative PCR was performed using a dye method and a QuantStudio 5 real-time PCR instrument. The reaction detection system is as follows: 4 μL of reverse transcription sample cDNA, 5 μL of qPCR mix, 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), and 0.6 μL of water, for a total of 10 μL; the reaction program is as follows: ① 95℃, 30s, ② 40 cycles: 95℃, 5s; 60℃, 30s; ③ 95℃, 15s; 60℃, 60s; 95℃, 15s. The primers used are shown in Table 6.

[0121] Experimental results: The results are as follows Figure 5 As shown, after coupling with the same delivery system, both modification scheme 1 (MITFSil-20-M1) and modification scheme 3 (MITFSil-20-M3) can effectively knock down the expression level of MITF in mouse skin, with no significant difference between the two groups.

[0122] Experimental conclusion: For MITFSil-20, modification scheme 1 and modification scheme 3 are both preferred chemical modification schemes. Modification scheme 1 was selected for subsequent experiments.

[0123] Example 6: Validation of the efficacy of modified candidate sequences in an animal model of melasma. Experimental Methods: The optimal chemical modification scheme was selected and applied to the efficacy verification experiment for melasma. All small nucleic acid molecules used in this experimental system were pre-modified chemically and coupled with functional delivery molecules. Twenty-four 6-8 week old female C57BL / 6J mice were selected and divided into a normal group (Ctrl., N=6) and a model group. Modeling began in the model group, and on the first day, the mice were regrouped into Group-2: model control group (Mel., N=6); Group-3: hydroquinone cream group (HQ, N=6); and Group-4: MITFSil-20 group (using MITFSil-20-M1 for the experiment, hereinafter referred to as MITFSil-20, N=6). Group G3 received topical application of the drug daily (10 μL / cm²). 2 ), lasting 28 days; G4 intradermal injection (0.5 mg / cm²) 2 The mice were given intradermal injections three times a week, one at each of the left and right ears, for 28 days. The model group was established by selecting animals with intact skin and subjecting them to daily UVB (wavelength 290-320nm) irradiation (60mJ / cm²) starting 24 hours later. 2 Combined with progesterone (0.4%), intramuscular injection of 5 mL / kg into the hind limb was administered for 28 consecutive days. Observations were conducted daily during the experiment, and photographs were taken before and once a week after administration. At the endpoint, bilateral ear tissue was collected, and the histopathology of the ear was assessed by staining with Masso-Fontana melanin staining solution to quantitatively analyze the melanin content. Changes in TYR content in the ear skin were detected by ELISA.

[0124] Experimental results: Apparent data as follows Figures 6-9 As shown, after animal modeling, the degree of pigmentation of the ear skin continued to worsen; the hydroquinone cream group and the MITFSil-20 group showed varying degrees of improvement in pigmentation compared to the model group.

[0125] Staining results of Masso-Fontana melanin staining solution on ear skin showed that melanin deposition in the ear was significantly increased after animal modeling (p<0.001). Compared with the model group, melanin granules in the hydroquinone cream group and the MITFSil-20 group were significantly reduced (p<0.001), with the hydroquinone cream group and the MITFSil-20 group showing the best effects. This indicates that MITFSil-20 can effectively treat melasma, and its efficacy is comparable to that of the clinical drug hydroquinone.

[0126] The content of TYR protein, a downstream target gene of MITF, was further detected by ELISA. The results showed that, compared with the control group, the TYR content in the model group was significantly increased (p<0.001), while the TYR content in the hydroquinone cream group and the MITFSil-20 group was significantly decreased (p<0.01, p<0.001).

[0127] Experimental conclusion: Injection of MITFSil-20 can effectively reduce the intradermal melanin content in mice with melasma, with effects comparable to hydroquinone. MITFSil-20 shows promise as a new treatment for melasma.

Claims

1. A double-stranded siRNA molecule, characterized in that, The double-stranded siRNA molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.39 or a nucleotide sequence that is at least 90% identical to the sequence of SEQ ID NO.39, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.40 or a nucleotide sequence that is at least 90% identical to the sequence of SEQ ID NO.

40.

2. The double-stranded siRNA molecule according to claim 1, characterized in that, The length of the sense strand is 19-27 nucleotides, and / or the length of the antisense strand is 19-29 nucleotides.

3. The double-stranded siRNA molecule according to claim 2, characterized in that, The length of the sense strand is 21-23 nucleotides, and / or the length of the antisense strand is 21-23 nucleotides.

4. The double-stranded siRNA molecule according to claim 1, characterized in that, One or more nucleotides in the sense strand and / or the antisense strand are modified nucleotides.

5. The double-stranded siRNA molecule according to claim 4, characterized in that, The modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or any combination thereof, and the sense strand and / or the antisense strand contains one or more phosphate thioester modified nucleotide inter-bonds.

6. The double-stranded siRNA molecule according to claim 5, characterized in that, The sense strand contains one or more nucleotides modified with 2'-fluorination at positions 4 to 14 from the 5' end, and / or the antisense strand contains one or more nucleotides modified with 2'-fluorination at positions 2 to 20 from the 5' end.

7. The double-stranded siRNA molecule according to claim 5, characterized in that, The 5' end of the positive strand contains two nucleotide inter-linked bonds modified with phosphate thioester, the 3' end of the positive strand contains two nucleotide inter-linked bonds modified with phosphate thioester, and the remaining nucleotides of the positive strand are nucleotides modified with 2'-O-methyl.

8. The double-stranded siRNA molecule according to claim 6, characterized in that, The 5' end of the antisense strand contains two phosphate-thioester-modified nucleotide inter-bonds, the 3' end of the antisense strand contains two phosphate-thioester-modified nucleotide inter-bonds, and the remaining nucleotides of the antisense strand are nucleotides modified with 2'-O-methyl.

9. The double-stranded siRNA molecule according to claim 8, characterized in that, The 3' end of the sense chain is modified with a targeting ligand, and / or the 5' end of the antisense chain is modified with a 5'-vinylphosphonate.

10. The double-stranded siRNA molecule according to claim 9, characterized in that, The targeting ligand is selected from one of the following: cholesterol, fatty acid, cyclic RGD peptide, N-acetylgalactosamine, or folic acid.

11. The double-stranded siRNA molecule according to any one of claims 1 to 10, characterized in that, The nucleotide sequence of the sense strand of the double-stranded siRNA molecule includes the sequence shown in SEQ ID NO. 53, 55, 57, 59 or 61, and / or the nucleotide sequence of the antisense strand of the double-stranded siRNA molecule includes the sequence shown in SEQ ID NO. 54, 56, 58, 60 or 62.

12. A coupling, characterized in that, The structure of the coupling is shown in formula (I): R1—B—R2, Formula (I), Wherein R1 is a targeting ligand, delivery enhancer, or hydrophobic modifier, B is an optional linker, and R2 is selected from the double-stranded siRNA molecule described in any one of claims 1 to 10.

13. The coupling according to claim 12, characterized in that, The R1 is selected from one or more of cholesterol, fatty acids, cyclic RGD peptides, N-acetylgalactosamine, folic acid, steroids, lithocholic acid, open-ring steroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, eicosapentaenoic acid, docosahexaenoic acid, or docosuccinic acid.

14. The coupling according to claim 12, characterized in that, The connector is selected from one of the following structural formulas: , , , , , , , , or Where * represents the linker to the target ligand, and ** represents the linker to the double-stranded siRNA molecule; Where n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

15. A delivery formulation or cell, characterized in that, It contains the double-stranded siRNA molecule as described in any one of claims 1 to 11 or the conjugate as described in any one of claims 12 to 14.

16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and an active ingredient, characterized in that, The active ingredients are selected from: (a) The double-stranded siRNA molecule according to any one of claims 1 to 11; or (b) The coupling as described in any one of claims 12 to 14.

17. The use of the double-stranded siRNA molecule of any one of claims 1 to 11, the conjugate of any one of claims 12 to 14, the delivery formulation or cell and pharmaceutically acceptable carrier or excipient of claim 15, or the pharmaceutical composition of claim 16 in the preparation of a medicament or cosmetic for the prevention and / or treatment and / or improvement of MITF gene-mediated diseases or conditions.

18. The application according to claim 17, characterized in that, The diseases or conditions mediated by the MITF gene include those that improve MITF gene-mediated pigmentary skin diseases.

19. The application according to claim 18, characterized in that, The pigmentary skin conditions mentioned include melasma.

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