CN117866955A - Interference RNA for inhibiting expression of melanin avidin (MLPH) gene and application thereof - Google Patents

Interference RNA for inhibiting expression of melanin avidin (MLPH) gene and application thereof Download PDF

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CN117866955A
CN117866955A CN202311313825.4A CN202311313825A CN117866955A CN 117866955 A CN117866955 A CN 117866955A CN 202311313825 A CN202311313825 A CN 202311313825A CN 117866955 A CN117866955 A CN 117866955A
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antibodies
interfering rna
seq
antibody
group
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贾宏丽
林美娜
杨脉
陈晓萌
郭军
王庆彬
赵宣
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Jenkem Technology Co Ltd
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • C07—ORGANIC CHEMISTRY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Abstract

The invention relates to an interference RNA for inhibiting MLPH expression, which comprises any one or more than two nucleotide sequences shown in SEQ ID NO: 1-20 or SEQ ID NO: 27-38, and the siRNA has good activity for inhibiting MLPH mRNA expression, and the inhibition rate is 70-80%. The chemically modified interfering RNA sequences have better activity in inhibiting the expression of MLPH mRNA. In addition, the invention provides a lipid nano delivery system which delivers siRNA to a proper position under skin to exert biological effect, and has wide application in whitening products. The invention also provides a medicament comprising an interfering RNA antibody-nucleic acid conjugate for preventing and/or treating pigmentation or diseases associated with increased MLPH expression.

Description

Interference RNA for inhibiting expression of melanin avidin (MLPH) gene and application thereof
Technical Field
The invention relates to the technical field of molecular biology and biological medicine, in particular to an interfering RNA for effectively inhibiting the expression of a melanin avidin (MLPH) gene and application thereof.
Background
Skin tone is determined by the content of melanin in epidermis, which is produced by melanocytes located in the stratum spinosum, is encapsulated in the melanosome, is transported into keratinocytes by the processes of melanocytes, and then is degraded by digestion of keratinocytes, and is peeled off with exfoliation of keratinocytes, and also is deposited in keratinocytes to form color spots.
The transport mechanism of melanin is currently unknown. It was found that Rab27a, myosin Va and Melanocortin (MLPH) together constitute a transport complex. Wherein, after binding GTP, rab27a binds to melanosomes, the Slp Homology Domain (SHD) of MLPH binds to Rab27a, and its C-terminal end binds to the globular tail of myosin Va. The head domain of Myosin Va resembles a motor, interacting with the microfilaments of actin (actin) to form a triple protein complex. Here, MLPH plays a role of molecular mounting, and Myosin Va is a molecular motor, allowing the carried melanosomes to move along actin to the outer periphery of the cell. This triple protein complex is necessary for capturing melanosomes, moving locally around the actin-rich cell periphery. It has been postulated by the scholars that phosphorylation of MLPH determines the direction of movement of the transport complex. Thus, MLPH plays an important role in melanin transport.
In the hair-color-reduced rabbits, the transcripts of MLPH were significantly decreased. MLPH splice variation may cause exon skipping, resulting in protein truncation, manifested as a lightening of rabbit hair. In primary melanocytes, melanosomes are uniformly distributed in the cytoplasm, with a distinct melanosome distribution also at the cell's processes; in the leaden genotype (mlph mutant) melanocytes, however, melanosomes are mainly surrounded by the nucleus, and there is no melanosome distribution at the cell processes. When melanocytes are transfected with MLPH siRNA, MLPH expression is inhibited and melanosomes in the cells are no longer transported to the peripheral processes of the cells but accumulate around the nucleus. These results indicate that the presence or absence of MLPH is closely related to the transport profile of melanosomes. Therefore, inhibition of MLPH expression may be a potential target for achieving freckle removal and whitening.
In addition, the MLPH gene has been shown to be significantly increased in the case of bladder cancer, prostate cancer and other diseases, and is expected to be developed as a target or marker.
RNA interference technology is an important means of regulating gene expression. A short double-stranded RNA molecule homologous to the target gene is used and introduced into cells, the antisense strand of the siRNA can bind to the Argonaute protein, and then the Argonaute protein breaks the mRNA and prevents translation as a template, thereby preventing synthesis of the relevant protein. Therefore, according to the principle of RNA inhibition, siRNA sequences aiming at specific gene sequences are designed, the siRNA sequences are obtained by a chemical synthesis method, and sequences with higher inhibition activity are screened out, so that the siRNA sequences can be developed into siRNA medicaments. Five siRNA drugs are currently approved for sale (Patisiran, givosiran, lumasiran, inclisiran, vutrisiran, respectively).
siRNA is easily degraded due to the presence of nucleases in blood and interstitial fluid. To improve the stability of siRNA, chemical modifications to the siRNA backbone and groups are typically employed. Common modifications are methylation, fluorination, thiophosphorylation, pseudouracil, etc.
siRNA molecules are large, carrying a large number of negative charges, and are difficult to pass through the cell membrane, which is also negatively charged. Finding a suitable delivery system is therefore an important part of the process of developing siRNA drugs. The most studied delivery systems are currently liposomes/lipid nanoparticles. The liposome/lipid nanoparticle is an artificial membrane, and can be used for delivering drugs into cells by utilizing the characteristic that the liposome/lipid nanoparticle can be fused with cell membranes. Meanwhile, besides siRNA, various components beneficial to skin can be wrapped in the liposome/lipid nanoparticle, and the components can be directly delivered into cells to play a role, so that expected effects can be produced more quickly and better.
Patent US 11,187,696 B2 discloses a method for treating obesity by inhibiting mlph, wherein siRNA that inhibits mlph expression is disclosed, but no suitable delivery system thereof nor the whitening effect of siRNA is disclosed.
The prior art (Kyung Rhim Lee, cheol Hwann Myung, and Jae Sung Hwang, inhibitory Effect of Haplamine on Melanosome Transport and Its Mechanism of Action, korean Journal of Cosmetic Science Vol.1, no.1, december 2019,31-43) discloses siRNAs that inhibit mlph expression, but does not disclose its whitening efficacy nor its suitable delivery system.
In summary, there is little disclosure in the prior art about the application of MLPH siRNA in whitening and freckle removing and preventing and/or treating diseases related to the increased expression of MLPH.
Disclosure of Invention
In order to solve the above problems, the first aspect of the present invention provides an interfering RNA that inhibits MLPH expression.
The target site sequence of the interfering RNA comprises: nucleotide sequences shown in any one or more than two of SEQ ID NO: 1-20 or SEQ ID NO: 27-38.
Specifically, the nucleotide sequence: (1) SEQ ID NO: 1 and/or SEQ ID NO: 2, (2) SEQ ID NO: 3 and/or SEQ ID NO: 4, (3) SEQ ID NO: 5 and/or SEQ ID NO: 6, (4) SEQ ID NO: 7 and/or SEQ ID NO: 8, (5) SEQ ID NO: 9 and/or SEQ ID NO: 10, (6) SEQ ID NO: 11 and/or SEQ ID NO: 12, (7) SEQ ID NO: 13 and/or SEQ ID NO: 14, (8) SEQ ID NO: 15 and/or SEQ ID NO: 16, (9) SEQ ID NO: 17 and/or SEQ ID NO: 18, (10) SEQ ID NO: 19 and/or SEQ ID NO: 20.
Specifically, the interfering RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 pairs of nucleotide sequences;
further, the interfering RNA comprises: (2) SEQ ID NO: 3 and/or SEQ ID NO: 4, (3) SEQ ID NO: 5 and/or SEQ ID NO: 6, (6) SEQ ID NO: 11 and/or SEQ ID NO: 12, (7) SEQ ID NO: 13 and/or SEQ ID NO: 14.
Further, the interfering RNA also includes a dangling base; preferably, the interfering RNA contains 1-10 hanging bases.
Wherein, the hanging bases can be the same or different deoxynucleosides (such as deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU) and the like), and the number of the hanging bases can be 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;
preferably, when the number of the dangling bases is 2, the dangling bases may be dTdT, dTdC or dUdU.
Further, the overhang base is located at the 3' end of the sense strand and/or the antisense strand of the interfering RNA.
Preferably, the interfering RNA molecule may further comprise at least one modified nucleotide, and the modified interfering RNA has better properties than the corresponding non-modified interfering RNA, such as higher stability, lower immunostimulatory properties, etc.
The interfering RNA may also comprise at least one modification, including modifications made to the base, sugar ring, and/or phosphate backbone;
preferably, modifications of the base include, but are not limited to, pyrimidine modifications at position 5, purine modifications at position 8, pseudouracil modifications, and/or 5-bromouracil substitutions;
preferably, the modification of the sugar ring includes, but is not limited to, 2' -OH quilt H, OZ, Z, halo, SH, SZ, NH 2 、NHZ、NZ 2 Or CN, wherein Z is an alkyl group;
the alkyl group represents a hydrocarbon chain radical which is straight or branched and does not contain an unsaturated bond, and the hydrocarbon chain radical is connected with other parts of the molecule by a single bond. Typical alkyl groups contain 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and the like.
Preferably, the phosphate backbone modification includes, but is not limited to, phosphorothioate modification.
The modifications also include nucleotides having inosine, pigtail, xanthine, 2' -methyl ribose, unnatural phosphodiester linkages (e.g., methylphosphonate, thiophosphonate), and/or peptides.
In one embodiment of the invention, the modification may be methylation, fluorination, thiophosphorylation, pseudouracil, or the like.
Further, the interfering RNA further comprises: (11) SEQ ID NO: 27 and/or SEQ ID NO: 28, (12) SEQ ID NO: 29 and/or SEQ ID NO: 30, (13) SEQ ID NO: 31 and/or SEQ ID NO: 32, (14) SEQ ID NO: 33 and/or SEQ ID NO: 34, (15) SEQ ID NO: 35 and/or SEQ ID NO: 36, (16) SEQ ID NO: 37 and/or SEQ ID NO: 38.
Specifically, the interfering RNA is selected from: siRNA, dsRNA, shRNA, aiRNA, miRNA, or a combination of two or more thereof.
In one embodiment of the present invention, the interfering RNA is siRNA.
In one embodiment of the invention, the interfering RNA is chemically synthesized.
The specific interfering RNA of the invention can reduce the expression of MLPH mRNA, inhibit melanin transport, and enable the MLPH mRNA to gather around the melanocyte nucleus, and not be transported into keratinocytes.
In a second aspect of the invention, there is provided a delivery system for interfering RNA comprising the interfering RNA of the first aspect and a vector.
Preferably, the vector is a viral vector or a non-viral vector.
Specifically, the above-mentioned vector may employ any vector suitable for delivering the above-mentioned interfering RNA of the present invention to a target tissue or a target cell or the like;
the vector is a viral vector or a non-viral vector;
preferably, the viral vector is selected from: one or a combination of two or more of lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors or herpesviral vectors.
More preferably, the vector is a non-viral vector selected from the group consisting of: any one or a combination of two or more of liposome, lipid Nanoparticle (LNP), polymer, polypeptide, antibody, aptamer, N-acetylgalactosamine (GalNAc).
The polymer is selected from: either or a combination of synthetic polymers and natural polymers;
the synthetic polymer is selected from: either one or the combination of two of polyethylenimine and cyclodextrin;
the natural polymer is selected from: either or a combination of chitosan and terminal collagen;
The polypeptide is a cell penetrating peptide selected from the group consisting of: any one or a combination of two or more of protamine, tat peptide, transport peptide, peneartin peptide and oligoarginine peptide;
more preferably, the non-viral vector is selected from the group consisting of: liposomes or lipid nanoparticles.
Preferably, the antibody may be a single chain antibody (e.g., scFv-tp, scFv-9R, etc.).
In particular, the liposome or lipid nanoparticle is selected from: any one or a combination of two or more of cationic lipid, polyethylene glycol lipid, neutral lipid, steroid lipid and anionic lipid.
In one embodiment of the invention, the lipid nanoparticle comprises the interfering RNA as described above, as well as polyethylene glycol lipid compounds, cationic lipids (excluding steroid-cationic lipid compounds), steroid lipids and neutral lipids.
Preferably, the molar ratio of the polyethylene glycol lipid compound, the cationic lipid, the steroid lipid and the neutral lipid in the lipid nanoparticle is (0.5-5): (30-55): (30-55): (5-20). Preferably, the molar ratio of the polyethylene glycol lipid compound, the cationic lipid, the steroid lipid and the neutral lipid is (1-5): (35-50): (40-50): (8-15).
In one embodiment of the invention, the lipid nanoparticle comprises the interfering RNA as described above, as well as a steroid-cationic lipid compound, a second lipid and a polyethylene glycol lipid. Wherein the second lipid is selected from the group consisting of: neutral lipids, zwitterionic lipids or anionic lipids.
Preferably, said steroid-cationic lipid in said lipid nanoparticle: second lipid: the molar ratio of polyethylene glycol lipid is 10-30:60-80:10-25, preferably 20-30:60-70:10-20.
The lipid nanoparticle can also encapsulate various components beneficial to human body, and can directly deliver the components to cells to play a role, so that expected effects can be produced faster and better.
The lipid nanoparticle can be prepared by adopting a lipid nanoparticle preparation method conventional in the art, such as a high-pressure homogenization method, an emulsion precipitation method, an ultrasonic dispersion method and the like.
Further, the cationic lipid comprises: octadecyl amide (SA), lauryl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, dimethyl Dioctadecyl Ammonium Bromide (DDAB), [ (4-hydroxybutyl) azadialkyl ] bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP), 1, 2-bis- (9Z-octadecyl) -3-trimethylammonio-propane and 1, 2-di-hexadecyl-3-trimethylammonio-propane, 3β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (DC cholesterol), dimethyl Dioctadecyl Ammonium (DDA), 1, 2-dimyristoyl-3-trimethylammonio-propane (AP), dipalmitoyl (C16: 0) trimethylammonio-propane (DPP), diacyl trimethylammonio-propane (DSTAP), N- [1- (2, 3-propionyloxy) -3-trimethylammonio-propane and 1, 2-dioleoyl-3-dioleoyl-N, DMT-carbamoyl ] cholesterol (DC cholesterol), dimethyl dioctadecyl ammonium chloride (DDA), 1, 2-dioleoyl-Dioctadecyl Ammonium Chloride (DACs), dimethyl dioctadecyl ammonium chloride (DCAC) and dimethyl dioctadecyl ammonium chloride (DCAC), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-dioleyloxy-3-dimethylaminopropane (DLinDMA), dioleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA), 1, 2-ditetradecanoyl-3-dimethylammonium-propane and 1, 2-dioctadecanoyl-3-dimethylammonium-propane, 1, 2-dioleoyl-c- (4' -trimethylammonium) -butyryl-sn-glycerol (DOTB), dioctadecanoyl-alanyl spermine, SAINT-2, the polycationic lipid 2, 3-dioleoyloxy-N- [2 (spermine-carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA),
(SM-102)、
(JK-102-CA)、
(JK-0315-CA) or a combination of two or more thereof.
Further, the neutral lipid comprises: one or more of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), and the like.
Further, the polyethylene glycol lipid comprises: 2- [ (polyethylene glycol) -2000]-N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol)](PEG-DSPE), PEG-distosterylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA),
One or a combination of two or more of the following;
wherein n is selected from integers of 20-300, such as 20, 30, 50, 80, 100, 150, 200, 250, 300, etc.
Further, the polyethylene glycol lipid is a polyethylene glycol lipid with a single molecular weight, preferably, the polyethylene glycol lipid comprises:
further, the cationic lipid is a steroid-cationic lipid compound:
the structure of the compound is as follows:
further, the anionic lipid comprises: one or more of dioleoyl phosphatidyl glycerol, dioleoyl phosphatidyl ethanolamine and the like.
Further, the steroid lipid includes: oat sterol, beta-sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterol, dehydrocholesterol, desmosterol, dihydroergocalcitol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, algae sterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.
In a third aspect, the invention provides a cell, the cell comprising the interfering RNA of the first aspect or the delivery system of the second aspect.
The expression level of MLPH in the cells is suppressed.
The cells may be tumor cells or non-tumor cells.
In a fourth aspect, the invention provides a method of preparing a cell, said method comprising introducing into a cell an interfering RNA as described in the first aspect or a delivery system as described in the second aspect.
In a fifth aspect, the invention provides a medicament or kit comprising an interfering RNA according to the first aspect, a delivery system according to the second aspect or a cell according to the third aspect.
The medicine also comprises pharmaceutically acceptable auxiliary materials.
Preferably, the pharmaceutically acceptable excipients include, but are not limited to, carriers, diluents, binders, lubricants, wetting agents, and the like.
Preferably, the drug is administered by a method including, but not limited to, subcutaneous injection, transdermal administration, and the like.
Preferably, the pharmaceutical dosage form includes, but is not limited to, emulsions, microemulsions, sub-microemulsions, nanoparticles, gels, powders, suspoemulsions, creams, jellies, sprays, and the like. The various dosage forms of the medicament can be prepared according to the conventional production method in the pharmaceutical field.
Preferably, the mass content of the interfering RNA, the delivery system or the cells in the medicament may be 1% -100%, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 100%.
In a sixth aspect the invention provides an antibody-nucleic acid conjugate medicament comprising an interfering RNA as described in the first aspect or a delivery system as described in the second aspect.
The antibody-nucleic acid conjugate medicament comprises one or more interfering RNAs.
The antibody and the nucleic acid in the antibody-nucleic acid conjugate medicament can be directly connected or connected through a connecting group or a connecting peptide.
Further, the general formula (I) of the antibody-nucleic acid conjugate medicine is:
wherein R is the interfering RNA of the first aspect;
x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2;
ab is an antibody, a protein or a polypeptide;
l is a linking unit linking Ab and R.
Further, the L moiety has the structure of formula (II): the L part is characterized by having a structure of a general formula (II):
Wherein,
in the formula II, x3 is selected from integers of 1-12; preferably 1 to 3;
in the formula II, x4 is selected from integers of 1-12; preferably 1 to 3;
P 1 、P 2 polyethylene glycol residues, which may be the same or different;
L 1 to connect Ab and P 1 A connecting unit therebetween;
L 2 to connect P 2 A linking unit with R;
A 1 to connect P 1 And P 2 A connecting unit therebetween;
preferably, the P 1 、P 2 Independently selected from linear, Y-type, multi-branched polyethylene glycol residues;
preferably, when said P 1 、P 2 Polyethylene glycol of single molecular weight, having a molecular weight of 88-4400Da, more preferably, said P 1 、P 2 Molecular weight is 176-1056Da;
preferably, when said P 1 、P 2 Polyethylene glycol with non-single molecular weight and molecular weight of 1000Da-40kDa;
more preferably, the P 1 、P 2 The molecular weight is 2000Da-10kDa.
Further, the L 1 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、/>one or a combination of two or more groups;
the straight or branched chain C 1-12 Chain alkylene, C 6-12 Arylene or C of (2) 3-12 Any H atom on the cycloalkylene group is represented by-H, -F-Cl, -Br, -I, -O-, -S-, -SO 2 、-NO 2 、C 1-12 Chain alkyl, C 3-12 Cycloalkyl, C 6-12 Aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more than two groups ofSubstitution of the group;
the L is 2 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、/>one or a combination of two or more groups;
the straight or branched chain C 1-12 Chain alkylene, C 6-12 Arylene or C of (2) 3-12 Any H atom on the cycloalkylene group is represented by-H, -F-Cl, -Br, -I, -O-, -S-, -SO 2 、-NO 2 、C 1-12 Chain alkyl, C 3-12 Cycloalkyl, C 6-12 Aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more than two groups are substituted;
preferably, the L 1 Is an amide bond, a hydrazone bond, and a mercapto-maleimide bond;
more preferably, the L 1 Is an amide bond;
preferably, the L 2 Is a disulfide bond and a thiol-maleimide bond;
more preferably, the L 2 Is a disulfide bond.
Further, the A 1 To connect P 1 And P 2 A linking group therebetween selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-, -S-S-/>one or a combination of two or more groups;
the straight or branched chain C 1-12 Chain alkylene, C 6-12 Arylene or C of (2) 3-12 Any H atom on the cycloalkylene group is represented by-H, -F-Cl, -Br, -I, -O-, -S-, -SO 2 、-NO 2 、C 1-12 Chain alkyl, C 3-12 Cycloalkyl, C 6-12 Aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more groups.
Further, the antibody-nucleic acid conjugate drug has the general formula (IV):
the L is 1 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、/>one or a combination of two or more groups;
preferably, P 1 Polyethylene glycol residues which are the same or different;
preferably, when said P 1 Polyethylene glycol of single molecular weight, having a molecular weight of 88-4400Da, more preferably, said P 1 Molecular weight is 176-1056Da;
preferably, when said P 1 Polyethylene glycol with non-single molecular weight and molecular weight of 1000Da-40kDa; more preferably, the P 1 The molecular weight is 2000Da-10kDa.
The L is 2 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、one or a combination of two or more groups;
x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.
Further, the antibody-nucleic acid conjugate drug has the following structure:
Said n 1 An integer selected from 4 to 100, preferably 4 to 24; n1 may be a constant value or an average value.
In one embodiment of the invention, the antibody-nucleic acid conjugate drug has the following structure (V):
said n 1 、n 2 Independently selected from integers of 4-100, for example: 4. 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99,100。
Preferably n 1 、n 2 Independently selected from integers from 4 to 24; n is n 1 、n 2 Either constant or mean.
Further, the Ab is selected from monoclonal antibodies, polyclonal antibodies, antibody fragments and antibody fusion fragments;
the antibody may be a single domain antibody or a single chain antibody.
Further preferred, ab is a monoclonal antibody;
more preferably, the monoclonal antibody is reactive with an antigen or epitope thereof associated with cancer, malignant cells, infectious organisms, or autoimmune diseases.
In a specific embodiment of the invention, the Ab is selected from: anti-HER 2 antibodies, anti-EGFR antibodies, anti-PMSA antibodies, anti-VEGFR antibodies, anti-CD 30 antibodies, anti-CD 22 antibodies, anti-CD 56 antibodies, anti-CD 29 antibodies, anti-GPNMB antibodies, anti-CD 138 antibodies, anti-CD 74 antibodies, anti-ENPP 3 antibodies, anti-Nectin-4 antibodies, anti-EGFR viii antibodies, anti-SLC 44A4 antibodies, anti-mesothelin antibodies, anti-ET 8R antibodies, anti-CD 37 antibodies, anti-CEACAM 5 antibodies, anti-CD 70 antibodies, anti-MUC 16 antibodies, anti-CD 79b antibodies, anti-MUC 16 antibodies, anti-MUC 1 antibodies, anti-CD 3 antibodies, anti-CD 28 antibodies, anti-CD 38 antibodies, anti-CD 19 antibodies, anti-PD-L1 antibodies, anti-4-1 BB antibodies, and the like.
In a seventh aspect, the invention provides a composition comprising an interfering RNA as described in the first aspect or a delivery system as described in the second aspect and an adjuvant.
Preferably, the composition is selected from: any one of medicines, cosmetics, and medical instruments;
preferably, when the composition is a pharmaceutical product, the auxiliary material comprises one or more of a filler, a wetting agent, a dispersing agent, a diluent, an anti-adhesion agent, an adhesive and a pigment.
Preferably, when the composition is a pharmaceutical product, the pharmaceutical dosage form is selected from the group consisting of: any one of liquid dosage forms, injections, aerosols, films and ointments;
preferably, the injection comprises: including sterile or sterilized solutions, injections, oil injections, and powder injections;
preferably, the ointment comprises: oleaginous base ointment, emulsion base ointment and water-soluble base ointment.
The ointment is mainly used for skin and mucous membrane surfaces.
Preferably, when the composition is a cosmetic, the auxiliary materials include: antioxidants, binders, biological additives, buffers, colorants, thickeners, polymers, astringents, fragrances, humectants, diluents, dispersants, opacifying agents, conditioning agents, exfoliants, pH adjusters, preservatives, natural extracts, essential oils, skin soothing agents, and skin healing agents;
Preferably, when the composition is a cosmetic, the carrier of said cosmetic is selected from: any one of an emulsion, lotion, cream, foam, gel, soap bar, stick, mask, cotton pad or patch.
In an eighth aspect, the present invention provides a composition comprising an interfering RNA as described in the first aspect or a delivery system as described in the second aspect for use in the manufacture of a medicament for the prevention and/or treatment of a disease associated with MLPH.
Such MLPH related diseases include, but are not limited to: tumor, skin pigmentation;
such tumors include, but are not limited to: rectal cancer, vulvar cancer, prostate cancer, breast cancer, lung squamous carcinoma and adenocarcinoma.
Such pigmentation includes, but is not limited to: senile plaque, acne marks, pigmented spots, freckle, cyasma or pathological pigment changes caused by sunlight, inflammation, medicines and the like.
In a ninth aspect, the present invention provides a composition comprising the interfering RNA of the first aspect or the delivery system of the second aspect for whitening, removing spots.
Preferably, the whitening and freckle removing are for non-therapeutic purposes;
the whitening and freckle removing steps comprise: reducing skin pigment content and pigmentation, and removing skin spots, such as senile plaque, acne mark, pigmented spots, freckle, cyasma or other pigment changes caused by sunlight, inflammation, medicine, etc.
The sequence applicable object of the present invention is a human.
The invention has the beneficial effects that:
the invention designs specific siRNA aiming at the mRNA of MLPH, and delivers the specific siRNA into target cells, thereby realizing the inhibition of MLPH expression, reducing MLPH synthesis, further inhibiting the transportation of melanosomes and realizing the purposes of skin whitening and freckle removal; or by reducing MLPH expression, to treat MLPH-related diseases (e.g., partial tumors). The siRNA of the invention has good activity of inhibiting MLPH, and the activity can reach about 80 percent at most. In addition, the present invention provides a suitable Lipid Nanoparticle (LNP) delivery system that can deliver siRNA to a suitable location under the skin, exert its biological effect, and is expected to have wide application in whitening products.
Drawings
FIGS. 1A-1J show mass spectral identification of sense and antisense strands of each sequence of siMLPH.
FIG. 2 shows the relative expression levels of MLPH mRNA in each of the 24h and 48h groups of cells after the transfection of the siMLPH.
FIG. 3 shows cytotoxicity evaluation of siMLPH.
FIGS. 4A to 4F show the results of mass spectrometry identification of the modified sequences siMLPH265, siMLPH-m1 to siMLPH-m 6.
FIG. 5 shows the relative expression levels of MLPH mRNA in groups of cells 48h after transfection of the modified sequence of siMLPH265, siMLPH-m1 to siMLPH-m 6.
FIG. 6 shows cytotoxicity evaluation of the modified sequences siMLPH265, siMLPH-m1 to siMLPH-m 6.
FIG. 7 shows the melanin distribution in cells after transfection of siMLPH.
FIG. 8 shows the relative expression levels of MLPH mRNA in groups of cells 48h after LNP-siMLPH265 transfection.
FIG. 9 shows the cytotoxicity evaluation of LNP-siMLPH 265.
Fig. 10 shows representative photographs of the slice groups after treatment of the 3D melanin skin model.
Fig. 11 shows the area ratios of each group of melanin positive areas after treatment with the 3D melanin skin model.
FIG. 12 shows the relative expression levels of pMLPH mRNA in groups of cells 24h after transfection of porcine MLPH siRNA.
FIG. 13 shows cytotoxicity evaluation 72h after transfection of porcine MLPH siRNA.
FIG. 14 shows the results of staining skin sections of the Bama pig control group.
FIG. 15 shows the staining results of skin sections of the group of Ba Ma Zhu LNP-si-pMLPH 265.
FIG. 16 shows the percentage of melanin in the epidermis layer of each group of sections of Bama pigs.
Figure 17 shows the body weight change of Bama pigs following administration.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.
The term "interfering RNA" or "RNAi" or "interfering RNA sequence" as used herein includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that is capable of reducing or inhibiting expression of a target gene or sequence (e.g., by mediating degradation and inhibiting translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. An interfering RNA is thus a single-stranded RNA complementary to the target mRNA sequence or a double-stranded RNA formed from two complementary strands or from a single self-complementary strand. Specifically, interfering RNA molecules are chemically synthesized.
The interfering RNA includes a "small interfering RNA" or "siRNA", where each strand of the siRNA molecule comprises nucleotides of about 15 to about 60 in length (e.g., nucleotides of about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 in length, or nucleotides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 in length). In a specific embodiment, the siRNA is chemically synthesized. The siRNA molecules of the invention are capable of silencing expression of a target sequence in vitro and/or in vivo. In other embodiments, the siRNA comprises at least one modified nucleotide, e.g., the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in the double-stranded region.
As used herein, the term "dsRNA" or "precursor RNAi molecule" is intended to include any precursor molecule that is processed in vivo by an endonuclease to produce an active siRNA.
In the present invention, the term "therapeutically effective amount" refers to the amount of a subject compound that will elicit the biological or medical response of a tissue, system or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes the amount of the active ingredient: when administered, it is sufficient to prevent the development of, or to alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the active ingredient, the disease to be treated and its severity, the age, weight, sex, etc. of the subject.
In the present invention, the subject may be a mammal, such as a human, monkey, dog, rabbit, mouse, rat, or the like.
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application.
EXAMPLE 1 siRNA design and Synthesis
For the melanin avidin (MLPH) sequence (GenBank: NM_ 000372.5), the applicant has creatively worked to select and design 10 siRNA sequences according to the comprehensive consideration of the structure, the targeting position and the combination of early fumbling experience of the sequences, and the sequences are synthesized by Beijing qing department biotechnology Co., ltd for screening, and the sequence information is as follows (table 1):
TABLE 1 siRNA sequences
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In addition, a pair of unrelated sequences (siNC) were synthesized at the same time, the sense strand: 5' -UUC UCC GAA CGU GUC ACG UdTdT (SEQ ID NO: 21), antisense strand: 5' -ACG UGA CAC GUU CGG AGA AdTdT (SEQ ID NO: 22) was designed and synthesized by the division of Biotechnology (Shanghai).
EXAMPLE 2 inhibition of siRNA in melanocytes
1. Cell culture and transfection
(1) A375 cells were cultured in DMEM medium (100U/mL Penicillin, 100. Mu.g/mL Streptomycin) containing 10% FBS at 37℃with 5% CO 2 Culturing in a saturated humidity incubator. 1X 10 per well 24 hours before the experiment 5 Cells were seeded in 24-well plates and cultured overnight.
(2) mu.L of OPTI-MEM medium was diluted to 2.5. Mu.L of either siMLPH or siNC (mother liquor concentration was 10. Mu.M) of 10 strips (SEQ ID NO: 1-20) described in example 1, 23.5. Mu.L of OPTI-MEM medium was diluted to 1.5. Mu. L Lipofectamine RNAiMAX of transfection reagent, and the two were mixed, gently shaken and allowed to stand for 5min. Further, a blank group and a lipofectamine RNAiMAX group are provided.
(3) Cells in each well of the cell plate were changed, and an antibiotic-free medium (90% DMEM+10% FBS) was added thereto in a volume of 450. Mu.L per well, and then 50. Mu.L of the above-mentioned mixed solution was added thereto per well, to thereby obtain a total volume of 500. Mu.L per well. The final concentration of the siMLPH (or siNC) transfection was 50nM.
(4) 24h and 48h after transfection 24 well plates were incubated from 37℃with 5% CO 2 Is removed from the incubator and the cells are collected for subsequent detection of RNA extraction.
2. RNA extraction
(1) RNA was extracted using Promega RNA extraction kit. After washing each group of cells in PBS 1, 300. Mu.L of lysate was added, the cells were blown off with a pipette, after sufficient lysis, 300. Mu.L of diluent was added, and then water bath was performed at 70℃for 3min. Centrifuge at 14000rpm for 10min, transfer the supernatant to a new 1.5mL Ep tube, add 300 μl absolute ethanol, mix well and add to the column. Centrifuge at 14000rpm for 1min, discard filtrate, add 600 μl wash and centrifuge again for 1min. Discarding the filtrate, adding 50 mu L of the ready-prepared DNase reaction solution into each hole, and standing at room temperature for 15min; adding 600 mu L of washing liquid, and centrifuging for 1min; removing filtrate, adding 600 μl of washing solution, and centrifuging for 1min again; the filtrate was discarded, centrifuged for 2min, 50. Mu.L of nuclease-free water was added to each well, and the mixture was allowed to stand at room temperature for 5min, followed by centrifugation to collect the eluted RNA.
(2) RNA quality inspection, RNA content and purity detection, and 1% agarose gel electrophoresis detection of RNA integrity.
3. Q-PCR detection flow
(1) RNA reverse transcription
Using the total RNA extracted in the step 2 as a template and using a Promega reverse transcription kit, establishing a reaction system as follows:
TABLE 2 RNA reverse transcription System
Mixing the above systems, centrifuging to collect liquid to the bottom of the tube, and standing at 42deg.C for 60min and at 72deg.C for 10min; the product is the cDNA template.
(2) Fluorescent quantitative PCR detection
Using TB green Premix Ex Taq II (Tli RNaseH Plus) (Takara) reagent and the primers in Table 3, a reaction system was established as follows (Table 4):
TABLE 3 primer sequences
TABLE 4 fluorescent quantitative PCR reaction System
PCR amplification was performed as follows
Pre-denatured at 95 ℃ for 10min, and then enters the following circulation
*95℃10s
60℃20s
70℃10s
Reading board
Return is performed for a total of 40 cycles.
Preparing a melting curve: plates were read between 65℃and 95℃and stopped for 5s every 0.5 ℃.
4. Evaluation of inhibitory Effect
GAPDH as internal standard gene, pass 2 -ΔΔCt Calculating the relative expression quantity of MLPH mRNA by a method; each group of mRNA expression levels were normalized to 100% of the blank group expression levels.
FIG. 2 shows the relative expression levels of the cells of each group after transfection of the sMLPH in A375 cells, showing that the selected MLPH siRNA has a remarkable and sustained effect of inhibiting the expression of the MLPH gene, and the relative expression levels of the mRNA of each group of cells are shown in Table 5 or FIG. 2. The results show that the relative expression level of MLPH mRNA in other groups except siMLPH1238 in siRNA-treated cells is obviously reduced at 24 hours relative to that of blank groups and siNC groups, and the expression inhibition of the MLPH mRNA can be up to about 80 percent. At 48h post-transfection, siMLPHSiMLPH262, siMLPH265, siMLPH427 and siMLPH1144siMLPH still show obvious inhibition effect, and the inhibition effect is about 70%, which indicates that the screened MLPH siRNA has obvious and continuous effect of inhibiting MLPH gene expression.
TABLE 5 relative expression levels of MLPH mRNA in groups of cells 24h and 48h after transfection of siMLPH in example 1
EXAMPLE 3 siRNA cytotoxicity assay
A375 cells in logarithmic growth phase were plated in 96-well plates with 200. Mu.L of 5X 10 cells per well 3 Culture medium of individual cells at 37℃with 5% CO 2 The incubator was cultured overnight. The next day, the medium was discarded from each well, and transfection reagents containing each siMLPH were prepared, and 180. Mu.L of antibiotic-free medium and 20. Mu.L of transfection reagent were added to each well at a final siMLPH concentration50nM, continue 37 ℃,5% CO 2 The incubator was cultured for 48 hours. The plates were removed and 10. Mu.L of 5mg/mL MTT solution was added to each well and the incubation was continued for 4h. The culture solution was aspirated, 200. Mu. LDMSO was added to each well, and the mixture was shaken for 3min with a microplate reader to detect absorbance at 490 nm. The absorbance values of each group were normalized to the blank group and the cell viability of each group of samples was calculated (see fig. 3 and table 6).
Cell viability= (a Treatment of -A 0 )/(A con -A 0 )×100％
TABLE 6 cytotoxicity evaluation of siMLPH
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As can be seen from fig. 3 and table 6, none of the siMLPH treatments caused significant cytotoxic performance and had good potential for use.
EXAMPLE 4 modified siRNA design and Synthesis
The following modification sequences were designed according to simph 265 (see table 7), and synthesized by Shanghai biosciences, inc. The mass spectrum is shown in figure 4.
TABLE 7 modified sequences designed for siMLPH265
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Note that: m is methyl modification, f is fluoro modification, -phosphorothioate modification, ψ is pseudouracil substitution.
Example 5 inhibition of modified siRNA in melanocytes
The experimental procedure is as in example 2, and the results are shown in FIG. 5 and Table 8.
TABLE 8 relative expression levels of MLPH mRNA in groups of cells 48h after transfection of the siMLPH265 modification sequence
As can be seen from FIG. 5 and Table 8, the modified 6 modified sequences of siMLPH265 were capable of significantly inhibiting the expression of MLPH mRNA except for siMLPH-m1, wherein siMLPH-m4, siMLPH-m5 and siMLPH-m6 can inhibit the expression of MLPH mRNA by 80% -90%.
EXAMPLE 6 modified siRNA cytotoxicity assay
The cytotoxicity evaluation test method was the same as in example 3 except that each siRNA was siMLPH265, siMLPH265m1-m6, and the results are shown in FIG. 6 and Table 9.
TABLE 9 cytotoxicity evaluation of siMLPH265 modified sequences
As can be seen from FIGS. 6 and 9, the modified sequences of siMLPH265 were not significantly cytotoxic in each of the other groups except for the slight cytotoxicity of siMLPH265-m4 and siMLPH265-m 6.
EXAMPLE 7 melanosome distribution observations
The distribution of melanosomes after siMLPH treatment was observed using the human melanoma cell line MNT-1. Briefly, MNT-1 cells were cultured in DMEM medium containing 20% FBS and 1% diabody at 37℃and 5% CO 2 Culturing in a saturated humidity incubator, and passaging every 3 d. Before the experiment, each hole is 2 to 5 multiplied by 10 for 24 hours 5 Cells were seeded in 8-well chamber cover slips and cultured overnight. Cell transfection procedure referring to example 2, a blank set (BK), a lipofectamine RNAiMAX +siMLPH265 set (abbreviated as lipo+siMLPH265 set), an LNP mock set, an LNP-siMLPH265 set, and an LNP-siMLPH265-m4 set were set, respectively. The final concentration of the siMLPH transfection was 50nM. Laser confocal microscopy was performed 24h, 48h, 72h after transfection.
The results show that: in cells not treated with siMLPH (BK, LNP mock), melanin is distributed at the cell edges and melanin aggregates are present in individual cells; in siMLPH-treated cells (Lipo + siMLPH265, LNP-siMLPH265-m 4), melanin was concentrated mainly around the nucleus and with increasing time, the phenomenon of melanin aggregation became more and more evident, and the number of cells in which melanin corpuscle aggregation occurred increased (fig. 7), indicating that the transfer of melanin corpuscles from the nucleus to the outside was inhibited, resulting in that the produced melanin corpuscles were mostly only present around the nucleus, not outside.
EXAMPLE 8 preparation of LNP-siMLPH265
Weighing SM-10271.02mg, adding 10mL of EtOH, and dissolving to obtain SM-102 mother liquor;
Weighing M-DMG-2000249.5mg, and dissolving 10mL of anhydrous EtOH to obtain PEG component mother liquor;
weighing 79.02mg of DSPC, and dissolving 10mL of anhydrous EtOH to obtain a DSPC mother solution;
38.66mg of cholesterol was weighed out, 10mL of absolute EtOH was used, and the resulting cholesterol mother liquor was dissolved.
Taking 64.8 mu L, DSPC mu L of SM-102 mother liquor 2160 mu L, PEG component mother liquor and 1664 mu L of cholesterol mother liquor respectively, and uniformly mixing to obtain LNP/EtOH solution;
taking the simlPH265150nmol, adding 6.5mL of 50mM citric acid buffer solution (pH=4.0) to obtain a simlPH265/buffer solution;
microfluidic preparation: LNP/EtOH solution was used in passageway 1, solution volume 2mL, flow rate 5mL/min; the siMLPH265/buffer solution uses a passage 2, the volume of the solution is 6mL, and the flow rate is 15mL/min;
4mL of the microfluidic preparation solution was dialyzed against PBS (pH=7.4) for 16h to give LNP-siMLPH265, and the final concentration of siMLPH265 was 0.2mg/mL.
Example 9 inhibition of LNP-siMLPH265 in melanocytes
The experimental procedure was the same as in example 2, using MNT-1 cells in place of A375 cells, and the experimental results are shown in FIG. 8 and Table 10.
TABLE 10 relative expression levels of MLPH mRNA in groups of cells 48h after LNP-siMLPH265 transfection
As can be seen from fig. 8 and table 10, LNP can effectively deliver simph 265 into cells and allow it to function, significantly inhibiting the expression of MLPH mRNA.
EXAMPLE 10 LNP-siMLPH265 cytotoxicity assay
The experimental procedure is as in example 3, using MNT-1 cells to replace A375 cells, and the results are shown in FIG. 9 and Table 11.
TABLE 11 cytotoxicity evaluation of LNP-siMLPH265
As can be seen from fig. 9 and table 11, the LNP-siMLPH265 group was less cytotoxic than lipofectamine RNAiMAX + siMLPH265 group without affecting the survival status of the cells.
Example 11 3D skin tissue experiment
Using Guangdong Boxi Biotechnology Co., ltdThe 3D melanin skin model was subjected to experiments. />The keratinocyte and the melanocyte separated from the skin tissue of Chinese are taken as seed cells, and the keratinocyte and the melanocyte are developed into a 3D melanocyte skin model containing a melanocyte multi-layered structure in vitro. />Has melanin granule distribution, epidermis multi-layered structure, physiological and metabolic functions which are highly similar to natural skin, and is widely used for detecting the field of in-vitro whitening efficacyAnd (5) measuring.
The 3D melanin skin model was used for experiments after adaptation at 37 ℃ for 24h (day 0) and transfected on day 1 and day 4, respectively. Before the start of the daily experiment, three groups were irradiated with UVB at 311nM for 5min and then with melagrow medium containing 100 nM. Alpha. MSH, except that the blank group 1 was changed daily to medium without αMSH and without other treatments. The experiment was set up in 4 groups, respectively blank 1, blank 2, LNP mock, LNP-siMLPH-m4 (500 nM), and the experiment was continued for 7 days. The same UVB irradiation and fluid exchange was performed at morning, tissue was fixed in 4% paraformaldehyde at afternoon overnight, and tissue embedding, sectioning, masson-Fontana melanin staining and eosin counterstaining were performed the next day.
The area of the melanin region in the sections (fig. 10) was counted, and the results are shown in fig. 11 and table 12:
table 12 statistics of melanin proportion in 3d melanin model slices (n=3 to 6)
As can be seen from fig. 11 and table 12, the group of the blank2 treated with UVB irradiation and αmsh had a significantly higher melanin positive area than the untreated group of the blank1, while the group of LNP-simph-m 4 and kojic acid had significantly lower melanin positive area, even lower than the group of the blank1, despite the UVB irradiation and αmsh treatment, showing that the LNP-simph had a similar or even better whitening effect as the kojic acid.
EXAMPLE 12 inhibition of porcine homologous siMLPH (si-pMLPH) in porcine MLPH over-expression stable transgenic cell lines
Pig MLPH sequences (NM-001098594.1) were retrieved from NCBI, sequences homologous to human MLPH were selected, and the corresponding siRNA sequences were synthesized (Table 13). The corresponding LNP-si-pMLPH was prepared as in example 9.
TABLE 13 human and porcine siMLPH265 (si-pMLPH) sequences
The inhibitory effect of si-pMLPH on pMLPH gene expression was verified using HEK293T cell line (HEK-293T-pMLPH) stably transfected with porcine MLPH (pMLPH) sequence. The experimental procedure is as in example 2. Primer sequences are shown in Table 14.
TABLE 14 pig MLPH sequence amplification primers
qPCR results (Table 15, FIG. 12) show that the relative expression level of pMLPH mRNA was around 60% in three groups of cells delivered 24h,lipofectamine RNAiMAX after transfection of HEK-293T-pMLPH cells with si-pMLPH. The relative expression level of pMLPH mRNA was 20% -30% in the LNP-si-pMLPH group at 3 doses. The result shows that the screened pMLPH siRNA has obvious effect of inhibiting pMLPH gene expression. Meanwhile, LNP can effectively deliver pMLPH siRNA into cells to make the cells function, and obviously inhibit the expression of pMIPH mRNA.
TABLE 15 relative expression levels of pMLPH mRNA in groups of cells 24h after transfection of si-pMLPH
Example 13 cytotoxicity assay of si-pMLPH sequence
Toxicity of si-pMLPH in HEK-293T-pMLPH cells was verified using a HEK293T cell line (HEK-293T-pMLPH) stably transfected with porcine MLPH sequences. The experimental procedure is as in example 3. The results are shown in Table 16 and FIG. 13.
As can be seen from table 16 and fig. 13, the viability of each group of cells transfected by Lipofectamine RNAiMAX is 50% -60%, which is slightly lower than lipofectamine RNAiMAX +sinc group, and has certain cytotoxicity; the survival rate of LNP-si-pMLPH group cells at three doses was 77% -95%, indicating that LNP containing si-pMLPH has no obvious toxicity to MLPH cells.
Table 16 cytotoxicity of porcine MLPH siRNA after 72h transfection
Example 14 effect of LNP-si-pMLPH265 on porcine skin
The Bama pig is used as a model animal, and light black skin at the boundary of black spots and white skin at the back is selected for experiments. Two groups are: control, LNP-si-pMLPH265 injection group, three regions per group, with the three regions of the same group being spaced about 1cm apart, different groups being assigned to different extra-black corresponding regions. The administration part is shaved, cleaned and administered. LNP-si-pMLPH265 stock solution was 2mg/mL, diluted 10-fold, and subcutaneously injected using 9-needle microneedles, 100. Mu.L per zone. Administration was performed at day0, 8, 15, 22 and 29, respectively, and skin tissue was taken at 52d for fixation, sectioning and melanin staining analysis, and the results are shown in fig. 14 and 15. The area ratio of melanin granules in the epidermis was counted in this area and compared with the control group, and the results are shown in table 17 and fig. 16.
During the whole experiment, the LNP-si-pMLPH265 group test area only has transient slight swelling phenomenon at the second week, and then returns to normal soon after the transient slight swelling phenomenon; the weight of the Bama pig decreased slightly, and the administration was stopped and the upward trend was recovered, while the state, activity, etc. were not significantly affected (see fig. 17). The results of the tissue sections show (see fig. 14 and 15), and in the tissue sections of the control group, the epidermis melanin is widely distributed, and the melanin distributed along the hair follicle is obviously seen at the hair follicle; in the LNP-si-pMLPH265 group, little melanin was seen in the epidermis layer with little scattered melanin distribution in the basal layer. Indicating that LNP-si-pMLPH265 can significantly inhibit melanin production and transport.
Table 17 ratio of melanin positive areas in administration area of bama pigs (mean±sd, n=6)
As can be seen from table 17 and fig. 16, the melanin-positive areas in the epidermis of the LNP-si-pMLPH265 treated group were significantly reduced to 41.4% of the control group as compared to the control group. The LNP-si-pMLPH265 treatment can obviously reduce the melanin content in epidermis, and has the whitening effect.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
The foregoing embodiments and methods described herein may vary based on the capabilities, experience, and preferences of those skilled in the art.
In the present invention, the steps of the method are listed in a certain order only and do not constitute any limitation on the order of the steps of the method.

Claims (33)

1. An interfering RNA that targets MLPH expression, wherein the target site sequence of the interfering RNA comprises: nucleotide sequences shown in any one or two or more of SEQ ID NO: 1-20 or SEQ ID NO: 27-38.
2. The interfering RNA of claim 1 wherein the interfering RNA comprises: SEQ ID NO: 3 and/or SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6, SEQ ID NO: 11 and/or SEQ ID NO: 12, SEQ ID NO: 13 and/or SEQ ID NO: 14.
3. The interfering RNA of any one of claims 1-2 wherein the interfering RNA further comprises a dangling base; preferably, the interfering RNA comprises 1-10 dangling bases, the dangling bases are deoxynucleosides.
4. The interfering RNA of claim 3 wherein the overhang base is located at the 3' -end of the sense strand and/or the antisense strand of the interfering RNA.
5. The interfering RNA of claim 1 wherein the interfering RNA further comprises at least one modification, the modification comprising a modification on a base, sugar ring, and/or phosphate backbone;
Preferably, modifications of the base include, but are not limited to, pyrimidine modifications at position 5, purine modifications at position 8, pseudouracil modifications, and/or 5-bromouracil substitutions;
preferably, the modification of the sugar ring includes, but is not limited to, 2' -OH quilt H, OZ, Z, halo, SH, SZ, NH 2 、NHZ、NZ 2 Or CN, wherein Z is an alkyl group;
preferably, the phosphate backbone modification includes, but is not limited to, phosphorothioate modification.
6. The interfering RNA of claim 5 wherein the modification further comprises a nucleotide having inosine, plaitin, xanthine, 2' -methylribose, non-natural phosphodiester linkage, and/or peptide.
7. The interfering RNA of claim 5 or 6, wherein the interfering RNA further comprises: SEQ ID NO: 27 and/or SEQ ID NO: 28, SEQ ID NO: 29 and/or SEQ ID NO: 30, SEQ ID NO: 31 and/or SEQ ID NO: 32, SEQ ID NO: 33 and/or SEQ ID NO: 34, SEQ ID NO: 35 and/or SEQ ID NO: 36, SEQ ID NO: 37 and/or SEQ ID NO: 38.
8. The interfering RNA of claim 1 wherein the interfering RNA is selected from the group consisting of: siRNA, dsRNA, shRNA, aiRNA, miRNA, or a combination of two or more thereof;
preferably, the interfering RNA is siRNA.
9. A delivery system for interfering RNA, characterized in that it comprises the interfering RNA of any one of claims 1-8 and a vector;
the vector is a viral vector or a non-viral vector;
preferably, the viral vector is selected from: one or a combination of two or more of lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors or herpesviral vectors;
preferably, the vector is a non-viral vector;
more preferably, the non-viral vector is selected from the group consisting of: any one or a combination of two or more of liposome, lipid nanoparticle, polymer, polypeptide, antibody, aptamer, N-acetylgalactosamine (GalNAc);
the polymer is selected from: either or a combination of synthetic polymers and natural polymers;
the synthetic polymer is selected from: either one or the combination of two of polyethylenimine and cyclodextrin;
the natural polymer is selected from: either or a combination of chitosan and terminal collagen;
the polypeptide is a cell penetrating peptide selected from the group consisting of: any one or a combination of two or more of protamine, tat peptide, transport peptide, peneartin peptide and oligoarginine peptide; more preferably, the non-viral vector is selected from the group consisting of: liposomes or lipid nanoparticles.
10. The delivery system of claim 9, wherein the liposome or lipid nanoparticle is selected from the group consisting of: any one or a combination of two or more of cationic lipid, polyethylene glycol lipid, neutral lipid, steroid lipid and anionic lipid.
11. The delivery system of claim 10, wherein the cationic lipid comprises: octadecyl amide (SA), lauryl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, dimethyl Dioctadecyl Ammonium Bromide (DDAB), [ (4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP), 1, 2-bis- (9Z-octadecyl) -3-trimethylammonio-propane and 1, 2-di-hexadecyl-3-trimethylammonio-propane, 3β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (DC cholesterol), dimethyl Dioctadecyl Ammonium (DDA), 1, 2-dimyristoyl-3-trimethylammonio-propane (AP), dipalmitoyl (C16: 0) trimethylammonio-propane (DPP), diacyl trimethylammonio-propane (DSTAP), N- [1- (2, 3-propionyloxy) -3-trimethylammonio-propane and 1, 2-dioleoyl-3-dioleoyl-N, DMT-carbamoyl ] cholesterol (DC cholesterol), dimethyl dioctadecyl ammonium chloride (DDA), 1, 2-dioleoyl-Dioctadecyl Ammonium Chloride (DACs), dimethyl dioctadecyl ammonium chloride (DCAC) and dimethyl dioctadecyl ammonium chloride (DCAC), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-dioleyloxy-3-dimethylaminopropane (DLinDMA), dioleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA), 1, 2-ditetradecanoyl-3-dimethylammonium-propane and 1, 2-dioctadecanoyl-3-dimethylammonium-propane, 1, 2-dioleoyl-c- (4' -trimethylammonium) -butyryl-sn-glycerol (DOTB), dioctadecanoyl-alanyl spermine, SAINT-2, the polycationic lipid 2, 3-dioleoyloxy-N- [2 (spermine-carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA),
One or a combination of two or more of them.
12. The delivery system of claim 10, wherein the neutral lipid comprises: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE).
13. The delivery system of claim 10, wherein the polyethylene glycol lipid comprises: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159) 1, 2-dimyristoyl-sn-glycerylmethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA),
One or a combination of two or more of them;
wherein n is an integer from 20 to 300.
14. The delivery system of claim 10, wherein the polyethylene glycol lipid is a single molecular weight polyethylene glycol lipid, preferably wherein the polyethylene glycol lipid comprises:
15. the delivery system of claim 10, wherein the cationic lipid is a steroid-cationic lipid compound:
the structure of the compound is as follows:
16. the delivery system of claim 10, wherein the anionic lipid comprises: one or more of dioleoyl phosphatidyl glycerol and dioleoyl phosphatidyl ethanolamine.
17. The delivery system of claim 10, wherein the steroid lipid comprises: oat sterol, beta-sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterol, dehydrocholesterol, desmosterol, dihydroergocalcitol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, algae sterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.
18. A cell comprising the interfering RNA of any one of claims 1-8 or the delivery system of any one of claims 9-17.
19. A method of preparing a cell, comprising introducing into the cell an interfering RNA according to any one of claims 1-8 or a delivery system according to any one of claims 9-17.
20. A medicament or kit comprising the interfering RNA of any one of claims 1-8, the delivery system of any one of claims 9-17, or the cell of claim 18.
21. An antibody-nucleic acid conjugate comprising the interfering RNA of any one of claims 1-8 or the delivery system of any one of claims 9-17.
22. The antibody-nucleic acid conjugate of claim 21, wherein the antibody-nucleic acid conjugate has the general formula (I):
wherein R is the interfering RNA of any one of claims 1-8;
x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2;
Ab is an antibody, a protein or a polypeptide;
l is a linking unit linking Ab and R.
23. The antibody-nucleic acid conjugate of claim 22, wherein the L moiety has the structure of formula (ii):
wherein,
in the formula II, x3 is selected from integers of 1-12; preferably 1 to 3;
in the formula II, x4 is selected from integers of 1-12; preferably 1 to 3;
P 1 、P 2 polyethylene glycol residues which are the same or different;
L 1 to connect Ab and P 1 A connecting unit therebetween;
L 2 to connect P 2 A linking unit with R;
A 1 to connect P 1 And P 2 A connecting unit therebetween;
preferably, the P 1 、P 2 Independently selected from linear, Y-type, multi-branched polyethylene glycol residues;
preferably, when said P 1 、P 2 Polyethylene glycol of single molecular weight, having a molecular weight of 88-4400Da, more preferably, said P 1 、P 2 Molecular weight is 176-1056Da;
preferably, when said P 1 、P 2 Polyethylene glycol with non-single molecular weight and molecular weight of 1000Da-40kDa;
more preferably, the P 1 、P 2 The molecular weight is 2000Da-10kDa.
24. The antibody-nucleic acid conjugate of claim 23, wherein said L 1 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-, -S-S-、/> one or a combination of two or more groups;
The straight or branched chain C 1-12 Chain alkylene, C 6-12 Arylene or C of (2) 3-12 Any H atom on the cycloalkylene group is represented by-H, -F-Cl, -Br, -I, -O-, -S-, -SO 2 、-NO 2 、C 1-12 Chain alkyl, C 3-12 Cycloalkyl, C 6-12 Aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more groups of the group are substituted;
the L is 2 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、/>one or a combination of two or more groups;
the straight or branched chain C 1-12 Chain alkylene, C 6-12 Arylene or C of (2) 3-12 Any H atom on the cycloalkylene group is represented by-H, -F-Cl, -Br, -I, -O-, -S-, -SO 2 、-NO 2 、C 1-12 Chain alkyl, C 3-12 Cycloalkyl, C 6-12 Aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more than two groups are substituted;
preferably, the L 1 Is an amide bond, a hydrazone bond, and a mercapto-maleimide bond;
more preferably, the L 1 Is an amide bond;
preferably, the L 2 Is a disulfide bond and a thiol-maleimide bond;
more preferably, the L 2 Is a disulfide bond.
25. The antibody-nucleic acid conjugate of claim 23 or 24, wherein a is 1 To connect P 1 And P 2 A linking group therebetween selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -0-,-S-S-、/>one or a combination of two or more groups;
the straight or branched chain C 1-12 Chain alkylene, C 6-12 Arylene or C of (2) 3-12 Any H atom on the cycloalkylene group is represented by-H, -F-Cl, -Br, -I, -O-, -S-, -SO 2 、-NO 2 、C 1-12 Chain alkyl, C 3-12 Cycloalkyl, C 6-12 Aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more groups.
26. The antibody-nucleic acid conjugate of any of claims 22-25, wherein the antibody-nucleic acid conjugate is of formula (IV):
the L is 1 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、one or a combination of two or more groups;
preferably, P 1 Polyethylene glycol residues which are the same or different;
preferably, when said P 1 Polyethylene glycol of single molecular weight, having a molecular weight of 88-4400Da, more preferably, said P 1 Molecular weight is 176-1056Da;
preferably, when said P 1 Polyethylene glycol with non-single molecular weight and molecular weight of 1000Da-40kDa; more preferably, the P 1 The molecular weight is 2000Da-10kDa;
the L is 2 Is a linking group selected from linear or branched C 1-12 Alkylene, C 6-12 Arylene group, C 3-12 Cycloalkylene, -S-, -O-,-S-S-、/>one or two or more of the groupsA combination of clusters;
x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;
x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.
27. The antibody-nucleic acid conjugate of any of claims 22-26, having the structure:
said n 1 An integer selected from 4 to 100, preferably 4 to 24; n1 may be a constant value or an average value.
28. The antibody-nucleic acid conjugate of any of claims 22-27, wherein the Ab is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment, and an antibody fusion fragment; preferably, ab is a monoclonal antibody;
more preferably, the Ab is selected from: anti-HER 2 antibodies, anti-EGFR antibodies, anti-PMSA antibodies, anti-VEGFR antibodies, anti-CD 30 antibodies, anti-CD 22 antibodies, anti-CD 56 antibodies, anti-CD 29 antibodies, anti-GPNMB antibodies, anti-CD 138 antibodies, anti-CD 74 antibodies, anti-ENPP 3 antibodies, anti-Nectin-4 antibodies, anti-EGFR viii antibodies, anti-SLC 44A4 antibodies, anti-mesothelin antibodies, anti-ET 8R antibodies, anti-CD 37 antibodies, anti-CEACAM 5 antibodies, anti-CD 70 antibodies, anti-MUC 16 antibodies, anti-CD 79b antibodies, anti-MUC 16 antibodies, anti-MUC 1 antibodies, anti-CD 3 antibodies, anti-CD 28 antibodies, anti-CD 38 antibodies, anti-CD 19 antibodies, anti-PD-L1 antibodies, anti-4-1 BB antibodies.
29. The antibody-nucleic acid conjugate of any of claims 22-28, having the structure:
said n 1 、n 2 Independently selected from integers of 4 to 100, preferably 4 to 24; n is n 1 、n 2 Either constant or mean.
30. A composition comprising the interfering RNA of any one of claims 1-8 or the delivery system of any one of claims 9-17 and an adjuvant;
preferably, the composition is selected from: one or more of medicines, cosmetics and medical instruments;
preferably, when the composition is a medicine, the auxiliary materials comprise one or more than two of a filler, a wetting agent, a dispersing agent, a diluent, an anti-adhesion agent, an adhesive and a pigment;
preferably, when the composition is a cosmetic, the auxiliary materials include: antioxidants, binders, biological additives, buffers, colorants, thickeners, polymers, astringents, fragrances, humectants, diluents, dispersants, opacifying agents, conditioning agents, exfoliants, pH adjusters, preservatives, natural extracts, essential oils, skin soothing agents, and skin healing agents.
31. The composition of claim 30, wherein the pharmaceutical dosage form is selected from the group consisting of: any one of liquid dosage forms, injections, aerosols, films and ointments;
Preferably, the injection comprises: including sterile or sterilized solutions, injections, oil injections, and powder injections;
preferably, the ointment comprises: oleaginous base ointment, emulsion base ointment and water-soluble base ointment;
the cosmetic carrier is selected from: any one of an emulsion, lotion, cream, foam, gel, soap bar, stick, mask, cotton pad or patch.
32. Use of an interfering RNA according to any one of claims 1 to 8, a delivery system according to any one of claims 9 to 17, a composition according to any one of claims 30 to 31 for the manufacture of a medicament for the prevention and/or treatment of a MLPH-related disease;
the MLPH-related diseases include: tumor, skin pigmentation;
the tumor comprises: rectal cancer, vulvar cancer, prostate cancer, breast cancer, lung squamous carcinoma and adenocarcinoma;
the pigmentation comprises: senile plaque, acne marks, pigmented spots, freckle, cyasma or pathological pigment changes caused by sunlight, inflammation and medicinal factors.
33. Use of the interfering RNA of any one of claims 1 to 8, the delivery system of any one of claims 9 to 17, the composition of any one of claims 30 to 31 for whitening, spot-removing;
Preferably, the whitening and freckle removing are for non-therapeutic purposes;
the whitening and freckle removing steps comprise: reducing skin pigment content and pigmentation, and removing skin spots, such as senile plaque, acne mark, pigmented spots, freckle, cyasma or other pigment changes caused by sunlight, inflammation and medicinal factors.
CN202311313825.4A 2022-10-12 2023-10-11 Interference RNA for inhibiting expression of melanin avidin (MLPH) gene and application thereof Pending CN117866955A (en)

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