SiRNA for inhibiting PLK1 as well as modifier and application of siRNA

Abstract

The invention relates to siRNA for inhibiting PLK1 as well as a modifier and application thereof, and belongs to the technical field of biology. The invention provides siRNA (small interfering Ribonucleic Acid) for inhibiting PLK1 (PLK1). The siRNA has high inhibitory activity on the PLK1. The invention also provides the modified siRNA for inhibiting the PLK1, the four siRNA sequences all have good inhibition efficiency, and the knock-down effects of the PLK1-6M and the PLK1-10M are outstanding.

Description

Technical Field

[0001] The present invention relates to siRNAs for inhibiting PLK1, their modifications and applications, and belongs to the field of biotechnology. Background Art

[0002] PLK1 is a serine / threonine protein kinase that plays multiple roles in the cell cycle, such as mitotic entry and G2 / M checkpoint, coordinating centrosomes and the cell cycle, regulating spindle assembly and chromosome segregation, and promoting DNA replication.

[0003] Studies have found that the expression level of PLK1 in tumor tissues is higher than that in normal tissues. In addition, the expression level of PLK1 is usually associated with poor prognosis, tumorigenicity and invasiveness, so PLK1 expression is used as a prognostic marker.

[0004] Small interfering RNA (siRNA), usually a double-stranded RNA with a length of 20 to 25 nucleotides, mainly regulates gene expression in a specific manner through the RNA interference (RNAi) mechanism to achieve the purpose of treating diseases. Therefore, developing an siRNA to inhibit the generation of PLK1 will be an effective way to treat tumors (such as breast cancer, liver cancer, lung cancer and colon cancer, etc.) and their various complications. Summary of the Invention

[0005] To solve the above problems, the present invention provides an siRNA for inhibiting PLK1, the siRNA contains a sense strand and an antisense strand, and the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region.

[0006] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.2, or,

[0007] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.4, or,

[0008] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.6, or,

[0009] Wherein, the sense strand contains the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.8, or,

[0010] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.10, or,

[0011] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.11, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.12, or,

[0012] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.14, or,

[0013] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.16, or,

[0014] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.18, or,

[0015] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.19, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.20, or,

[0016] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.22, or,

[0017] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.23, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.24, or,

[0018] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.25, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.26, or,

[0019] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.27, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.28, or,

[0020] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.29, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.30, or,

[0021] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 31, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 32, or,

[0022] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 33, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 34, or,

[0023] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 35, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 36, or,

[0024] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 37, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 38, or,

[0025] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 39, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 40, or,

[0026] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 41, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 42, or,

[0027] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 43, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 44, or,

[0028] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 45, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 46, or,

[0029] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 47, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 48, or,

[0030] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 49, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 50, or,

[0031] Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO. 51, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 52.

[0032] In one embodiment of the present invention, taking the sense strand and the antisense strand shown in SEQ ID NO.1 and SEQ ID NO.2 as examples, the sense strand comprises: a nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.1 and retaining the biological function of the sequence from which it is derived, and the antisense strand comprises: a nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO.2 and retaining the biological function of the sequence from which it is derived. The nucleotide sequences of the remaining sense strand and antisense strand are nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity and retaining the biological function of the sequence from which they are derived.

[0033] In one embodiment of the present invention, the siRNA is prepared by solid-phase synthesis or liquid-phase synthesis.

[0034] In one embodiment of the present invention, the nucleotides in the siRNA are each independently modified or unmodified nucleotides.

[0035] In one embodiment of the present invention, each nucleotide in the siRNA is an unmodified nucleotide.

[0036] In one embodiment of the present invention, some or all of the nucleotides in the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNA of the present disclosure in inhibiting PLK1 gene expression.

[0037] In one embodiment of the present invention, at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

[0038] In one embodiment of the present invention, at least one phosphate group in the sense strand or the antisense strand of the siRNA is a phosphate group with a modifying group.

[0039] In one embodiment of the present invention, at least a part of the phosphate groups and / or ribose groups in the phospho-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA are phosphate groups with modifying groups and / or ribose groups with modifying groups.

[0040] In one embodiment of the present invention, all nucleotides in the sense strand and / or the antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA of the present disclosure in inhibiting PLK1 gene expression.

[0041] In one embodiment of the present invention, each nucleotide in the sense strand and the antisense strand of the siRNA is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0042] In one embodiment of the present invention, the modification is a chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluorine modification or phosphorothioate linkage.

[0043] In one embodiment of the present invention, a "fluorinated modified nucleotide" refers to a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with fluorine, and it has the structure shown in the following formula (1). The non-fluorinated modified nucleotide is independently selected from a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of the nucleotide with a non-fluorine group or a nucleotide analogue.

[0044] In one embodiment of the present invention, the nucleotides formed by substituting the hydroxyl group at the 2'-position of the ribose group with a non-fluorine group are well-known to those skilled in the art, and these nucleotides can be selected from one of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxynucleotides.

[0045] In one embodiment of the present invention, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2); the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3), the 2'-amino (2'-NH2) modified nucleotide is as shown in formula (4), and the 2'-deoxynucleotide (DNA) is as shown in formula (5):

[0046]

[0047] In one embodiment of the present invention, the fluorinated modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated modified nucleotides.

[0048] In one embodiment of the present invention, the fluorine-modified nucleotides are located in the sense strand and the antisense strand. The number of fluorine-modified nucleotides in the sense strand is no more than 5. And, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides. The number of fluorine-modified nucleotides in the antisense strand is no more than 7. And, at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides.

[0049] In one embodiment of the present invention, in the direction from the 5'-end to the 3'-end, in the sense strand, the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorine-modified nucleotides. In the antisense strand, the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides.

[0050] In one embodiment of the present invention, the methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotides of the sense strand are methoxy-modified nucleotides, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st nucleotides of the antisense strand are methoxy-modified nucleotides.

[0051] In one embodiment of the present invention, the nucleotide analog refers to a group that can replace nucleotides in nucleic acids but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide, or thymidine deoxyribonucleotide.

[0052] In one embodiment of the present invention, the nucleotide analog can be a non-natural nucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0053] In one embodiment of the present invention, the bridged nucleic acid (BNA) refers to a constrained or inaccessible nucleotide. BNA can contain a bridged structure with a "fixed" C3'-endo sugar puckering of a five-membered ring, a six-membered ring, or a seven-membered ring. Usually, this bridge is incorporated into the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide.

[0054] In one embodiment of the present invention, the BNA may be LNA, ENA, cET BNA, etc. Among them, LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8):

[0055]

[0056] In one embodiment of the present invention, at least a part of the phosphate ester groups in the phospho-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA is a phosphate ester group with a modifying group.

[0057] In one embodiment of the present invention, the phosphate ester group with a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0058] In one embodiment of the present invention, the phosphate ester group with a modifying group is a phosphorothioate group having the structure shown in formula (9):

[0059]

[0060] In one embodiment of the present invention, the connection of the phosphorothioate group exists in at least one of the positions consisting of the following groups: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above.

[0061] In one embodiment of the present invention, the connection of the phosphorothioate group exists at all the above positions except at the 5'-end of the sense strand.

[0062] In one embodiment of the present invention, the connection of the phosphorothioate group exists at all the above positions except at the 3'-end of the sense strand.

[0063] In one embodiment of the present invention, the connection of the phosphorothioate group exists in at least one of the following positions:

[0064] between the first and second nucleotides at the 5'-end of the sense strand;

[0065] between the second and third nucleotides at the 5'-end of the sense strand;

[0066] between the first and second nucleotides at the 3'-end of the sense strand;

[0067] between the second and third nucleotides at the 3'-end of the sense strand;

[0068] between the first and second nucleotides at the 5'-end of the antisense strand;

[0069] between the second and third nucleotides at the 5'-end of the antisense strand;

[0070] between the first and second nucleotides at the 3'-end of the antisense strand; and

[0071] between the second and third nucleotides at the 3'-end of the antisense strand.

[0072] In one embodiment of the present invention, the phosphorothioate-linked nucleotides are located in both the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the first and second positions, and the second and third positions of the sense strand are nucleotides linked by phosphorothioate groups; in the direction from the 5'-end to the 3'-end, at least the first and second positions, the second and third positions, the 19th and 20th positions, and the 20th and 21st positions of the antisense strand are nucleotides linked by phosphorothioate groups.

[0073] In one embodiment of the present invention, the siRNA is introduced by using nucleotide monomers with corresponding modifications to introduce modified nucleotides.

[0074] The present invention also provides a product for inhibiting PLK1, the product comprising an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the above siRNA or the above modified siRNA.

[0075] In one embodiment of the present invention, the product is a pharmaceutical composition or a kit.

[0076] In one embodiment of the present invention, the product is a pharmaceutical composition, and the pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives.

[0077] In one embodiment of the present invention, there is no particular requirement for the contents of siRNA and the pharmaceutically acceptable carrier, and they can be the conventional contents of each component.

[0078] In one embodiment of the present invention, in the pharmaceutical composition, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier is 1:(1 - 500).

[0079] In one embodiment of the present invention, in the pharmaceutical composition, the weight ratio of the active ingredient to the pharmaceutically acceptable carrier is 1:(1 - 50).

[0080] In one embodiment of the present invention, the pharmaceutical composition may further contain other pharmaceutically acceptable excipients, and the excipient can be one or more of various preparations or compounds commonly used in the art.

[0081] In one embodiment of the present invention, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0082] In one embodiment of the present invention, the pH buffer may be a tris (hydroxymethyl) aminomethane hydrochloride buffer with a pH value of 7.5 to 8.5 and / or a phosphate buffer with a pH value of 5.5 to 8.5.

[0083] In one embodiment of the present invention, the cryoprotectant may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0084] In one embodiment of the present invention, based on the total weight of the pharmaceutical composition, the content of the cryoprotectant may be 0.01 to 30% by weight.

[0085] In one embodiment of the present invention, the osmotic pressure regulator may be sodium chloride and / or potassium chloride.

[0086] In one embodiment of the present invention, the content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200 to 700 milliosmoles per liter (mOSM / L). Those skilled in the art can easily determine the content of the osmotic pressure regulator according to the required osmotic pressure.

[0087] In one embodiment of the present invention, the pharmaceutical composition may be a liquid preparation, such as an injection; or it may be a freeze-dried powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation.

[0088] In one embodiment of the present invention, the liquid preparation may be but is not limited to being administered subcutaneously, intramuscularly, or intravenously, and may also be but is not limited to being administered to the lungs by spraying, or being administered to other organ tissues (such as the liver) through the lungs by spraying.

[0089] In one embodiment of the present invention, the pharmaceutical composition is used for intravenous injection.

[0090] In one embodiment of the present invention, the pharmaceutical composition may be in the form of a liposomal preparation.

[0091] In one embodiment of the present invention, the pharmaceutically acceptable carrier used in the liposomal preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid, and / or a polyethylene glycolylated lipid.

[0092] In one embodiment of the present invention, the organic amine, co-lipid, and polyethylene glycolylated lipid may be respectively selected from one or more of the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, co-lipids, and polyethylene glycolylated lipids described in CN108220295B (which is incorporated herein by reference in its entirety).

[0093] In one embodiment of the present invention, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc). N-acetylgalactosamine is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. The asialoglycoprotein receptor is an endocytic receptor specifically expressed in hepatocytes. N-acetylgalactosamine is used as a targeting molecule to deliver small RNAs to the liver.

[0094] In one embodiment of the present invention, the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. The monovalent, divalent, trivalent, and tetravalent respectively refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule containing the coupling group as the targeting group in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4 after the formation of the siRNA conjugate.

[0095] In one embodiment of the present invention, when the siRNA is conjugated to the coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent.

[0096] In one embodiment of the present invention, when the siRNA is conjugated to the coupling group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0097] In one embodiment of the present invention, the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.

[0098] In one embodiment of the present invention, for the types of the linker, the targeting group, and the connection mode with the siRNA, reference can be made to the disclosure of WO2015006740A2, and its entire content is incorporated herein by reference.

[0099] In one embodiment of the present invention, the siRNA conjugate formed by GalNAc and the siRNA molecule has the structure shown in the following formula (10):

[0100]

[0101] In one embodiment of the present invention, the kit further comprises a pharmaceutically acceptable carrier and / or excipient.

[0102] In one embodiment of the present invention, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit can exist alone, exist in the form of a mixture of two or more of them, or exist in the form of a final pharmaceutical composition.

[0103] In one embodiment of the present invention, in the kit, the pharmaceutically acceptable carrier is an amine-containing compound, a helper lipid and a polyethylene glycolylated lipid.

[0104] In one embodiment of the present invention, in the kit, the pharmaceutically acceptable carrier is a mixture or exists independently.

[0105] In one embodiment of the present invention, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit are provided in liquid form, dry form or lyophilized form.

[0106] In one embodiment of the present invention, the siRNA, pharmaceutically acceptable carrier and / or excipient in the kit are substantially pure and / or sterile.

[0107] In one embodiment of the present invention, in the kit, it includes a container for providing siRNA, one or more containers for providing an amine-containing compound, a helper lipid and a polyethylene glycolylated lipid, and optionally, a container for providing an excipient.

[0108] In one embodiment of the present invention, the kit further includes one or more components that are necessary or beneficial for a specific application, and the components are selected from:

[0109] One or more components for achieving the desired cell transfection;

[0110] One or more components for achieving the diagnosis, treatment or prevention of a specific disease or physical disorder;

[0111] One or more buffers;

[0112] Positive or negative control samples;

[0113] Excipients, stabilizers or preservatives.

[0114] In one embodiment of the present invention, the one or more components for achieving the diagnosis, treatment or prevention of a specific disease or physical disorder are one or more additional therapeutic compounds or compositions, one or more diagnostic reagents.

[0115] In one embodiment of the present invention, the kit further includes one or more of sterile water, physiological saline and PBS.

[0116] The present invention also provides the use of the above siRNA or the above product in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by PLK1.

[0117] In one embodiment of the present invention, the disease related to PLK1 is a tumor, such as breast cancer, liver cancer, lung cancer, colon cancer, etc.

[0118] The technical solution of the present invention has the following advantages:

[0119] The present invention provides a modified siRNA for inhibiting PLK1, and the modifications include methoxy modification, fluorine modification and phosphorothioate linkage. Activity detection was carried out in 293T cells. At a concentration of 100 nM, most of them could significantly inhibit the expression of the PLK1 gene. siRNAs with an inhibition rate > 70% were screened out. Activity detection was carried out again in A549, Hela, Hep3B, and SK-BR3 cells. The knockdown efficiencies of PLK1-6M, PLK1-9M, PLK1-10M, PLK1-17M, and PLK1-26M were all good in the five cell lines of 293T, A549, Hela, Hep3B, and SK-BR3.

[0120] Moreover, the dose-effect relationship of the four modified sequences of PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M was obvious, and they had good inhibitory effects. By diluting the siRNA with gradient concentrations, verification was carried out again in Hep3B and SK-BR3 cell lines. All four siRNA sequences had good inhibitory efficiencies, and among them, the knockdown effects of PLK1-6M and PLK1-10M were the best.

[0121] IC 50 Experiments showed that in A549, Hela, and Hep3B cells, the cytotoxicity of PLK1-6M and PLK1-10M was relatively high. It was fully proved that the gene knockdown efficiency of PLK1 siRNA was positively correlated with its cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 : Activity detection of modified sequences (PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M) for inhibiting PLK1 in Hep3B cells (siRNA concentrations were 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM). Figure A shows the activity detection of different concentrations of PLK1-6M in Hep3B cells; Figure B shows the activity detection of different concentrations of PLK1-9M in Hep3B cells; Figure C shows the activity detection of different concentrations of PLK1-10M in Hep3B cells; Figure D shows the activity detection of different concentrations of PLK1-26M in Hep3B cells.

[0123] Figure 2 : Modification sequences for inhibiting PLK1 (PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M) were assayed for their activities in SK-BR3 cells (siRNA concentrations were 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM). Figure A shows the activity assay of different concentrations of PLK1-6M in SK-BR3 cells; Figure B shows the activity assay of different concentrations of PLK1-9M in SK-BR3 cells; Figure C shows the activity assay of different concentrations of PLK1-10M in SK-BR3 cells; Figure D shows the activity assay of different concentrations of PLK1-26M in SK-BR3 cells. Detailed implementation manners

[0124] The following embodiments are provided to better further understand the present invention. They are not limited to the described optimal implementation manners, and do not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0125] In the following embodiments, PLK1 mRNA refers to the mRNA having the sequences shown in GeneBank accession numbers NM_005030.6, NM_01112, and NM_001284023. Further, unless otherwise specified, the term "target gene" used in this disclosure refers to the gene that transcribes the above PLK1 mRNA, and the term "target mRNA" refers to the above PLK1 mRNA.

[0126] For those not specifying specific experimental steps or conditions in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained commercially.

[0127] In the following embodiments, the capital letters C, G, U, and A represent ribonucleotides; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluoro group; the lowercase letter s indicates that there is a phosphorothioate modification between the two nucleotides adjacent to the left and right of the letter s.

[0128] In the following embodiments, "modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2'-position of the ribose of the nucleotide is replaced by another group, or a nucleotide in which the base on the nucleotide is a modified base. "Fluorine-modified nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribose of the nucleotide with fluorine, and "non-fluorine-modified nucleotide" refers to a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2'-position of the ribose of the nucleotide with a non-fluorine group. "Nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide or thymidine deoxyribonucleotide. Such as isonucleotide, bridged nucleic acid (abbreviated as BNA) or acyclic nucleotide. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose with a methoxy group.

[0129] In the following embodiments, the terms "complementary" or "reverse complementary" can be used interchangeably and have the meanings well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand.

[0130] In the following embodiments, especially when describing the preparation methods of the siRNA, pharmaceutical composition or siRNA conjugate of the present disclosure, unless otherwise specified, nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites) used in the phosphoramidite solid-phase synthesis according to the types and sequences of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method well-known to those skilled in the art for RNA synthesis. The nucleoside monomers used in the present disclosure are all commercially available.

[0131] In the following embodiments, "coupling" means that two or more chemical moieties each having a specific function are connected to each other in a covalent linkage; correspondingly, "conjugate" means a compound formed by covalent linkage between these respective chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties having specific functions to siRNA. The siRNA conjugate should be understood, depending on the context, as the general term for multiple siRNA conjugates or the siRNA conjugate represented by a certain chemical formula. In the context of the present disclosure, "coupling molecule" should be understood as a specific compound that can be coupled to siRNA through a reaction to ultimately form the siRNA conjugate of the present disclosure.

[0132] In the following embodiments, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes the case where the event or condition occurs and the case where it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitutions or substitution patterns that are spatially unrealistic, synthetically infeasible, and / or inherently unstable.

[0133] In the following embodiments, "treat", "alleviate", or "ameliorate" may be used interchangeably herein. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits. "Therapeutic benefit" means eradicating or ameliorating the underlying disorder being treated. In addition, a therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0134] In the following embodiments, "prevent" and "prophylaxis" may be used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to prophylactic benefits. To obtain a "prophylactic benefit", the composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more pathological symptoms of the disease, even if a diagnosis of the disease may not have been made.

[0135] Unless otherwise specified, the reagents and culture media used in the following embodiments are all commercially available products, and operations such as nucleic acid electrophoresis and real-time PCR are carried out according to the methods described in "Molecular Biology (Fourth Edition)" (edited by Alexander McLennan et al., 2019).

[0136] The experimental cells involved in the following embodiments are 293T, A549, and Hela, which are purchased from Frontier Biopharma Co., Ltd. (Nanjing), and Hep3B and SK-BR3 are purchased from the Cell Bank of the Chinese Academy of Sciences.

[0137] The siRNA involved in the following examples is the siRNA sequence synthesized by phosphoramidite solid-phase synthesis.

[0138] When the siRNA, siRNA conjugate targeting the PLK1 gene or the siRNA, siRNA conjugate used as a negative control involved in the following examples were transfected into cells, Lipo3000 or RNAiMAX (purchased from Invitrogen) was used as the transfection reagent, and the specific operation was referred to the instruction manual provided by the manufacturer. When performing qPCR detection, HiScript III RT SuperMix for qPCR (purchased from Vazyme) was used as the reverse transcription reagent, and the specific operation was referred to the instruction manual provided by the manufacturer.

[0139] Unless otherwise specified, the reagent ratios provided below are calculated by volume ratio (v / v).

[0140] Example 1: An siRNA for inhibiting PLK1

[0141] This example provides an siRNA for inhibiting PLK1. The nucleotide sequence of the siRNA was designed based on the target mRNA, as shown in Table 1. The siRNA molecule with the following sequence was synthesized by Suzhou GenePharma Co., Ltd.

[0142] Table 1. Nucleotide sequence of siRNA for inhibiting PLK1

[0143]

[0144]

[0145] Example 2: A modified siRNA for inhibiting PLK1

[0146] This embodiment provides a modified siRNA for inhibiting PLK1. The modified siRNA includes PLK1-1M to PLK1-26M. The sense strands of PLK1-1M to PLK1-26M are respectively obtained by chemically modifying the sequences shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, SEQ ID NO.47, SEQ ID NO.49, and SEQ ID NO.51. In the direction from the 5'-end to the 3'-end, the nucleotides at the 1st and 2nd positions, and the 2nd and 3rd positions are linked by phosphorothioate groups. The nucleotides at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions are nucleotides modified with methoxy groups, and the nucleotides at the 7th, 8th, and 9th positions are nucleotides modified with fluoro groups.The antisense strands of PLK1-1M to PLK1-26M are respectively obtained by chemically modifying the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44, SEQ ID NO.46, SEQ ID NO.48, SEQ ID NO.50 and SEQ ID NO.52. In the direction from the 5'-end to the 3'-end, the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, and the 20th and 21st positions are linked by phosphorothioate groups. The nucleotides at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, and 21st positions are nucleotides modified with methoxy groups, and the nucleotides at the 2nd, 6th, 14th, and 16th positions are nucleotides modified with fluoro groups.

[0147] Experimental Example 1: Activity Detection of Modified siRNA for Inhibiting PLK1 in 293T Cells

[0148] This experimental example provides the determination of the relative inhibitory level of the siRNA compound of the present invention on PLK1 mRNA in 293T cells by quantitative real-time PCR (hereinafter referred to as qPCR).

[0149] The experimental procedure for the activity detection of the modified siRNA for inhibiting PLK1 in 293T cells is as follows:

[0150] 1. Culture 293T cells in DMEM medium (purchased from TransgenBiotech, product number FI101-01) containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin-streptomycin (Gibco, Invitrogen). Dilute the cultured cells with DMEM medium to 6.67×10 4 / ml to obtain a 293T cell suspension, which was plated in a 24-well plate, and 450 μL of the cell suspension was added to each well.

[0151] 2. The dry powder of the siRNA to be tested prepared in Example 1 was centrifuged at low temperature and high speed, and then dissolved in Opti-MEM to prepare a 50 μM siRNA mother solution.

[0152] 3. Prepare a 100 nM siRNA transfection dilution

[0153] Preparation of 100 nM siRNA transfection dilution: Take 2 μl of the 50 μM siRNA mother solution and add 48 μl of Opti-MEM to obtain a 2 μM siRNA dilution; Take 25 μl of the above 2 μM siRNA dilution and an equal volume of Lipo3000 transfection reagent dilution (0.75 μl of Lipo3000 + 24.25 μl of Opti-MEM) and add them to the 293T cells cultured in the 24-well plate to obtain a final siRNA concentration of 100 nM.

[0154] 4. The cells were cultured for 48 hours after transfection, and two replicate wells were set. In addition, NC was set as a control. The NC group was a negative control siRNA unrelated to PLK1, and its sense strand was (5'-3'): CfsAms CfUmUfAmCfGmCfUmGfAmGfUmAfCmUfUmCfGmAf, and the antisense strand was (5'-3'): UmsCfsGmAfAmGfUmAfCmUfCmAfGmCfGmUfAmAfGmUfGmsAfsUm.

[0155] 5. Use a magnetic bead method cell total RNA extraction kit (purchased from Suzhou GenePharma Co., Ltd., product number E31008) to extract the total RNA in the cells of each well according to the method described in the kit instructions.

[0156] 6. RNA reverse transcription

[0157] Use HiScript III RT SuperMix for qPCR (purchased from Novoprotein Co., Ltd., product number R323-01), and the experimental steps refer to the product manual; Prepare a 20 μL reverse transcription reaction system according to the reverse transcription operation steps in the kit instructions to reverse transcribe the total RNA of the cells; The reverse transcription conditions were: incubate the reverse transcription reaction system at 37 °C for 15 min, then incubate at 85 °C for 5 s, and add 80 μL of DEPC water to each reverse transcription reaction system to obtain a solution containing cDNA;

[0158] 7. Configuration of qPCR reaction system

[0159] For each reverse transcription reaction system, 4 μL of the above cDNA-containing solution was taken as a template respectively, and the reagents provided by the AceQ Universal SYBR qPCR MasterMix kit (purchased from Vazyme, product number Q511-02) were used to prepare a 20-μL qPCR reaction system on an ice box according to Table 2. Among them, Primer1 and Primer2 are the PCR primer sequences for amplifying the target gene PLK1 and the internal reference gene GAPDH respectively (as shown in Table 3). Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using the three-step method. The amplification program was pre-denaturation at 95 °C for 10 min, then denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. After repeating the above denaturation, annealing, and extension processes 40 times, a product W containing the amplified target gene PLK1 and internal reference gene GAPDH was obtained. The product W was then incubated at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s in sequence. The real-time fluorescence quantitative PCR instrument collected the melting curves of the target gene PLK1 and the internal reference gene GAPDH in the product W respectively, and the Ct values of the target gene PLK1 and the internal reference gene GAPDH were obtained.

[0160] Table 2. DNA Amplification Reaction System

[0161] Reagent Name Volume per Well (μL) 2×AceQUniversalSYBRqPCRMasterMix 10 Primer1 (10μM) 0.4 Primer2 (10μM) 0.4 Template DNA / cDNA 2 <![CDATA[ddH2O]]> 7.2 Total Volume 20

[0162] Table 3. Primer Information

[0163]

[0164] The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculation on the target gene PLK1 in each test group. The calculation method is as follows:

[0165] ΔCt (test group) = Ct (test group target gene) – Ct (test group internal reference gene)

[0166] ΔCt (control group) = Ct (control group target gene) – Ct (control group internal reference gene)

[0167] ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group average)

[0168] ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group average)

[0169] Among them, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) of each control group sample; thus, each sample in the test group and the control group corresponds to a ΔΔCt value.

[0170] Normalize the expression level of PLK1 mRNA in the test group based on the control group, and define the expression level of PLK1 mRNA in the control group as 100%.

[0171] Relative expression level of PLK1 mRNA in the test group = 2 -ΔΔCt(测试组) × 100%

[0172] Inhibition rate of PLK1 mRNA in the test group = 1 - Relative expression level of PLK1 mRNA in the test group.

[0173] Compare the PLK1 mRNA level with the internal reference gene GAPDH, normalize this value to the average of the saline control group, represent the data as a percentage relative to the saline control group, and present it as the average plus the standard deviation.

[0174] The remaining activity results of 26 siRNAs in cells 293T are shown in Table 4. It can be seen that the modified siRNAs all have a high inhibitory effect, and the siRNAs shown as PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, PLK1-14M, PLK1-17M, PLK1-18M, PLK1-21M, and PLK1-26M have a good inhibitory effect at a concentration of 100 nM, with an inhibition rate > 70%. Among them, the inhibition rates of PLK1-9M, PLK1-13M, PLK1-14M, PLK1-18M, PLK1-21M, and PLK1-26M are > 80%.

[0175] Table 4. Remaining activity of modified siRNAs in cells 293T

[0176]

[0177]

[0178] Experimental Example 2: Detection of the activity of modified siRNAs for inhibiting PLK1 in cells A549

[0179] This experimental example provides the determination of the relative inhibition level of the siRNA compound of the present invention on PLK1 mRNA in cells A549 by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR, hereinafter referred to as qPCR).

[0180] Using the siRNA sequences with an inhibition rate > 70% screened in Experimental Example 1: PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, PLK1-14M, PLK1-17M, PLK1-18M, PLK1-21M, and PLK1-26M, the activity was detected at a concentration of 100 nM in A549 cells. The experimental procedure was referred to Experimental Example 1.

[0181] The remaining activity results of the 10 siRNAs in A549 cells are shown in Table 5. It can be seen that the modified siRNAs all have high inhibitory activity, and the siRNAs shown as PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, PLK1-14M, PLK1-17M, and PLK1-26M have good inhibitory effects at a concentration of 100 nM, with an inhibition rate > 80%. Among them, the inhibition rate of PLK1-10M > 90%.

[0182] Table 5. Remaining activity of the modified siRNAs in A549 cells

[0183]

[0184] Experimental Example 3: Activity detection of the modified siRNA for inhibiting PLK1 in Hela cells

[0185] This experimental example provides the determination of the relative inhibition level of the siRNA compound of the present invention on PLK1 mRNA in Hela cells by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR, hereinafter referred to as qPCR).

[0186] Using the siRNA sequences with an inhibition rate > 70% screened in Experimental Example 1: PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, PLK1-14M, PLK1-17M, PLK1-18M, PLK1-21M, and PLK1-26M, the activity was detected at a concentration of 100 nM in Hela cells. The experimental procedure was referred to Experimental Example 1.

[0187] The remaining activity results of the 10 siRNAs in Hela cells are shown in Table 6. It can be seen that the modified siRNAs all have high inhibitory activity, and the siRNAs shown as PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, and PLK1-26M have good inhibitory effects at a concentration of 100 nM, with an inhibition rate > 80%. Among them, the inhibition rates of PLK1-9M, PLK1-10M, and PLK1-13M > 90%.

[0188] Table 6. Residual Activity of Modified siRNA in Hela Cells

[0189]

[0190] Experimental Example 4: Activity Detection of Modified siRNA for Inhibiting PLK1 in Hep3B Cells

[0191] This experimental example provides the determination of the relative inhibition level of the siRNA compound of the present invention on PLK1 mRNA in Hep3B cells by quantitative real-time PCR (hereinafter referred to as qPCR).

[0192] The siRNA sequences with an inhibition rate > 70% screened in Experimental Example 1: PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, PLK1-14M, PLK1-17M, PLK1-18M, PLK1-21M, and PLK1-26M were used for activity detection at a concentration of 100 nM in Hep3B cells. The experimental procedure refers to Experimental Example 1, retaining steps 2 to 7, and step 1 is as follows:

[0193] Hep3B cells were cultured using MEM medium (purchased from Gibco, catalog number 11095-080) containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin-streptomycin (Gibco, Invitrogen). The cultured cells were diluted with MEM medium to 1.56×10 5 / ml to obtain a Hep3B cell suspension, which was plated into a 24-well plate, and 450 μL of the cell suspension was added to each well.

[0194] The residual activities of the 10 siRNAs in Hep3B cells are shown in Table 7. It can be seen that the modified siRNAs all have high inhibitory activities, and the siRNAs represented by PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M have good inhibitory effects at a concentration of 100 nM, with an inhibition rate > 80%.

[0195] Table 7. Residual Activity of Modified siRNA in Hep3B Cells

[0196]

[0197]

[0198] Experimental Example 5: Activity Detection of Modified siRNA for Inhibiting PLK1 in SK-BR3 Cells

[0199] This experimental example provides the determination of the relative inhibition level of the siRNA compound of the present invention on PLK1 mRNA in SK-BR3 cells by quantitative real-time PCR (hereinafter referred to as qPCR).

[0200] The siRNA sequences with an inhibition rate > 70% screened in Experimental Example 1: PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, PLK1-13M, PLK1-14M, PLK1-17M, PLK1-18M, PLK1-21M, and PLK1-26M were used for activity detection at a concentration of 100 nM in SK-BR3 cells. The experimental procedure referred to Experimental Example 1, retaining steps 2 to 7, and step 1 is as follows:

[0201] SK-BR3 cells were cultured using McCoy's 5A medium (purchased from Wuhan Pusaisai Life Science and Technology Co., Ltd., product number PM150710) containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin-streptomycin (Gibco, Invitrogen). The cultured cells were diluted with McCoy's 5A medium to 2.22×10 5 / ml to obtain an SK-BR3 cell suspension, which was seeded into a 24-well plate, and 450 μL of the cell suspension was added to each well.

[0202] The remaining activity results of the 10 siRNAs in SK-BR3 cells are shown in Table 8. It can be seen that the modified siRNAs all have high inhibitory activity, and the siRNAs shown as PLK1-5M, PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M have good inhibitory effects at a concentration of 100 nM, with an inhibition rate > 70%, and the inhibition rate of PLK1-10M > 80%.

[0203] Table 8. Remaining Activity of Modified siRNA in SK-BR3 Cells

[0204]

[0205]

[0206] In summary, from Experimental Example 1 to Experimental Example 5, it can be obtained that the knockdown efficiencies of PLK1-6M, PLK1-9M, PLK1-10M, PLK1-17M and PLK1-26M are all good in the five cell lines of 293T, A549, Hela, Hep3B and SK-BR3.

[0207] Experimental Example 6: Activity Detection of Modified siRNA for Inhibiting PLK1 in Hep3B Cells

[0208] This experimental example provides the determination of the relative inhibition level of the siRNA compound of the present invention on PLK1 mRNA in Hep3B cells by Quantitative Real-Time PCR (hereinafter referred to as qPCR).

[0209] The siRNA sequences with good knockdown efficiencies in the five cell lines of 293T, A549, Hela, Hep3B and SK-BR3 screened in Experimental Examples 1 to 5: PLK1-6M, PLK1-9M, PLK1-10M and PLK1-26M were used for activity detection at lower concentrations (100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM) in Hep3B cells. The experimental procedure refers to Experimental Example 4, and Steps 1 to 7 are retained. In Step 3, in addition to preparing a 1 μM concentration of siRNA dilution, Step 3 needs to prepare siRNA dilution with concentrations of 100 nM, 10 nM, 1 nM and 0.1 nM. The detailed steps are as follows:

[0210] Preparation of 100 nM siRNA transfection dilution: Take 2 μl of 100 μM siRNA mother solution, add 98 μl of Opti-medium to obtain a 2 μM siRNA dilution; then add an equal volume of Lipofectaine RNAiMax transfection reagent dilution (add 3 μl of Lipofectaine RNAiMax transfection reagent to 97 μl of Opti-medium to obtain Lipofectaine RNAiMax transfection reagent dilution) to obtain a 1 μM siRNA dilution. Starting from the 1 μM siRNA dilution as the initial concentration, use Opti-medium for gradient dilution to dilute into siRNA dilutions with concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM. Take 10 μl of the above 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM siRNA dilutions and add them to the Hep3B cells transfected and cultured in a 96-well plate to obtain a final siRNA concentration of 100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM.

[0211] The results of the relative mRNA expression levels of 4 siRNAs in Hep3B cells are as follows Figure 1 As shown, it can be seen that the modified siRNAs all have high inhibitory activity. The siRNAs represented by PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M have good inhibitory effects at concentrations of 100 nM, 10 nM, 1 nM, and 0.1 nM.

[0212] Experimental Example 7: IC of modified siRNA inhibiting PLK1 gene expression 50 Determination

[0213] According to the results of Experimental Example 6, the IC of modified siRNA inhibiting PLK1 gene expression was determined. 50 Determination.

[0214] Taking the log value of the siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the "log(inhibitor) vs. response – variable slope" function module of the analysis software GraphPad Prism 8 was used to fit the dose-effect curve, so as to obtain the IC of each siRNA. 50 Value.

[0215] The fitting formula is: Y = Bottom + (Top – Bottom) / (1 + 10^((LogIC50 – X)×HillSlope))

[0216] Among them: Top represents the percentage inhibition rate at the top plateau. The Top standard of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau. The Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0217] The experimental results are shown in Table 9. The siRNAs of the present application have a low IC 50 and have high inhibitory activity against the PLK1 gene in Hep3B cells.

[0218] Table 9 IC of siRNA 50 (nM)

[0219] Hep3B Experimental Grouping <![CDATA[IC 50 <!-- 18 -->]]> PLK1-6M 0.04484 nM PLK1-9M 0.1059 nM PLK1-10M 0.01117 nM PLK1-26M 0.1942 nM

[0220] Experimental Example 8: Activity detection of modified siRNA for inhibiting PLK1 in SK-BR3 cells

[0221] This experimental example provided the determination of the relative inhibition level of the siRNA compound of the present invention on PLK1 mRNA in the SK-BR3 cells by quantitative real-time PCR (hereinafter referred to as qPCR).

[0222] The siRNA sequences with good knockdown efficiency in the five cell lines of 293T, A549, Hela, Hep3B and SK-BR3 screened in Experimental Examples 1 to 5: PLK1-6M, PLK1-9M, PLK1-10M and PLK1-26M were used for activity detection at lower concentrations (100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM) in Hep3B cells. The experimental procedure referred to Experimental Example 5, retaining Steps 1 to 7, and Step 3 referred to Step 3 of Experimental Example 6.

[0223] The results of the relative mRNA expression levels of the 4 siRNAs in the SK-BR3 cells are as Figure 2 shown. It can be seen that the modified siRNAs all have high inhibitory activities. The siRNAs shown by PLK1-6M, PLK1-9M, PLK1-10M and PLK1-26M have good inhibitory effects at the concentrations of 100 nM, 10 nM and 1 nM. Among them, the siRNA shown by PLK1-6M also has an inhibitory effect at the concentration of 0.1 nM.

[0224] Experimental Example 9: IC of the modified siRNA in inhibiting PLK1 gene expression 50 Determination

[0225] According to the results of Experimental Example 8, the IC of the modified siRNA in inhibiting PLK1 gene expression was determined. The procedure referred to Experimental Example 7. 50 Determination. The procedure referred to Experimental Example 7.

[0226] The experimental results are shown in Table 10. The siRNA of the present application has a lower IC 50 and has a high inhibitory activity on the PLK1 gene in SK-BR3 cells.

[0227] Table 10 IC of siRNA 50 (nM)

[0228] SK-BR3 Experimental Grouping <![CDATA[IC 50 > PLK1-6M 0.03405 nM PLK1-9M 0.05735 nM PLK1-10M 0.05137 nM PLK1-26M 0.6862 nM

[0229] In summary, from Experimental Examples 6 to 9, it can be obtained that by diluting siRNA with gradient concentrations, it was verified again in the Hep3B and SK-BR3 cell lines. All four siRNA sequences had good inhibitory efficiencies, and the knockdown effects of PLK1-6M and PLK1-10M were the best.

[0230] Experimental Example 10: IC after transfection of siRNA at gradient concentrations into A549 cells 50 Detection

[0231] This experimental example provides the IC after transfection of siRNA at gradient concentrations into A549 cells 50 Detection. The steps are as follows:

[0232] 1) Cell seeding and cell transfection. The experimental steps refer to Example 1 of the experimental example. Among them, in step 3, the prepared 100 nM siRNA is used as the starting concentration, and 3-fold serial dilution is carried out to obtain a total of eight concentrations (100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM, and 0.05 nM).

[0233] 2) According to the number of wells actually to be detected in the 96-well plate, prepare the volume of the detection solution to be required by mixing 90 μL of complete culture medium per well with 10 μL of the CCK8 stock solution (Enhanced Cell Counting Kit-8, purchased from Beyotime, catalog number C0043) in the dark.

[0234] 3) After the cells transfected with siRNA are incubated in the cell culture incubator for 48 h and 96 h, aspirate the original cell culture medium in the 96-well plate, add 100 μL of the CCK8 detection solution to each well in the dark, and after incubating in the cell culture incubator for 1 hour, measure the absorbance at 450 nM with an enzyme-linked immunosorbent assay (ELISA) reader.

[0235] Based on the cell viability of PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M after transfection of siRNA at gradient concentrations into A549 cells at 96 h, follow the steps of Example 7 to obtain the IC for inhibiting the expression of the PLK1 gene by PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M 50 Determination. The experimental results are shown in Table 11.

[0236] Table 11. IC of siRNA 50 (nM)

[0237] A549 Experimental Grouping <![CDATA[IC 50 > PLK1-6M-96h 10.19 nM PLK1-9M-96h N / A PLK1-10M-96h 3.174 nM PLK1-26M-96h 56.36 nM

[0238] Since in the CCK8 results detected at 96 h, the highest concentration of 100 nM PLK1-9M did not reach the half-maximal inhibitory concentration of the cells, there is no IC 50 Result.

[0239] Experimental Example 11: IC after transfection of siRNA at gradient concentrations into Hela cells 50 Detection

[0240] This experimental example provides the IC 50 detection after transfection of siRNA at gradient concentrations in Hela cells. The experimental procedures refer to Experimental Example 10.

[0241] Based on the cell viability of PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M after transfection of siRNA at gradient concentrations in Hela cells at 96 h, the IC 50 for inhibiting the expression of the PLK1 gene was obtained, and the experimental results are shown in Table 12.

[0242] Table 12. IC of siRNA 50 (nM)

[0243] Hela Experimental Grouping <![CDATA[IC 50 > PLK1-6M-96h 7.513 nM PLK1-9M-96h 9.736 nM PLK1-10M-96h 2.145 nM PLK1-26M-96h 8.673 nM

[0244] Experimental Example 12: IC 50 detection after transfection of siRNA at gradient concentrations in Hep3B cells

[0245] This experimental example provides the IC 50 detection after transfection of siRNA at gradient concentrations in Hep3B cells. The experimental procedures refer to Experimental Example 10.

[0246] Based on the cell viability of PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M after transfection of siRNA at gradient concentrations in Hep3B cells at 96 h, the IC 50 for inhibiting the expression of the PLK1 gene was obtained, and the experimental results are shown in Table 13.

[0247] Table 13. IC of siRNA 50 (nM)

[0248] Experimental Grouping <![CDATA[IC 50 > PLK1-6M-96h 0.9244 nM PLK1-9M-96h 0.7107 nM PLK1-10M-96h 0.6667 nM PLK1-26M-96h 3.502 nM

[0249] In summary, considering the transfection toxicity results of PLK1-6M, PLK1-9M, PLK1-10M, and PLK1-26M in A549, Hela, and Hep3B cells respectively, PLK1-10M has the strongest cytotoxicity, followed by PLK1-6M.

[0250] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. An siRNA for inhibiting PLK1, characterized in that, The siRNA contains a sense strand and an antisense strand, and the sense strand can be at least partially reverse complementary to the antisense strand to form a double-stranded region. Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.2, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.4, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.6, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.8, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.10, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.11, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.12, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.14, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.16, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.18, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.19, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.20, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.22, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.23, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.24, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.25, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.26, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.27, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.28, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.29, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.30, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.31, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.32, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.33, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.34, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.35, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.36, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.37, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.38, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.39, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.40, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.41, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.42, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.43, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.44, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.45, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.46, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.47, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.48, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.49, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.50, or, Among them, the sense strand contains the nucleotide sequence shown in SEQ ID NO.51, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.

52.

2. The siRNA according to claim 1, wherein At least one nucleotide in the sense strand or antisense strand of the siRNA is a modified nucleotide.

3. The siRNA according to claim 2, wherein The modification is a chemical modification, and the chemical modification is selected from one or more of methoxy modification, fluoro modification, and phosphorothioate modification.

4. The siRNA according to any one of claims 2 to 3, wherein The fluorine-modified nucleotides are located in the antisense strand and sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorine-modified nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorine-modified nucleotides.

5. The siRNA according to any one of claims 2 to 3, characterized in that, The methoxy-modified nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides at least at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions of the sense strand are methoxy-modified nucleotides, and the nucleotides at least at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions of the antisense strand are methoxy-modified nucleotides.

6. The siRNA according to any one of claims 2 to 3, characterized in that, The phosphorothioate-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence. And, in the direction from the 5'-end to the 3'-end, the nucleotides between at least the 1st and 2nd positions, and the 2nd and 3rd positions of the sense strand are linked by a phosphorothioate group, and the nucleotides between at least the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, and the 20th and 21st positions of the antisense strand are linked by a phosphorothioate group.

7. The siRNA according to claim 2 to 3, characterized in that, The 3'-end of the sense strand of the siRNA is conjugated with a ligand, and the ligand is GalNAc.

8. A product for inhibiting PLK1, characterized in that, The product contains an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the siRNA according to any one of claims 1 to 7.

9. The product according to claim 8, wherein The product is a pharmaceutical composition or a kit.

10. Use of the siRNA according to any one of claims 1 to 7 or the product according to any one of claims 8 to 9 in the preparation of a product for preventing, diagnosing and / or treating a pathological condition or disease caused by PLK1.

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