WO2024169306A1 - 一种用于抑制细胞程序性死亡-配体1基因表达的siRNA、其缀合物和药物组合物及用途 - Google Patents

一种用于抑制细胞程序性死亡-配体1基因表达的siRNA、其缀合物和药物组合物及用途 Download PDF

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WO2024169306A1
WO2024169306A1 PCT/CN2023/133563 CN2023133563W WO2024169306A1 WO 2024169306 A1 WO2024169306 A1 WO 2024169306A1 CN 2023133563 W CN2023133563 W CN 2023133563W WO 2024169306 A1 WO2024169306 A1 WO 2024169306A1
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sirna
nucleotide
compound
nucleotides
antisense strand
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PCT/CN2023/133563
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French (fr)
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刘楠
张红丽
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苏州时安生物技术有限公司
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Publication of WO2024169306A1 publication Critical patent/WO2024169306A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present invention belongs to the field of biomedicine technology, and specifically relates to a siRNA for inhibiting the expression of programmed cell death-ligand 1 gene, a conjugate and a pharmaceutical composition thereof and uses thereof.
  • PD-L1 Programmed cell death 1 ligand 1
  • CD274 surface antigen cluster of differentiation 274
  • B7-H1 B7 homolog 1
  • PD-L1 is expressed on different tissue cells such as T cells, epithelial cells, and endothelial cells, and its expression is upregulated in many tumors.
  • PD-L1 is the main ligand of PD-1.
  • the interaction between ligand PD-L1 and receptor PD1 can induce phosphorylation of intracellular immunoreceptor tyrosine-based switch motifs (ITSMs) and receptor tyrosine-based inhibitory motifs (ITIMs), thereby inhibiting T cell activation and cytokine production.
  • IRSMs immunoreceptor tyrosine-based switch motifs
  • ITIMs receptor tyrosine-based inhibitory motifs
  • HBV hepatitis B virus
  • Envolizumab antibodies or small molecules, such as Envolizumab, Sugemalimab, Durvalumab, APL-502, CA-170, etc.
  • the indications of such drugs under development are mostly in the field of tumors. They are systemic drugs with high adverse reaction rates and poor safety in clinical practice. They are rarely used in the treatment of hepatitis B.
  • Envolizumab ASC22
  • ASC22 Envolizumab
  • oligonucleotide (ASO) drug targeting PD-L1, RO-9191863, from Roche in the clinical stage of the treatment of hepatitis B.
  • ASO is generally inferior to siRNA drugs in terms of effectiveness and long-term effectiveness, and has the characteristics of systemic distribution.
  • the liver-specific delivery is not as good as the siRNA drug form conjugated with liver-targeted molecules.
  • siRNA drugs can be conjugated with liver-targeted molecules to specifically deliver siRNA drugs targeting PD-L1 to the liver, which can specifically downregulate the level of PD-L1 molecules in the liver and reduce the systemic side effects of small molecules, antibodies, and ASO drugs.
  • siRNA targeting PD-L1 Although there are some patents for siRNA targeting PD-L1, no siRNA drugs targeting PD-L1 have been launched on the market, and no siRNA drugs are even in the clinical research stage. Therefore, there is still a need to continue to develop siRNA drugs that can inhibit the expression of the PD-L1 gene.
  • the object of the present invention is to provide the following siRNA and its modified sequence that can specifically inhibit the expression of PD-L1 gene in cells.
  • the pharmaceutical composition and siRNA conjugate containing the siRNA of the present invention can effectively deliver the siRNA of the present invention to target tissues and/or cells, thereby showing high drug potential in the treatment or prevention of diseases related to PD-L1 expression.
  • the first aspect of the present invention provides an siRNA for inhibiting the expression of programmed cell death-ligand 1 gene, which includes a sense chain and an antisense chain, the sense chain includes a nucleotide sequence selected from one of the nucleotide sequences shown in SEQ ID NO.1 to 12 or a nucleotide sequence with no more than 3 base mutations in the above sequence, and the antisense chain includes a nucleotide sequence selected from one of the nucleotide sequences shown in SEQ ID NO.13 to 24 or a nucleotide sequence with no more than 5 base mutations in the above sequence.
  • the positive strand includes a nucleotide sequence with no more than 2 base mutations and no more than 1 base mutation compared to any one of the nucleotide sequences shown in SEQ ID NOs. 1 to 12.
  • the base mutation of the sense strand can be at any position of the nucleotide sequence, for example, at any one, two or three of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth positions.
  • the antisense chain includes a nucleotide sequence with no more than 5 base mutations, a nucleotide sequence with no more than 4 base mutations, a nucleotide sequence with no more than 3 base mutations, a nucleotide sequence with no more than 2 base mutations, and a nucleotide sequence with no more than 1 base mutation compared to any one of the nucleotide sequences shown in SEQ ID NO.13 to 24.
  • the base mutation of the antisense strand can be at any position of the nucleotide sequence, for example, any one, two, three, four or five of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and twenty-first positions.
  • the base mutation of the sense chain is at the 3' end of its nucleotide sequence
  • the base mutation of the antisense chain is at any one or more of the 5' end, positions 2 to 8, and the last two positions at the 3' end of its nucleotide sequence.
  • the base mutation includes base substitution, insertion or deletion.
  • R1 is CH 3 .
  • the number of modified nucleotides in the sense strand is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen.
  • the number of modified nucleotides in the antisense strand is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or twenty-one.
  • all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.
  • the nucleotides at positions 7, 8 and 9 of the sense strand are 2'-fluoro-modified nucleotides
  • the nucleotides at positions 2, 14 and 16 of the antisense strand are 2'-fluoro-modified nucleotides.
  • the nucleotides at other positions of the sense strand are 2'-methoxy-modified nucleotides.
  • the 6th position of the antisense strand is a 2'-fluoro-modified nucleotide or SAFE-01
  • the 7th position of the antisense strand is a 2'-methoxy-modified nucleotide or SAFE-01.
  • SAFE-01 is Ago, Ggo, Cgo or Ugo.
  • the nucleotides at other positions of the antisense strand are 2'-methoxy-modified nucleotides, wherein the other positions refer to positions other than positions 2, 6, 7, 14 and 16.
  • At least one of the following linkages between nucleotides in the siRNA is a phosphorothioate linkage:
  • the 3' end of the sense strand is a phosphorothioate group and the 5' end of the antisense strand is a VP-modified nucleotide.
  • the siRNA is selected from any sequence in Table 2 or Table 3, wherein a sense strand and an antisense strand of a double-strand number constitute one siRNA.
  • the second aspect of the present invention provides a siRNA conjugate, which includes one or more of the above-mentioned siRNAs, and a conjugated group conjugated to the siRNA.
  • the conjugated group is derived from compound SA51, the structural formula of compound SA51 is:
  • the number of compound SA51 in the conjugated group is 2 to 4, and the 2 to 4 compounds SA51 are sequentially connected by chemical bonds.
  • the conjugate group is attached to the 3' end and/or the 5' end of the sense strand.
  • the siRNA conjugate has the following structural formula:
  • X O or S.
  • the third aspect of the present invention provides a pharmaceutical composition, which includes the above-mentioned siRNA or the above-mentioned siRNA conjugate, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is used to inhibit programmed cell death-ligand 1 gene expression.
  • the present invention provides use of the above siRNA, or the above siRNA conjugate, or the above pharmaceutical composition for preparing a medicament for treating and/or preventing diseases related to PD-L1 gene expression.
  • the disease is viral hepatitis or cancer.
  • the viral hepatitis is caused by infection with hepatitis B virus, hepatitis C virus, or hepatitis D virus.
  • the above siRNA, or the above siRNA conjugate, or the above pharmaceutical composition is formulated and administered to a subject according to a desired dose.
  • the above siRNA, or the above siRNA conjugate, or the above pharmaceutical composition is administered by subcutaneous injection, intravenous injection, intrathecal injection, or intramuscular injection.
  • the present invention has the following advantages compared with the prior art:
  • the siRNA disclosed in the present invention is a human PD-L1 siRNA sequence, and its conjugate and pharmaceutical composition have good stability and high PD-L1mRNA inhibitory activity.
  • This siRNA drug has liver targeting specificity and has better safety than small molecule drugs and antibody drugs. It is also long-lasting and can achieve longer dosing intervals in clinical practice, resulting in better patient compliance.
  • FIG1 is a graph showing the IC50 test results of the siRNA of Example 5 in SNU-387 cells
  • FIG. 2 is a graph showing the activity results of the siRNA of Example 7 in HDI mice on the 7th and 28th days.
  • Phosphorothioate-modified nucleotides refer to nucleotides in which one of the oxygen atoms in the phosphodiester bond in the phosphate group of the nucleotide is replaced by a sulfur atom.
  • VP-modified nucleotides refer to nucleotides in which the phosphate group of the nucleotide is replaced by a vinyl phosphate group. In some embodiments, the 5'-terminal phosphate group of the antisense strand is replaced by VP.
  • LNA is shown in formula (1)
  • ENA is shown in formula (2)
  • cET BNA is shown in formula (3)
  • UNA is shown in formula (4)
  • GNA is shown in formula (5)
  • VP is shown in formula (6):
  • Base represents a natural nucleobase, a modified nucleobase, or a universal base, such as A, U, G, or C.
  • R is selected from H, OH, or alkoxy (O-alkyl).
  • R1 is H, OH or CH 3
  • Base is a natural nucleobase, a modified nucleobase, a universal base or an H atom.
  • R1 is H.
  • R1 is OH.
  • R1 is CH 3 .
  • the nucleoside monomer refers to the modified or unmodified nucleoside phosphoramidite monomer used in the solid phase phosphoramidite synthesis according to the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared.
  • Solid phase phosphoramidite synthesis is a method used in RNA synthesis known to those skilled in the art.
  • the nucleoside monomers used in the present disclosure are all commercially available.
  • conjugation refers to the covalent bonding of two or more chemical moieties each having a specific function to each other; accordingly, “conjugate” refers to a compound formed by covalent bonding of the chemical moieties.
  • siRNA conjugate refers to a compound formed by covalent bonding of one or more chemical moieties having a specific function to siRNA.
  • siRNA conjugate should be understood as a general term for a plurality of siRNA conjugates or a siRNA conjugate represented by a certain chemical formula, depending on the context.
  • conjuggated molecule should be understood as a specific compound that can be conjugated to siRNA through a reaction to ultimately form the siRNA conjugate of the present disclosure.
  • hydroxyl protecting groups can be used in the present disclosure.
  • protecting groups make chemical functional groups insensitive to specific reaction conditions and can be added and removed from the functional group in the molecule without substantially damaging the rest of the molecule.
  • Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety.
  • the protecting group is stable under alkaline conditions but can be removed under acidic conditions.
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox).
  • non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).
  • the pharmaceutically acceptable carrier described in the present disclosure can be a carrier conventionally 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, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), PLL, chitosan (c hitosan), 1,2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D- or L-lactic/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer, PLGA), poly(aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA) and
  • the excipients can be one or more of various preparations or compounds conventionally used in the art.
  • the other pharmaceutically acceptable excipients can include at least one of a pH buffer, a protective agent and an osmotic pressure regulator.
  • subject refers to any animal, such as a mammal or marsupial.
  • Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and poultry of any kind.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cows, rabbits, sheep, rats, and poultry of any kind.
  • treatment refers to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
  • prevention refers to an approach to obtaining beneficial or desired results, including but not limited to a prophylactic benefit.
  • a prophylactic benefit siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.
  • such reagent can be obtained from any supplier of molecular biology reagents and has the quality/purity standards that meet the molecular biology application standards.
  • the PD-L1 siRNA sequence was synthesized at 200 nanomoles (nmol) on a Dr.Oligo48 synthesizer (Biolytic) using solid support-mediated phosphoramidite chemistry.
  • the solid support was a universal solid support (Shenzhen Comma Biotech). Nucleoside monomer raw materials 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Shanghai Zhaowei or Suzhou Jima.
  • the coupling time of all phosphoramidites was 6 minutes (min)
  • 5-ethylthio-1H-tetrazole (ETT) was used as an activator
  • 0.22M PADS dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine was used as a sulfurization reagent
  • the sulfurization reaction time was 3 minutes (min)
  • iodine pyridine/ An aqueous solution Karloma was used as an oxidant
  • the oxidation reaction time was 2 minutes (min).
  • the oligoribonucleotides were cleaved from the solid support and soaked in a 3:1 28% ammonia and ethanol solution at 50°C for 16 hours. Then, the supernatant was transferred to another centrifuge tube, concentrated and evaporated to dryness, and purified by C18 reverse chromatography with a mobile phase of 0.1MTEAA and acetonitrile, and DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotides were collected and freeze-dried, identified as the target product by LC-MS, and then quantified by UV (260nm).
  • the resulting single-stranded oligonucleotides were annealed according to the two complementary sequences in an equal molar ratio, and the resulting double-stranded siRNA was dissolved in 1X PBS and adjusted to the required concentration for the experiment. These monomers are linked to each other into oligonucleotides through 5'-3'-phosphodiester bonds.
  • N-benzyloxycarbonyl-L-serine (commercially available, purchased from Shanghai Titan Technology Co., Ltd.) (15.0 mmol, 3.58 g) was placed in a clean dry reaction bottle, 100 mL of dichloromethane was added, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 equiv, 22.5 mmol, 8.53 g), compound 2-2 (1.1 equiv, 16.5 mmol, 3.31 g) and N,N-diisopropylethylamine (3.0 equiv, 45.0 mmol, 5.78 g) were added at room temperature, followed by stirring at room temperature for 1 hour.
  • compound 2-5 (1.1 equiv, 5.08 mmol, 3.0 g) and N,N-diisopropylethylamine (3.0 equiv, 13.86 mmol, 17.91 g) were added to the reaction system, and then stirring was continued at room temperature for 1 hour. After the reaction, 150 mL of dichloromethane was added to the reaction solution, and the mixture was washed with 150 mL of saturated sodium bicarbonate solution and 150 mL of saturated brine. The organic phase was dried, filtered and concentrated.
  • the solid phase phosphoramidite method was used to initiate the cycle using compound SA51 prepared in the above steps, and nucleoside monomers were connected one by one from the 3'-5' direction according to the nucleotide arrangement order.
  • Each connection of a nucleoside monomer included four steps of deprotection, coupling, capping, oxidation or sulfurization. The same synthesis conditions were used for the sense strand and the antisense strand.
  • the nucleoside monomer was provided in an acetonitrile solution with a concentration of 0.05 M.
  • the conditions of the deprotection reaction in each step were the same, namely, the temperature was 25° C., the reaction time was 3 minutes, the deprotection reagent was DCA, and the injection volume was 180 ⁇ L.
  • reaction conditions for each coupling step were the same, including a temperature of 25°C, a reaction time of 3 minutes, a nucleoside monomer injection volume of 90 ⁇ L, and a catalyst ACT injection volume of 110 ⁇ L.
  • the capping conditions were the same for each step, including a temperature of 25°C and a reaction time of 2 minutes.
  • the capping reagent injection volume was 180 ⁇ L.
  • the oxidation reaction conditions were the same in each step, including a temperature of 25°C, a reaction time of 3 minutes, and an injection volume of 180 ⁇ L of the oxidation reagent OXD.
  • the reaction conditions for each step of the sulfurization reaction were the same, including a temperature of 25 °C, a reaction time of 4 minutes, and a sulfurization reagent of 0.05 M PADS in pyridine acetonitrile.
  • the injection volume of the sulfurization reagent was 180 ⁇ L.
  • the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and the antisense chain, wherein:
  • Purification and desalting conditions are as follows: Desalting is performed using a C18 reverse phase column. Specific conditions include:
  • TEAA triethylamine acetate
  • Activation 0.8 mL of acetonitrile was passed through each well of a 96-well plate for activation;
  • the 96-well plate was rinsed three times with 0.8 mL 3% trifluoroacetic acid to remove DMT, and the adsorption layer was observed to turn orange-red;
  • the 96-well plate was rinsed twice with 0.8 mL of deionized water to remove trifluoroacetic acid and residual salt;
  • the detection method is as follows: Waters Acquity UPLC-LTQ LCMS (column: ACQUITY UPLC BEH C18) was used to detect the purity of the above-mentioned sense chain and antisense chain and analyze the molecular weight. The measured values are consistent with the theoretical values, indicating that the synthesized sense chain and antisense chain are conjugated with groups at the 3' end and/or 5' end.
  • Annealing operation is as follows: dissolve the sense strand and antisense strand synthesized in step 2 in water for injection, prepare 0.1 mg/mL-40 mg/mL solution, calibrate the mixture in equal molar ratio using a concentration meter, heat at 90°C for 5 minutes, and then slowly cool down naturally to form a double-stranded structure through hydrogen bonding, take samples and send to detect the SEC purity of the product.
  • the double-stranded sample is freeze-dried.
  • Dissolve compound 1 (9.44 g, 79.9 mmol, 9.17 mL, 1.0 equiv) in 175 mL of anhydrous tetrahydrofuran, cool to -70°C to -78°C, and replace with nitrogen, and add the solution obtained in the previous step dropwise (slowly dropwise, the dropwise addition process lasts for at least 10 minutes).
  • the reaction solution is stirred at -70°C to -78°C for half an hour.
  • hexamethylphosphoric acid triamide (26.1 g, 146 mmol, 25.6 mL, 1.82 equiv) and benzyl chloromethyl ether (17.5 g, 112 mmol, 15.5 mL, 1.4 equiv) were slowly added dropwise to the previous reaction solution (slowly added dropwise, the addition process lasted for at least 10 minutes), and after the addition was completed, the temperature was raised to 0°C and stirred for 3 hours. TLC and LCMS detected the disappearance of compound 1. 600 mL of saturated ammonium chloride solution was added in two batches to quench the reaction, and the mixture was extracted with 200 mL of methyl tert-butyl ether.
  • reaction solution was dissolved in methyl tert-butyl ether (10.0 mL) and 1% sodium hydroxide solution (10.0 mL), and continued to stir at room temperature for 0.5 hours.
  • the remaining system was extracted with saturated sodium bicarbonate solution and dichloromethane for 3 times, and the organic phases were combined and spin-dried.
  • the crude product was used on a C18 reverse phase column to obtain a white solid compound SA000015 (1.5 g, reaction yield 55%).
  • reaction solution was extracted with saturated sodium bicarbonate solution and ethyl acetate for 3 times, the organic phases were combined and spin-dried, and the crude product was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate 10:1 to 8:1) to obtain a white solid compound 3-1 (16.1 g, reaction yield 99%).
  • the nucleotide position at the 6th or 7th position of the 5' region of the antisense chain contains a chemical modification represented by Ago, Cgo, or Ugo, and the modified siRNA is obtained by solid phase synthesis according to Example 2.
  • the inventors designed and synthesized a large number of PD-L1 siRNAs in the early stage, and verified the cell activity, eliminated some PD-L1 siRNAs with poor effects, and retained some PD-L1 siRNAs with good activity for further experiments.
  • the specific sequences are shown in Tables 2 to 4 below.
  • Table 2 below shows the unmodified sense strand and antisense strand of PD-L1 siRNA
  • Table 3 below shows the modified sense strand and antisense strand of PD-L1 siRNA (the table is the modified sequence corresponding to each siRNA in Table 1 from top to bottom)
  • Table 4 below shows the siRNA conjugate sequence.
  • capital letters A, C, G, and U represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, and uridine-3'-phosphate, respectively; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left is a 2'-fluoro-modified nucleotide; lowercase letter s in the middle of the capital letter indicates that the two nucleotides adjacent to the left and right of s are linked by a phosphorothioate group; When s is the first at the 3' end, it means that the nucleotide terminal adjacent to the left side of the letter s is a phosphorothioate group.
  • siRNA targeting PD-L1 was tested by dual luciferase assay.
  • the final concentration of siRNA for initial transfection was 10 nM, and compounds with a residual activity percentage of less than 35% at a concentration of 10 nM were screened for activity at two concentrations, 1 nM and 0.2 nM, and IC50 screening was performed on the preferred compounds screened at two concentrations.
  • HEK293 cells were cultured in DMEM high glucose medium (Gibco, C11995500BT) with 10% fetal bovine serum at 37 ° C and 5% CO 2 , grown to a confluence of about 90%, and then digested with trypsin (Thermo, 25200072) and resuspended.
  • the complete human PD-L1 reference sequence (NM_014143.4) was cloned into the dual luciferase psiCHECK2TM vector multi-cloning sites XhoI and NotI to construct the hs-psiCHECK plasmid.
  • the dual luciferase plasmid was co-transfected with siRNA into 1x10 4 cells using LipofectamineTM2000Transfection Reagent (Thermo, 11668500).
  • LipofectamineTM2000Transfection Reagent Thermo, 11668500.
  • Opti-MEM Opti-MEM
  • 20 ng of plasmid vector and siRNA per well 20 ng of plasmid vector and siRNA per well, and incubated at room temperature for 15 minutes, and the mixture was added to the 96 plate.
  • cells resuspended in 80 ⁇ L of fresh DMEM high glucose medium (Gibco, C11995500BT) containing 10% fetal bovine serum were added. The cells were incubated for 24 hours and luciferase (Yishen Bio, 11405ES80) was detected.
  • Renilla luciferase signal readings in each well were normalized to the firefly luciferase (control) signal and then compared with cells transfected with the same plasmid but without siRNA treatment to assess the activity of each siRNA. All transfections were performed in duplicate.
  • the results are expressed as the remaining percentage relative to the control group without siRNA (the control group is 100%), and the data are shown in Table 5. As can be seen from Table 5, except for SD003798 and SD003955, the experimental results of other groups are better, and the siRNA with better effect is further studied.
  • IC50 in vitro screening was performed in SNU-387 cells using 8 concentrations (starting from 10 nM and 3-fold dilutions, a total of 8 concentration points).
  • SNU-387 cells were cultured in RPMI-1640 medium (Thermo, 11875119) with 10% fetal bovine serum at 37°C and 5% CO2, and then digested with trypsin and the cells were resuspended. 2x10 4 cells were co-transfected with siRNA using RNAiMAX (Thermo, 13778150).
  • RNAiMAX was added to 19.7 ⁇ L Opti-MEM (Gibco, 31985070) containing different concentrations of siRNA per well, and incubated at room temperature for 15 minutes, the mixture was added to a 96-well plate, and then cells resuspended in 80 ⁇ L of fresh RPMI-1640 medium (Thermo, 11875119) containing 10% fetal bovine serum were added. The cells were incubated for 24 hours, RNA was extracted using a tissue cell extraction kit (Zhiang Biotechnology, MNTR/FX96), and reverse transcribed into cDNA (Takara, 6210B). The expression level of the PD-L1 gene was measured by SYBR Green qPCR (Vazyme, Q711). For specific operation methods, please refer to the corresponding instructions.
  • Target gene PD-L1 primers and probes
  • Reverse primer CCTTGGGAACCGTGACAGTA (SEQ ID NO.30);
  • Reverse primer ACTGTGGTCATGAGTCCTTCCA (SEQ ID NO.32);
  • Dual luciferase reporter gene was used to detect the on-target and off-target activities of siRNAs with different chemical modifications.
  • HEK293 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2.
  • the reporter gene plasmid and siRNA of different concentrations starting from a final concentration of 90nM, 3-fold dilution, a total of 11 concentrations
  • Lipofectamine 2000 ThermoFisher, 11668019
  • the cells were tested using a dual luciferase assay kit (Yishen Bio, 11405ES80).
  • the Renilla luciferase signal readings of each well were normalized to the firefly luciferase (control) signal, and then compared with cells transfected with the same plasmid but not treated with siRNA to calculate the relative levels at different concentrations, and the IC50 values were calculated.
  • the results are shown in Table 7. The results showed that modification of the nucleotides at the 6th or 7th position of the 5' region of the antisense strand in some sequences did not affect the activity while improving the off-target effect, thereby improving the safety of siRNA.
  • siRNA was selected for conjugation synthesis, and the in vivo activity was evaluated in the HDI mouse model.
  • the siRNA conjugate was synthesized as in Example 2 by solid phase synthesis.
  • mice At least 14 days before the administration of the conjugate, six to eight-week-old female Balb/C mice (Zhejiang Weitong Lihua) were injected with high pressure tail vein to construct humanized mice expressing PD-L1, and 2 ⁇ g of plasmid containing PD-L1 mRNA sequence was injected into the mice through the tail vein within 5-7 seconds to produce PD-L1-SEAP model mice (Zhang G et al., "High levels of foreign gene expression in hepatocytes after tail vein injection of naked plasmid DNA.” Human Gene Therapy 1999 Vol. 10, p1735-1737.).
  • the inhibition of PD-L1 expression by PD-L1 siRNA conjugates leads to the inhibition of secretory alkaline phosphatase (SEAP) expression.
  • SEAP secretory alkaline phosphatase
  • One day before administration blood was collected by orbital bleeding, and the SEAP expression level in serum was measured using the Phospha-Light SEAP reporter gene analysis system (Invitrogen) according to the product instructions, and the mice were grouped according to the average SEAP level.
  • the mice in the experimental group were given the conjugate, and the mice in the vehicle group were given phosphate Buffered saline (PBS), according to each mouse given 3mg/kg conjugate dose for subcutaneous administration.
  • PBS phosphate Buffered saline
  • the results were expressed as the residual expression level of serum SEAP before and after administration of each group (the vehicle group was 100%).
  • the relative residual expression level results are shown in Table 8 and Figure 2.
  • the conjugates SD004177, SD004178, SD004179, SD004240, SD004180, SD004241, SD004242, SD004181, and SD004182 had good activity, with an inhibition rate of greater than 80%.
  • the SD004178 and SD004180 compounds still maintained greater than 80% activity, reflecting long-term effectiveness.
  • This embodiment uses the HDI model to detect human PD-L1 gene levels in vivo, thereby better predicting the in vivo activity of human PD-L1 mRNA and providing more and more reliable experimental data for clinical trials and drug marketing.
  • Example 8 In vivo efficacy of siRNA in the hPD-1/PD-L1 humanized mouse AAV-HBV (hepatitis B virus) model
  • siRNA conjugate was synthesized as in Example 2 by solid phase synthesis.
  • an hPD-1/PD-L1 humanized mouse AAV-HBV model was constructed.
  • a mouse animal model of persistent HBV infection was prepared by intravenous injection of rAAV8-1.3HBV into the tail vein of 6-8 week-old B6-hPD-1/PD-L1 female mice.
  • the AAV virus injection dose was 1E+11vg/mouse.
  • the animals were submandibularly blooded and centrifuged after blood collection. The centrifugation conditions were 5000rpm, 4°C, and centrifuged for 10 minutes.
  • the serum was diluted with PBS, and the dilution multiple should be determined according to the actual situation.
  • HBsAg hepatitis B surface antigen
  • the HBsAg quantification method is direct electrochemiluminescence.
  • the animals were grouped according to HBsAg levels. On day 0, they were injected subcutaneously with a dose of 7.5 mg/kg body weight once a week. Blood was collected on day 49 to detect HBsAg levels and evaluate the anti-HBV activity of siRNA.

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Abstract

本发明涉及一种用于抑制细胞程序性死亡-配体1基因表达的siRNA、其缀合物和药物组合物及用途，所述siRNA包括正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，所述正义链包括选自SEQ ID NO.1至12所示核苷酸序列中的一种或上述序列不超过3个碱基突变的核苷酸序列，所述反义链包括选自SEQ ID NO.13至24所示核苷酸序列中的一种或上述序列不超过5个碱基突变的核苷酸序列。

Description

一种用于抑制细胞程序性死亡-配体1基因表达的siRNA、其缀合物和药物组合物及用途 技术领域
本发明属于生物医药技术领域，具体涉及一种用于抑制细胞程序性死亡-配体1基因表达的siRNA、其缀合物和药物组合物及用途。
背景技术
细胞程序性死亡配体1(Programmed cell death 1ligand 1，PD-L1)也称为表面抗原分化簇274(cluster of differentiation 274，CD274)或B7同源体(B7homolog 1，B7-H1)，是大小为40kDa的I型跨膜蛋白，由CD274基因编码。PD-L1在T细胞、上皮细胞、内皮细胞等不同组织细胞上均有表达,并且其在包括在许多肿瘤中表达上调。
PD-L1是PD-1的主要配体，配体PD-L1与受体PD1的相互作用可以诱导细胞内免疫受体酪氨酸转换基序(ITSM)和受体酪氨酸抑制基序(ITIM)磷酸化，从而抑制T细胞的活化和细胞因子的产生。在正常组织感染或炎症期间，这种相互作用对于通过维持免疫反应稳态来预防自身免疫很重要。在肿瘤微环境中，这种相互作用可以促使效应T细胞失活，从而为肿瘤细胞提供免疫逃逸。
此外有文章报道(Sun Y等人，Am J Physiol Gastrointest Liver Physiol.2020Jan 1；318(1):G162-G173.)病毒调节PD-1/PD-L1途径以改善感染性。在乙型肝炎病毒(HBV)感染过程中，HBV可以通过PTEN/β-catenin/c-Myc信号通路增加PD-L1的表达，从而抑制T细胞反应，最终促进HBV免疫逃逸。靶向该信号通路是治疗慢性乙型肝炎的潜在策略。
目前靶向PD-L1的上市或临床药物类型大多数是抗体类或小分子类，如恩沃利单抗、舒格利单抗、度伐利尤单抗、APL-502、CA-170等。此类药物在研适应症多为肿瘤领域，为全身性用药，临床上不良反应率较高，安全性较差，较少有乙型肝炎治疗领域应用，目前上市药物中仅恩沃利单抗(ASC22)针对乙型肝炎适应症在美国开展临床试验，临床前研究表明在感染乙型肝炎病毒的土拨鼠模型中，ASC22与恩替卡韦联用可以降低表面抗原水平(Balsitis S等人，PLoS One.2018Feb 14；13(2):e0190058.)，IIa期临床数据显示单次给药0.3、1.0或2.5mg/kg ASC22后剂量依赖性HBsAg减少。
目前在核酸领域，在乙型肝炎治疗领域临床阶段仅罗氏一款针对PD-L1的寡核苷酸类(ASO)RO-9191863药物，通常ASO在有效性和长效性表现上稍逊于siRNA药物，具有全身分布的特性，肝部特异性递送不如肝靶向分子缀合的siRNA药物形式。siRNA药物可以通过肝靶向分子缀合的方式，特异地将靶向PD-L1的siRNA药物递送至肝脏，可以特异地下调肝部PD-L1分子的水平，降低小分子、抗体、ASO药物形式的全身副作用。
截至目前，虽有一些靶向PD-L1的siRNA的专利，但尚未有靶向PD-L1的siRNA药物上市，甚至未有siRNA药物处于临床研究阶段。因此，仍需持续开发能够抑制PD-L1基因表达的siRNA药物。
发明内容
本发明的目的在于提供如下能够特异性地抑制细胞中PD-L1基因的表达的siRNA及其修饰序列，包含本公开的siRNA的药物组合物和siRNA缀合物能够有效地将本公开的siRNA递送至目标组织和/或细胞，从而在治疗或预防与PD-L1表达相关的疾病中显示出高的成药潜力。
为达到上述目的，本发明采用的技术方案是：
本发明第一方面提供一种用于抑制细胞程序性死亡-配体1基因表达的siRNA，其包括正义链和反义链，所述正义链包括选自SEQ ID NO.1至12所示核苷酸序列中的一种或上述序列不超过3个碱基突变的核苷酸序列，所述反义链包括选自SEQ ID NO.13至24所示核苷酸序列中的一种或上述序列不超过5个碱基突变的核苷酸序列。
根据某些实施方式，所述正义链包括与SEQ ID NO.1至12所示核苷酸序列中的任一条不超过2个碱基突变的核苷酸序列，不超过1个碱基突变的核苷酸序列。
根据某些实施方式，所述正义链的碱基突变可以在核苷酸序列的任意位置，例如第一位、第二位、第三位、第四位、第五位、第六位、第七位、第八位、第九位、第十位、第十一位、第十二位、第十三位、第十四位、第十五位、第十六位、第十七位、第十八位、第十九位中的任意一位、两位或三位。
根据某些实施方式，所述反义链包括与SEQ ID NO.13至24所示核苷酸序列中的任一条不超过5个碱基突变的核苷酸序列，不超过4个碱基突变的核苷酸序列，不超过3个碱基突变的核苷酸序列，不超过2个碱基突变的核苷酸序列，不超过1个碱基突变的核苷酸序列。
根据某些实施方式，所述反义链的碱基突变可以在核苷酸序列的任意位置，例如第一位、第二位、第三位、第四位、第五位、第六位、第七位、第八位、第九位、第十位、第十一位、第十二位、第十三位、第十四位、第十五位、第十六位、第十七位、第十八位、第十九位、第二十位、第二十一位中的任意一位、两位、三位、四位或五位。
根据某些实施方式，按照5’到3’的方向，所述正义链的碱基突变在其核苷酸序列的3’末端，所述反义链的碱基突变在其核苷酸序列的5’末端、第2～8位、3’端最后两位中的任意一位或多位。
根据某些实施方式，所示碱基突变包括碱基的替换、插入或者缺失。
在某些实施方案中，所述修饰的核苷酸为2’-甲氧基修饰的核苷酸、2’-氟代修饰的核苷酸、2’-O-CH2-CH2-O-CH3修饰的核苷酸、2’-O-CH2-CH＝CH2修饰的核苷酸、2’-CH2-CH2-CH＝CH2修饰的核苷酸、2’-脱氧基核苷酸、2’‐甲氧基乙基修饰的核苷酸、硫代磷酸酯键修饰的核苷酸、VP修饰的核苷酸、LNA、ENA、cET BNA、UNA、GNA和SAFE-01中的一种或多种的组合，其中SAFE-01所示结构式中的R1为H、OH或CH3，Base为天然核碱基、修饰的核碱基、通用碱基或H原子。
进一步地，R1为CH3。
在某些实施方案中，所述正义链中的修饰核苷酸的个数为一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个、十四个、十五个、十六个、十七个、十八个或十九个。
在某些实施方案中，所述反义链中的修饰核苷酸的个数为一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个、十三个、十四个、十五个、十六个、十七个、十八个、十九个、二十个或二十一个。
在某些实施方案中，所述正义链和所述反义链中的全部的核苷酸为修饰的核苷酸。
在某些实施方案中，按照5’到3’的方向，所述正义链的第7、8和9位的核苷酸为2’-氟代修饰的核苷酸，所述反义链的第2、14和16位的核苷酸为2’-氟代修饰的核苷酸。
在某些进一步地实施方案中，所述正义链的其他位置的核苷酸为2’-甲氧基修饰的核苷酸。
在某些进一步地实施方案中，按照5’到3’的方向，所述反义链的第6位为2’-氟代修饰的核苷酸或SAFE-01，所述反义链的第7位为2’-甲氧基修饰的核苷酸或SAFE-01。
优选地，SAFE-01为Ago、Ggo、Cgo或Ugo。
在某些进一步地实施方案中，所述反义链的其他位置的核苷酸为2’-甲氧基修饰的核苷酸，其中，所述其他位置是指第2、6、7、14和16位以外的位置。
在某些进一步地实施方案中，所述siRNA中以下核苷酸之间的连接中至少一个为硫代磷酸酯基连接：
所述正义链的5’端第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的5’端第2个核苷酸和第3个核苷酸之间的连接；
所述正义链的3’端第1个核苷酸和第2个核苷酸之间的连接；
所述正义链的3’端第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的5’端第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的5’端第2个核苷酸和第3个核苷酸之间的连接；
所述反义链的3’端第1个核苷酸和第2个核苷酸之间的连接；
所述反义链的3’端第2个核苷酸和第3个核苷酸之间的连接。
在某些进一步地实施方案中，所述正义链的3’末端为硫代磷酸酯基，所述反义链的5’末端为VP修饰的核苷酸。
在某些实施方案中，所述siRNA选自表2或表3中的任意序列，其中一个双链编号的一条正义链和一条反义链组成一条siRNA。
本发明第二方面提供一种siRNA缀合物，其包括上述siRNA中的一条或多条，以及缀合至所述siRNA的缀合基团。
在某些实施方案中，所述缀合基团来自化合物SA51，所述化合物SA51的结构式为：
进一步地，所述缀合基团中的化合物SA51的个数为2～4个，2～4个所述化合物SA51通过化学键依次连接。
在某些实施方案中，所述缀合基团连接在所述正义链的3’末端和/或5’末端。
在某些实施方案中，所述siRNA缀合物的结构式如下：
其中，表示本公开的siRNA形成的双链体，X为O或S。
本发明第三方面提供一种药物组合物，其包括上述siRNA或上述siRNA缀合物，以及药学上可接受的载体或辅料。
在某些实施方案中，所述药物组合物用于抑制细胞程序性死亡-配体1基因表达。
本发明第四方面提供上述siRNA，或上述siRNA缀合物，或上述药物组合物用于制备治疗和/或预防PD-L1基因表达相关的疾病的药剂的用途。
在某些实施方案中，所述疾病为病毒性肝炎或癌症。
进一步地，所述病毒性肝炎为乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒感染所导致的。
在某些实施方案中，上述siRNA，或上述siRNA缀合物，或上述药物组合物按照所需剂量被配制施用于受试者。
在某些实施方案中，上述siRNA，或上述siRNA缀合物，或上述药物组合物通过皮下注射、静脉注射、鞘内注射或肌肉注射等形式施用。
由于上述技术方案运用，本发明与现有技术相比具有下列优点：
本发明公开的siRNA为人源PD-L1siRNA序列，其缀合物和药物组合物具有良好的稳定性，较高的 PD-L1mRNA抑制活性。该siRNA药物具有肝靶向特异性，相比于小分子药物和抗体药物具有更好的安全性，同时具有长效性，可在临床中实现更长给药间隔，患者的依从性更佳。
附图说明
图1为实施例5的siRNA在SNU-387细胞中的IC50测试结果图；
图2为实施例7的siRNA在HDI小鼠体内第7天和第28天的活性结果图。
具体实施方式
需要说明的是，除非另外定义，本申请使用的技术术语或者科学术语应当为所属领域的技术人员所理解的通常意义。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售产品。
定义
在上文及下文中，2’-甲氧基修饰的核苷酸指核苷酸的核糖基2'位的羟基被甲氧基取代形成的核苷酸；同理，2’-氟代修饰的核苷酸、2’-O-CH2-CH2-O-CH3修饰的核苷酸、2’-O-CH2-CH＝CH2修饰的核苷酸、2’-CH2-CH2-CH＝CH2修饰的核苷酸、2’-脱氧基核苷酸、2’‐甲氧基乙基修饰的核苷酸分别是指核苷酸的核糖基2'位的羟基被相应基团取代所形成的核苷酸。
硫代磷酸酯键修饰的核苷酸指核苷酸的磷酸酯基中的磷酸二酯键中的一个氧原子被硫原子取代。
VP修饰的核苷酸是指核苷酸的磷酸基团被乙烯基磷酸酯基取代形成的核苷酸，在一些实施方式中，反义链的5’末端磷酸基团被VP取代。
LNA如式(1)所示，ENA如式(2)所示，cET BNA如式(3)所示，UNA如式(4)所示，GNA如式(5)所示，VP如式(6)所示：
上述式(1)至式(6)中，Base表示天然核碱基、修饰的核碱基、通用碱基，例如A、U、G或C。上述式(4)至式(6)中，R选自H、OH或烷氧基(O-烷基)。
SAFE-01的结构式为其中，R1为H、OH或CH3，Base为天然核碱基、修饰的核碱基、通用碱基或H原子。根据一些实施方式，R1为H。根据一些实施方式，R1为OH。根据一些实施方式，R1为CH3。
在上文及下文中，特别是在描述本公开的siRNA、药物组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，固相亚磷酰胺合成中使用的修饰或未修饰的核苷亚磷酰胺单体。固相亚磷酰胺合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合 物。siRNA缀合物应根据上下文，理解为多个siRNA缀合物的总称或者某个化学式所示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的特定化合物。
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能团对特定的反应条件不敏感，并且可以在分子中的该官能团上添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2d ed，John Wiley&Sons，New York，1991中，以引用的方式将上述文献各自整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基黄嘌呤-9-基(Pixyl)和9-(对甲氧基苯基)黄嘌呤-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-二甲氧基三苯甲基)和TMTr(4,4',4”-三甲氧基三苯甲基)。
本公开中所述的药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbon nanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphate nanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethyl methacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。
如本说明书所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且所述描述包括事件或状况发生的情况和其中不发生的情况。
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、兔、绵羊、大鼠和任何种类的家禽。
如本文所使用的，“治疗”指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。
如本文所使用的“预防”指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将siRNA、siRNA缀合物或药物组合物给予有罹患特定疾病风险的受试者，或给予报告疾病的一种或多种生理症状的受试者，即便可能该疾病的诊断尚未作出。
下面结合具体的实施例对本发明提供的技术方案做进一步的描述。下述实施例仅用于对本发明进行说明，并不会对本发明的保护范围进行限制。
实施例1 siRNA的合成
本文中，若未给出实际的试剂来源，则该类试剂可以自任意分子生物学试剂的供应商获得；且具备满足用于分子生物学应用的质量/纯度标准。
使用固体支撑物介导的亚磷酰胺化学于Dr.Oligo48合成器(Biolytic)上以200纳摩尔(nmol)规格合成PD-L1 siRNA序列。该固体支撑物是通用固体支撑物(深圳逗点生物)。核苷单体原料2’-F RNA、2’-O-甲基RNA等核苷亚磷酰胺单体购自上海兆维或苏州吉玛。全部亚磷酰胺(50mM乙腈溶液)的偶合时间是6分钟(min)，采用5-乙基硫-1H-四唑(ETT)作为活化剂(0.6M乙腈溶液)，使用0.22M的PADS溶于1:1体积比的乙腈和三甲基吡啶(苏州柯乐玛)溶液作为硫化试剂，硫化反应时间是3分钟(min)，使用碘吡啶/ 水溶液(柯乐玛)作为氧化剂，氧化反应时间2分钟(min)。
固相合成完成后，寡核糖核苷酸自该固体支撑物裂解，采用3:1的28％氨水和乙醇溶液在50℃条件下浸泡16小时。然后高速离心，将上清液转移到另一个离心管中，浓缩蒸发干后，使用C18反向色谱纯化，流动相为0.1MTEAA和乙腈，并使用3％三氟乙酸溶液脱出DMTr。目标寡核苷酸收集后冻干，并经LC-MS鉴定为目标产物，再经过UV(260nm)定量。
所得到的单链寡核苷酸，根据等摩尔比，按照互补配对的两条序列，进行退火，最后所得到的双链siRNA溶于1X PBS中，并调整至实验所需浓度。这些单体通过5’-3’-磷酸二酯键相互连接成寡核苷酸中。
实施例2 siRNA缀合物的制备
一、Galnac靶头制备
本实施例中，化合物SA51(I-1-7)的合成路线如下所示：
1、中间体2-1的制备
将化合物(R)-(+)-N-苄基-3-羟基吡咯烷(市售，购买于上海泰坦科技股份有限公司)(16.9mmol,3.0g)和咪唑(3.0equiv,50.7mmol,3.45g)置于洁净干燥反应瓶中，加入50mL乙腈，室温下缓慢加入叔丁基二甲基氯硅烷(1.3equiv,21.9mmol,3.31g)，随后室温继续搅拌12小时。反应后向反应液中加入100mL乙酸乙酯，并用100mL饱和碳酸氢钠溶液和100mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝20/1-5/1),得到无色油状物化合物2-1(4.9g,16.8mmol,99％收率)。化合物2-1分子式:C17H29ONSi,分子量:291.2,LC-MS实测：292.4(M+H).
2.2中间体2-2的制备
将化合物2-1(16.8mmol,4.9g)置于洁净干燥反应瓶中，加入100mL甲醇，室温氢气下加入钯碳(湿基，10％Pd/C)(10％wt，490.0mg)，随后室温继续搅拌12小时。反应后过滤除去钯碳，滤液浓缩，得到粗产品白色固体化合物2-2(3.31g，16.5mmol，98％收率)，未纯化直接投入下一步反应。化合物2-2分子式:C10H23ONSi,分子量:201.1，LC-MS实测：202.3(M+H).
2.3中间体2-3的制备
将化合物N-苄氧羰基-L-丝氨酸(市售，购买于上海泰坦科技股份有限公司)(15.0mmol,3.58g)置于洁净干燥反应瓶中，加入100mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.5equiv,22.5mmol,8.53g)、化合物2-2(1.1equiv,16.5mmol,3.31g)和N,N-二异丙基乙胺(3.0equiv,45.0mmol,5.78g),随后室温搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝10/1-1/3),得到白色固体化合物2-3(4.5g,10.65mmol,63％两步收率)。化合物2-3分子式:C21H34O5N2Si,分子量:422.2,LC-MS实测：423.3(M+H).1H NMR(400MHz,CDCl3):δ7.35–7.29(m,5H),5.96(dd,J＝14.3,8.3Hz,1H),5.10(s,2H),4.61–4.40(m,2H),3.85–3.68(m,2H),3.65–3.49(m,2H),3.41(d,J＝12.7Hz,1H),3.31(s,1H),1.95(qdd,J＝15.0,11.8,5.3Hz,2H),1.77(s,1H),0.86(s,9H),0.06(d,J＝3.1Hz,6H).
2.4中间体2-4的制备
将化合物2-3(10.65mmol,4.5g)置于洁净干燥反应瓶中，加入100mL吡啶，室温下加入4,4'-双甲氧基三苯甲基氯(1.2equiv,12.78mmol,4.32g)，随后室温继续搅拌12小时。反应后向反应液中加入150mL乙酸乙酯，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝20/1-1/1),得到淡黄色油状物化合物2-4(7.56g,10.43mmol,98％收率)。化合物2-4分子式:C42H52O7N2Si,分子量:724.3，LC-MS实测：747.4(M+Na).1H NMR(400MHz,CDCl3):δ7.35–7.31(m,1H),7.28(d,J＝4.7Hz,4H),7.27–7.23(m,2H),7.23–7.14(m,5H),7.13–7.11(m,2H),6.80–6.78(m,1H),6.76(dd,J＝7.7,5.4Hz,4H),5.72(dd,J＝22.7,8.3Hz,1H),5.08–4.99(m,2H),4.69–4.59(m,1H),4.35–4.30(m,1H),3.73(dd,J＝4.5,3.7Hz,6H),3.65–3.44(m,2H),3.36–3.20(m,3H),1.86–1.81(m,1H),1.70(s,1H),0.80(d,J＝13.1Hz,9H),-0.02(dd,J＝14.9,4.2Hz,6H).
2.5中间体2-5的制备
将化合物2-4(10.43mmol,7.56g)置于洁净干燥反应瓶中，加入100mL甲醇，室温氢气下加入钯碳(湿基,10％Pd/C)(10％wt,750.0mg)，随后室温继续搅拌12小时。反应后过滤除去钯碳，滤液浓缩，得到粗产品白色固体化合物2-5(6.0g,10.22mmol,98％收率)，未纯化直接投入下一步反应。化合物2-5分子式:C34H46O5N2Si,分子量:590.3,LC-MS实测：591.6(M+H).
2.6中间体2-6的制备
将化合物5-(((2R,3R,4R,5R,6R)-3-乙酰氨基-4,5-二乙酰氧基-6-(乙酰氧基甲基)四氢-2H-吡喃-2-基)氧基)戊酸(市售，购买于诺甘林生物医药科技(苏州)有限公司)(2.06g,4.62mmol)置于洁净干燥反应瓶中，加入100mL二氯甲烷，室温下加入苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(1.5equiv,6.93mmol,2.63g)，搅拌十分钟，随后向反应体系中加入化合物2-5(1.1equiv,5.08mmol,3.0g)和N,N-二异丙基乙胺(3.0equiv,13.86mmol,17.91g)，随后室温继续搅拌1小时。反应后向反应液中加入150mL二氯甲烷，并用150mL饱和碳酸氢钠溶液和150mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝50/1-10/1),得到白色固体化合物2-6(3.58g,3.51mmol,76％收率)。化合物2-6分子式:C53H73O15N3Si,分子量:1019.3,LC-MS实测：1018.3(M-H).
2.7中间体2-7的制备
将化合物2-6(3.51mmol,3.58g)置于洁净干燥反应瓶中，加入50mL吡啶，室温下加入三乙胺氟化氢(5.0equiv,17.55mmol,2.97g)，随后室温继续搅拌12小时。反应后向反应液中加入100mL乙酸乙酯，并用100mL饱和碳酸氢钠溶液和100mL饱和食盐水洗涤，干燥有机相，过滤并浓缩，所得粗产品硅胶柱层析分离纯化(二氯甲烷/甲醇＝50/1-10/1),得到淡黄色油状物化合物2-7(2.4g,2.65mmol,76％收率)。化合物2-7分子式:C47H59O15N3,分子量:905.3,LC-MS实测：904.4(M-H).1H NMR(400MHz,CDCl3):δ7.30(dd,J＝4.2,1.9Hz,1H),7.29–7.25(m,5H),7.19–7.15(m,4H),6.85–6.81(m,4H),5.40–5.34(m,1H),5.30(s,1H),5.26–5.19(m,1H),5.13(ddd,J＝14.5,10.9,3.3Hz,1H),4.85–4.78(m,1H),4.75–4.71(m,1H),4.68–4.61(m,1H),4.50(d,J＝11.5Hz,1H),4.18–4.11(m,2H),4.06–3.97(m,1H),3.90(dt,J＝10.6,6.8Hz,2H),3.80(s,6H),3.67–3.46(m,4H),3.25–3.19(m,2H),2.34–2.23(m,1H),2.17–2.15(m,3H),2.10–2.03(m,5H),1.98(dt,J＝12.9,2.4Hz,4H),1.76–1.61(m,4H),1.37(t,J＝7.3Hz,3H).
2.8化合物I-1-7的制备
将化合物2-7(2.65mmol,2.4g)置于洁净干燥反应瓶中，加入50mL无水二氯甲烷(市售，购买于上海泰坦科技股份有限公司)。室温氩气保护下加入化合物2-氰乙基N,N,N’,N’-四异丙基亚磷酰二胺(2.0equiv,5.3mmol,1.6g)和4,5-二氰咪唑(1.5equiv,3.97mmol,470.0mg)，室温下继续搅拌一小时。反应后向反应液中加入50mL二氯甲烷，并用100mL饱和碳酸氢钠溶液洗涤，干燥有机相，过滤并浓缩，所得粗产品经C18反相柱(规格：30μm；市售，购买于上海博蕴生物科技有限公司)(MeCN:H2O＝75％:25％)制备得到白色固体I-1-7(2.03g,1.84mmol,70％收率)。化合物I-1-7分子式:C56H76O15N6P,分子量:1105.5,LC-MS实测：1128.4(M+Na).1H NMR(400MHz,DMSO-d6):δ8.12–8.06(m,1H),7.78(dd,J＝9.1,2.0Hz,1H),7.35–7.28(m,4H),7.23–7.17(m,5H),6.88(d,J＝7.5Hz,4H),5.21(d,J＝3.3Hz,1H),4.97(dd,J＝11.2,3.0Hz,1H),4.86–4.75(m,1H),4.52(s,1H),4.47(d,J＝8.5Hz,1H),4.04–3.98(m,3H),3.91–3.83(m,1H),3.74(s,6H),3.70–3.60(m,3H),3.58–3.47(m,3H),3.39(t,J＝11.7Hz,2H),3.31(d,J＝4.1Hz,1H),3.19(qd,J＝8.7,3.8Hz,1H),3.02(dt,J＝19.9,6.8Hz,1H),2.78–2.70(m,1H),2.60(td,J＝5.8,1.8Hz,1H),2.09(s,5H),1.98(s,4H),1.89(s,3H),1.73(s,3H),1.44(s,4H),1.20–0.88(m,14H).31P NMR(162MHz,DMSO-d6):δ148.22(s),146.82(t,J＝35.8Hz).
二、siRNA缀合物制备
通过固相亚磷酰胺法，利用上述步骤制备的化合物SA51起始循环，按照核苷酸排布顺序自3’-5’方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。正义链和反义链采用相同的合成条件。
仪器设备型号：Biolytic Dr.Oligo 48固相合成仪，逗点生物Embed CPG Frits通用合成柱DS0200，逗点生物96孔板脱盐柱DC189650(80mg)。表1为合成siRNA缀合物使用的试剂。
表1
合成条件如下：
核苷单体以0.05M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为25℃，反应时间为3分钟，脱保护试剂为DCA，进样体积180μL。
每一步偶联反应条件均相同，包括温度为25℃，反应时间为3分钟。核苷单体进样体积90μL，催化剂ACT进样体积110μL。
每一步盖帽条件均相同，包括温度为25℃，反应时间为2分钟。盖帽试剂溶液的摩尔比为1:1的CapA和CapB(CapB1：CapB2＝1:1)的混合溶液。盖帽试剂进样体积180μL。
每一步氧化反应条件均相同，包括温度为25℃，反应时间为3分钟，氧化试剂OXD进样体积为180μL。
每一步硫化反应条件均相同，包括温度为25℃，反应时间为4分钟，硫化试剂为0.05M PADS的吡啶乙腈溶液。硫化试剂进样体积180μL。
待最后一个核苷单体连接完成后，依次对固相载体上连接的核酸序列进行切割、脱保护、纯化、脱盐，随后冻干获得正义链和反义链，其中：
切割和脱保护条件如下：将合成的连接有载体的核苷酸序列加入氨水：乙醇＝3:1的混合溶液至体积为0.8mL。在50℃反应15h，过滤除去剩余载体，将上清液真空浓缩至干。
纯化和脱盐条件如下：利用C18反相色谱柱进行脱盐。具体条件包括：
(1)样品的准备
向寡核苷酸样品中加入0.1M的TEAA(三乙胺醋酸盐)至体积为0.8mL。
(2)96孔板的活化
活化：0.8mL乙腈通过96孔板的每个孔中进行活化；
平衡：用0.8mL TEAA(pH 7.0)溶液进行96孔板的平衡。
(3)纯化过程依次按照如下操作：
将0.8mL包含寡核苷酸的溶液通过脱盐柱；
用0.8mL 6.5％氨水洗涤96孔板2次，去除失败的序列；
用0.8mL去离子水冲洗96孔板2次，去除盐分；
用0.8mL 3％三氟乙酸冲洗96孔板3次，去除DMT，观察到吸附层变橙红色；
用0.8mL 0.1M TEAA冲洗96孔板；
用0.8mL去离子水冲洗96孔板2次，去除三氟乙酸和残余的盐分；
用0.6mL 20％乙腈进行洗脱，并收集冻干。
检测方法如下：使用Waters Acquity UPLC-LTQ LCMS(column：ACQUITY UPLC BEH C18)检测上述正义链和反义链纯度并分析分子量。实测值与理论值相符，表明所合成的是3’端和/或5’端缀合了基团的正义链以及反义链。
退火操作如下：将步骤2合成获得的正义链和反义链分别溶于注射用水中，配制0.1mg/mL-40mg/mL的溶液，用浓度仪标定等摩尔比混合，90℃加热5分钟，再缓慢自然降温，使它们通过氢键形成双链结构，取样送检测产品的SEC纯度。将双链样品冻干。
实施例3 Ago单体、Cgo单体、Ugo单体的合成及含该单体修饰的siRNA的制备
3.1.Ago单体的合成路线参见本申请人的专利申请号202310436173.7的实施例2，具体合成方法如下：
(1)将无水二异丙胺(16.2g,160.0mmol,22.6mL,2.0equiv)溶于350mL无水四氢呋喃中，降温至零下70℃至零下78℃之间，氮气置换保护，滴加正丁基锂溶液(2.5M,67.1mL)(缓慢滴加，滴加过程至少10分钟以上)，反应液在零下70℃至零下78℃之间继续搅拌半小时。将化合物1(9.44g,79.9mmol,9.17mL,1.0equiv)溶于175mL无水四氢呋喃中，降温至零下70℃至零下78℃之间，氮气置换保护下，滴加前一步所得溶液(缓慢滴加，滴加过程至少10分钟以上)。此反应液在零下70℃至零下78℃之间继续搅拌半小时。维持之前温度，将六甲磷酰三胺(26.1g,146mmol,25.6mL,1.82equiv)与苄基氯甲基醚(17.5g,112mmol,15.5mL,1.4equiv)缓慢滴加到此前反应液中(缓慢滴加，滴加过程至少10分钟以上)，滴加完毕，升温至0℃继续搅拌3小时。TLC与LCMS检测化合物1消失。分两批加入600mL饱和氯化铵溶液淬灭反应，200mL甲基叔丁基醚萃取混合物，收集有机相，饱和食盐水洗涤有机相，无水硫酸钠干燥有机相，过滤之后，浓缩滤液，所得粗产品用硅胶柱层析分离纯化(石油醚/乙酸乙酯＝100/1到1/1)，得到淡褐色化合物2(9.69g,40.7mmol,51％收率).1H NMR(400MHz,CDCl3):δ7.46-7.28(m,10H),4.58-4.46(m,2H),4.19-4.08(m,1H),3.82-3.67(m,5H),2.77(q,J＝6.1Hz,1H),1.24(d,J＝6.5Hz,4H).LC-MS:C13H18O4，分子量238.1,239.1(M+H).
(2)将化合物2(14.7g,61.7mmol,1.0equiv)溶于150mL二氯甲烷中，氮气保护，室温下加入咪唑(16.8g,247.0mmol,4.0equiv)与叔丁基二甲基氯硅烷(27.9g,185.0mmol,22.7mL,3.0equiv)。反应液在室温下搅拌一小时，TLC与LCMS检测显示化合物2消失。反应液中加入100mL二氯甲烷，400mL饱和食盐水洗涤反应液两次，干燥有机相，过滤后浓缩有机相，所得粗产品硅胶柱层析分离纯化(石油醚/乙酸乙酯＝100/1到10/1)，得到淡黄色油状物化合物3(12.0g,34.0mmol,55％收率)。1H NMR(400MHz,CDCl3):δ7.40-7.25(m,5H),4.52(d,J＝2.4Hz,2H),4.10(t,J＝6.3Hz,1H),3.75-3.65(m,4H),3.59(dd,J＝9.2,5.3Hz,1H),2.79(dt,J＝8.4,5.9Hz,1H),1.17(d,J＝6.2Hz,3H),0.86(s,9H),0.04(d,J＝8.6Hz,6H). LC-MS:C19H32O4Si，分子量352.1，353.2(M+H).
(3)将化合物3(11.1g,31.5mmol,1.0equiv)溶于四氢呋喃中，降温至零下70℃到零下60℃之间，氮气保护下滴加二异丁基氢化铝(1.0M,69.3mL,2.2equiv)。在此温度下继续搅拌两小时。LCMS检测显示化合物3消失，升温至0℃，加入20mL乙酸乙酯，再用100mL酒石酸钾钠溶液淬灭反应，继续搅拌半小时。此混合物用100mL饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得到淡黄色油状物化合物4(9.75g,30.0mmol,95％收率).1H NMR(400MHz,CDCl3):δ7.40-7.27(m,5H),4.59-4.45(m,2H),4.19(dd,J＝6.2,3.7Hz,1H),4.01(dd,J＝11.3,4.0Hz,1H),3.78-3.60(m,3H),1.78-1.68(m,1H),1.29-1.21(m,3H),0.95-0.87(m,9H),0.12-0.02(m,6H).LC-MS:C18H32O3Si，分子量324.1，325.2(M+H).
(4)将化合物4(9.75g,30.0mmol,1.0equiv)溶于四氢呋喃中，降温至0℃，氮气保护下滴加对甲苯磺酰氯(11.5g,60.1mmol,2.0equiv)与甲基咪唑(6.17g,75.1mmol,5.99mL,2.5equiv)，滴加完毕升温至室温继续搅拌16小时。LCMS检测显示化合物4消失。反应液中加入20mL乙酸乙酯，冰浴下用100mL酒石酸钾钠溶液淬灭反应，继续搅拌半小时。此混合物用100mL饱和食盐水洗涤后，无水硫酸钠干燥，过滤浓缩得到淡黄色油状物化合物5(13.7g,28.6mmol,95％收率)粗产品。1H NMR(400MHz,CDCl3):δ7.89-7.68(m,2H),7.42-7.17(m,10H),4.47-4.33(m,2H),4.23(dd,J＝9.6,5.1Hz,1H),4.13-4.06(m,1H),4.01-3.94(m,1H),3.52-3.34(m,2H),2.43(s,3H),2.02-1.89(m,1H),1.10(d,J＝6.3Hz,3H),0.81(s,8H),0.04--0.05(m,6H).LC-MS:C25H38O5SSi,分子量478.1,479.2(M+H).
(5)氮气保护下，向干燥的反应瓶中加入化合物5(13.7g,28.6mmol,1.0equiv)与35mL乙腈，35-40℃下蒸除乙腈，除去化合物5中水分。在氮气保护下，另一洁净干燥的反应瓶中加入80mL N,N-二甲基甲酰胺，化合物5-1(9H-嘌呤-6-胺)(4.25g,31.5mmol,1.1equiv)与碳酸钾(3.96g,28.6mmol,1.0equiv)，升温至95-100℃继续搅拌半小时。此温度下向反应液中滴加化合物5(13.7g,28.6mmol,1.0equiv)的N,N-二甲基甲酰胺(60mL)溶液，继续搅拌12小时。LCMS检测显示化合物5消失，反应液降至室温，加入200mL乙酸乙酯，此混合溶液依次用200mL碳酸氢钠溶液，100mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩滤液，得到的粗产品硅胶柱层析(石油醚/乙酸乙酯＝20/1到10/1)分离纯化，得到淡黄色油状物化合物6(6.2g,14.0mmol,49％收率)。1H NMR(400MHz,CDCl3):δ8.37(s,1H),7.75(s,1H),7.39-7.24(m,6H),5.66(br,s,2H),4.47-4.34(m,3H),4.21(dd,J＝14.1,8.8Hz,1H),4.07(dd,J＝6.2,4.9Hz,1H),3.42-3.34(m,2H),2.10-1.96(m,1H),1.19(d,J＝6.4Hz,3H),0.90(s,9H),0.06(d,J＝6.0Hz,6H).LC-MS:C23H35N5O2Si,分子量441.1,442.3(M+H)。
(6)室温下将钯碳(3.0g,10％纯度)溶于25mL甲醇中，加入25mL化合物6(5.2g,11.8mmol,1.0equiv)甲醇溶液和三氟乙酸(134.0mg,1.18mmol,87.2uL)。此反应液在50psi氢气压力下搅拌48小时。LCMS检测显示化合物6消失。将反应液过滤浓缩得到粗产物淡黄色油状物化合物7(3.9g,11.1mmol)。1H NMR(400MHz,CDCl3):δ8.21(s,1H),8.12(s,1H),4.50-4.36(m,1H),4.33-4.22(m,1H),4.15-4.02(m,1H),3.53(d,J＝5.9Hz,2H),2.21-2.09(m,1H),1.25(d,J＝6.4Hz,3H),0.88(s,9H),0.05(d,J＝3.1Hz,6H).LC-MS:C16H29N5O2Si,分子量351.1,352.2(M+1).
(7)将化合物7(3.9g,11.1mmol,1.0equiv)置于洁净干燥反应瓶中，氮气保护下加入10mL吡啶，升温至蒸除吡啶，重复一次此操作，以除去化合物7中水分。加入28mL吡啶，冰浴下加入三甲基氯硅烷(4.8g,44.4mmol,5.63mL,4.0equiv)，升温至室温，继续搅拌两小时，TLC检测显示化合物7消失，冰浴下加入苯甲酰氯(6.2g,44.4mmol,5.15mL,4.0equiv)，继续搅拌2小时，TLC检测显示原料反应消失，滴加60mL水与90mL浓氨水继续搅拌半小时。用250mL乙酸乙酯萃取反应液，有机相用饱和食盐水洗涤，干燥后抽滤浓缩，所得粗产品硅胶柱层析分离纯化(洗脱体系:二氯甲烷/甲醇＝50/1到10/1)，得到淡黄色油状物化合物8(4.0g,8.78mmol,79％收率).1H NMR(400MHz,CDCl3):δ9.22-8.90(m,1H),8.79(s,1H),8.07-8.04(m,2H),8.03(d,J＝1.4Hz,1H),7.65-7.59(m,1H),7.57-7.50(m,2H),4.62-4.50(m,1H),4.42(dd,J＝14.3,8.6Hz,1H),4.24-4.09(m,1H),4.05(d,J＝6.3Hz,1H),3.59-3.48(m,1H),3.47-3.35(m,1H),2.04-1.98(m,1H),1.31(d,J＝6.3Hz,3H),0.98-0.84(m,9H),0.11(d,J＝10.1Hz,5H).LC-MS: C23H33N5O3Si,分子量455.2,456.3(M+H).
(8)将化合物8(4.0g,8.78mmol,1.0equiv)置于洁净干燥反应瓶中，氮气保护下加入10mL吡啶，升温蒸除吡啶，重复一次此操作，以除去化合物8中水分。加入28mL吡啶，冰浴下加入4,4'-双甲氧基三苯甲基氯(3.27g,9.66mmol,1.1equiv)，升至室温继续搅拌1小时，TLC检测显示化合物8消失，反应混合物中加入150mL乙酸乙酯，并用200mL碳酸氢钠溶液，150mL饱和食盐水洗涤，干燥有机相，过滤有机相并浓缩滤液，所得粗产品硅胶柱层析分离纯化(梯度洗脱：石油醚/乙酸乙酯＝10/1到0/1)，得到淡黄色油状物化合物9(5.91g,7.8mmol,88％收率)。1H NMR(400MHz,CDCl3):δ9.07(br s,1H),8.82(s,1H),8.04(d,J＝7.1Hz,2H),7.80(s,1H),7.65-7.58(m,1H),7.57-7.50(m,2H),7.28-7.13(m,9H),6.81-6.69(m,4H),4.52-4.43(m,1H),4.25(dd,J＝14.2,8.6Hz,1H),4.07(dd,J＝6.3,4.2Hz,1H),3.76(d,J＝4.5Hz,6H),3.22(dd,J＝9.8,5.4Hz,1H),3.04(dd,J＝9.8,6.1Hz,1H),2.34(dd,J＝8.1,4.5Hz,1H),1.19(d,J＝6.3Hz,3H),0.90-0.77(m,9H),0.07--0.09(m,6H).LC-MS:C44H51N5O5Si,分子量757.3,758.4(M+H).
(9)将化合物9(3.00g,3.96mmol,1.0equiv)溶于四氢呋喃中，氮气保护、冰浴下加入氢氟酸吡啶盐(2.26g,79.2mmol,2.1mL,20.0equiv)和咪唑(10.8g,158.0mmol,40.0equiv)。升温至室温，继续搅拌2小时。LCMS检测显示化合物9消失。加入50mL乙酸乙酯，并用100mL碳酸氢钠溶液，50mL饱和食盐水洗涤，干燥后过滤，浓缩滤液，所得粗产品硅胶柱层析分离纯化(梯度洗脱：石油醚/乙酸乙酯＝10/1到0/1)，得到淡黄色油状物化合物10(2.44g,3.79mmol,96％收率)。1H NMR(400MHz,DMSO-d6):δ11.16(br s,1H),8.70(s,1H),8.33(s,1H),8.05(d,J＝7.4Hz,2H),7.69-7.61(m,1H),7.60-7.52(m,2H),7.25-7.12(m,5H),7.05(t,J＝8.9Hz,4H),6.78(dd,J＝8.9,3.1Hz,4H),4.79(d,J＝4.0Hz,1H),4.51-4.28(m,2H),3.81(d,J＝4.0Hz,1H),3.70(s,6H),3.10(dd,J＝9.8,4.9Hz,1H),2.89(dd,J＝9.7,5.1Hz,1H),2.37-2.23(m,1H),1.03(d,J＝6.3Hz,3H).LC-MS:C38H37N5O5,分子量643.2,644.2(M+H).
(10)将化合物10(1.5g,2.33mmol,1.0equiv)置于洁净干燥反应瓶中，氮气保护下加入4mL乙腈，升温至35-40℃蒸除乙腈，重复一次此操作，以除去化合物10中水分。室温下向上述反应瓶中加入15mL无水二氯甲烷。再加入化合物11-1(1.05g,3.5mmol,1.11mL,1.5equiv)与4,5-二氰咪唑(358mg,3.03mmol,1.3equiv)，室温下继续搅拌一小时。TLC检测显示化合物10消失。加入30mL二氯甲烷，用碳酸氢钠溶液(50mLx2)，饱和食盐水(50mL)洗涤，干燥有机相，过滤并浓缩滤液，所得粗产品溶于50mL甲基叔丁基醚中，加入100mL 1％的氢氧化钠水溶液，搅拌半小时。静置分层收集有机相，有机相用饱和食盐水洗涤，干燥后过滤浓缩，粗产品经C18反相柱制备得到淡黄色产物SA000001(1.1g,1.3mmol,56％收率)。1H NMR(400MHz,CD3CN):δ9.33(br s,1H),8.61(br s,1H),7.99(d,J＝11.0Hz,3H),7.67-7.60(m,1H),7.59-7.49(m,2H),7.32-6.99(m,9H),6.75(td,J＝9.2,6.5Hz,4H),4.50-4.38(m,1H),4.36-4.13(m,2H),3.72(dd,J＝4.8,1.5Hz,6H),3.66-3.45(m,3H),3.25(td,J＝9.6,5.4Hz,1H),3.01(ddd,J＝17.9,10.0,5.8Hz,1H),2.62(t,J＝5.9Hz,1H),2.53(t,J＝5.9Hz,1H),1.32-1.22(m,4H),1.17-1.05(m,12H).31P NMR(DMSO-d6,162MHz):δppm 147.7,146.8.LCMS:C47H54N7O6P,分子量843.3,844.5(M+H).
3.2.Cgo单体的合成路线参见本申请人的专利申请号202310436173.7的实施例4，具体合成方法如下：
(1)在室温条件下将化合物3-2(8.5g,20.3mmol，由实施例5制备)和1,2,4-三氮唑(19.5g,282.0mmol,13.9equiv)溶解于吡啶(128.0mL)中，随后在冰水浴条件下向反应体系中滴加4-氯苯基二氯磷酸酯(56.9mmol,9.25mL,2.8equiv)。将反应液在30℃条件下搅拌16小时。反应完毕减压蒸馏除去溶剂吡啶，剩余体系用水和乙酸乙酯萃取反应液3次，将有机相合并旋干得到黄色油状的粗产品化合物C-1(9.5g)，直接用于下一步反应。化合物C-1的质谱鉴定(C24H35N5O3Si，分子量469.1，[M+H]＝470.3).
(2)在室温条件下将化合物C-1(9.5g,20.2mmol)溶解于1,4-二氧六环(95.0mL)中，随后向反应体系中加入氨水(1.38mol,212.1mL,25％purity,68.0equiv)。将反应液在30℃条件下搅拌16小时。反应完毕减压蒸馏除去溶剂，剩余体系用水和二氯甲烷萃取反应液3次，将有机相合并旋干得到黄色固体的粗产品化合物C-2(8.5g)，直接用于下一步反应。化合物C-2的质谱鉴定(C22H35N3O3Si，分子量417.1，[M+H]＝418.2).
(3)在室温条件下将化合物C-2(8.5g,20.2mmol)溶解于N,N-二甲基甲酰胺(85.0mL)中，随后向反应体系中缓慢加入醋酸酐(3.1g,30.4mmol,2.85mL,1.5equiv)。将反应液在30℃条件下搅拌3小时。反应完毕减压蒸馏除去溶剂，剩余体系用饱和碳酸氢钠溶液和乙酸乙酯萃取反应液3次，将有机相合并旋干，得到的粗产品用硅胶柱层析(二氯甲烷/甲醇1:0至100:1)进行分离纯化，得到黄色固体化合物C-3(4.2g，反应收率43％)。化合物C-3的1H NMR数据:(400MHz,DMSO-d6)δ10.7(s,1H),7.79(d,J＝7.2Hz,1H),7.29(m,5H),7.08(d,J＝7.2Hz,1H),4.36(d,J＝1.2Hz,2H),4.05-3.99(m,2H),3.72-3.67(m,1H),3.39(dd,J＝6.4Hz,2H),2.15-2.08(m,1H),2.08(s,3H),1.15(d,J＝6.4Hz,3H),0.85(s,9H),0.02(d,J＝6.8Hz,6H)。化合物C-3的质谱鉴定(C24H37N3O4Si，分子量459.1，[M+H]＝460.2).
(4)在-78℃条件下将化合物C-3(4.3g,9.35mmol)溶解于二氯甲烷(46.0mL)中，随后向反应体系中缓慢加入三氯化硼(1.0M in DCM,46.8mL,5.0equiv)。随后将反应液在-78℃条件下搅拌4小时。反应完毕用三乙胺(40.0mL)和甲醇(88.0mL)淬灭反应，剩余体系用水和二氯甲烷萃取反应液3次，将有机相合并旋干，得到的粗产品用C18反相柱制备得到白色固体化合物C-4(0.96g，反应收率40％)。化合物C-4的1H NMR数据:(400MHz,DMSO-d6)δ10.7(s,1H),8.01(d,J＝7.2Hz,1H),7.12(d,J＝7.2Hz,1H),4.60(d,J＝4.8Hz,2H),4.53-4.50(m,1H),4.00-3.99(m,1H),3.75-3.70(m,2H),3.37-3.32(m,2H),2.08(s,3H),1.81-1.77(m,1H),1.01(d,J＝6.4Hz,3H)。化合物C-4的质谱鉴定(C11H17N3O4，分子量255.1，[M+H]＝256.2).
(5)在室温条件下将化合物C-4(1.5g,5.88mmol)溶解于吡啶(10.0mL)中，冰水浴条件下向反应体系中加入4,4'-双甲氧基三苯甲基氯(2.4g,7.05mmol,1.2equiv)。随后将反应液在室温条件下搅拌4小时。反应完毕减压蒸馏除去溶剂吡啶，剩余体系用饱和碳酸氢钠溶液和乙酸乙酯萃取反应液3次，将有机相合并旋干，得到的粗产品用硅胶柱层析(石油醚/乙酸乙酯4:1至0:1)进行分离纯化，得到白色固体化合物C-5(0.7g，反应收率70％)。化合物C-5的1H NMR数据:(400MHz,CDCl3)δ9.71(s,1H),7.39(d,J＝7.3Hz,3H),7.35-7.21(m,7H),7.06(d,J＝7.1Hz,1H),6.85(d,J＝8.8Hz,4H),4.71(d,J＝3.3Hz,1H),4.43(dd,J＝13.8,4.5Hz,1H),4.23-3.99(m,2H),3.80(s,6H),3.50-3.38(m,1H),3.31(dd,J＝10.0,4.1Hz,1H),2.69(t,J＝9.4Hz,1H),2.21(s,3H),1.08(d,J＝6.1Hz,3H)。化合物C-5的质谱鉴定(C32H35N3O6，分子量557.1，[M+H]＝558.4).
(6)在室温氮气条件下将化合物C-5(2.0g,3.59mmol)溶解于二氯甲烷(20.0mL)中，分别向反应体系中加入4,5-二氰基咪唑(466.0mg,3.95mmol,1.1equiv)和双(二异丙基氨基)(2-氰基乙氧基)膦(1.4g,4.66mmol,1.48mL,1.3equiv)。随后将反应液在室温条件下搅拌2小时。反应完毕将反应液溶解于甲基叔丁基醚(10.0mL)和1％氢氧化钠溶液(10.0mL)，室温条件下继续搅拌0.5小时。剩余体系用饱和碳酸氢钠溶液和二氯甲烷萃取反应液3次，将有机相合并旋干，得到的粗产品用C18反相柱制备得到白色固体化合物SA000015(1.5g，反应收率55％)。化合物SA000015的1H NMR数据:(400MHz,CD3CN)δ9.15(s,1H),7.50-7.18(m,11H),6.84-6.80(m,4H),4.22-4.08(m,2H),3.75-3.22(m,11H),3.23-3.22(m,2H),2.62-2.51(m,2H),2.38-2.35(m,1H),2.12(s,3H),1.022-1.07(m,15H).31P NMR数据:(400MHz,CD3CN)δ147.5,146.7。化合物SA000015的质谱鉴定(C41H52N5O7P，分子量757.1，[M+H]＝758.5).
3.3.Ugo单体的合成路线参见本申请人的专利申请号202310436173.7的实施例5，具体合成方法如下：
(1)在冰水浴条件下分别将化合物1-4(9.99g,30.8mmol)、化合物U-1(市售，购买于上海皓鸿生物医药科技有限公司)(9.33g,43.1mmol,1.4equiv)和三苯基膦(16.6g,63.2mmol,2.0equiv)溶解于干燥的四氢呋喃(350.0mL)中，随后向反应体系中滴加偶氮二甲酸二异丙酯(13.1g,64.7mmol,12.6mL,2.1equiv)。随后将反应液在室温条件下搅拌2小时。反应完毕用饱和碳酸氢钠溶液和乙酸乙酯萃取反应液3次，将有机相合并旋干，得到的粗产品用硅胶柱层析(石油醚/乙酸乙酯10:1至8:1)进行分离纯化，得到白色固体化合物3-1(16.1g，反应收率99％)。化合物3-1的1H NMR数据:(400MHz,DMSO-d6)δ7.89-7.74(m,4H),7.56-7.53(m,2H),7.33-7.29(m,5H),5.80-5.73(m,1H),4.46-4.38(m,2H),4.00-3.91(m,2H),3.60-3.50(m,1H),3.49-3.43(m,2H)2.17-1.98(m,1H),1.13(s,3H),0.83(s,9H),0.01(d,J＝6.0Hz,6H)。化合 物3-1的质谱鉴定(C29H38N2O5Si，分子量522.1，[M+H]＝523.2).
(2)室温条件下将化合物3-1(8.05g,15.4mmol)溶解于甲醇(160.0mL)中，随后向反应体系中缓慢加入甲醇钠(2.77g,15.4mmol,30％purity,1.0equiv)。随后将反应液在室温条件下搅拌12小时。反应完毕减压蒸馏除去溶剂甲醇，剩余体系用1M盐酸溶液和乙酸乙酯萃取反应液3次，将有机相合并旋干，得到的粗产品用硅胶柱层析(石油醚/乙酸乙酯10:1至0:1)进行分离纯化，得到白色固体化合物3-2(6.1g，反应收率94％)。化合物3-2的1H NMR数据:(400MHz,DMSO-d6)δ11.1(s,1H),7.48(d,J＝8.0Hz,1H),7.30-7.24(m,5H),5.47-5.44(m,1H),4.45-4.35(m,2H),3.98-3.94(m,1H),3.79-3.78(m,1H),3.63-3.57(m,1H),3.38-3.35(m,2H),2.04-1.95(m,1H),1.10(s,3H),0.83(s,9H),0.01(d,J＝6.0Hz,6H)。化合物3-2的质谱鉴定(C22H34N2O4Si，分子量418.1，[M+H]＝418.2).
(3)在-70℃条件下将化合物3-2(4.0g,9.56mmol)溶解于二氯甲烷(30.0mL)中，随后向反应体系中缓慢加入三氯化硼(1.0M in DCM,66.9mL,7.0equiv)。随后将反应液在-70℃条件下搅拌3小时。反应完毕用三乙胺(5.0mL)和甲醇(30.0mL)淬灭反应，剩余体系用水和二氯甲烷萃取反应液3次，将有机相合并旋干，得到的粗产品用C18反相柱制备得到白色固体化合物3-3(0.7g，反应收率33％)。化合物3-3的1H NMR数据:(400MHz,DMSO-d6)δ7.54(d,J＝8.0Hz,1H),5.51(d,J＝7.6Hz,1H),3.88-3.84(m,1H),3.72-3.70(m,1H),3.61-3.59(m,2H),1.75-1.68(m,1H),1.09(d,J＝6.4Hz,3H)。化合物3-3的质谱鉴定(C9H14N2O4，分子量214.1，[M+H]＝215.2).
(4)在室温条件下将化合物3-1(0.4g,1.87mmol)溶解于吡啶(4.0mL)中，冰水浴条件下向反应体系中加入4,4'-双甲氧基三苯甲基氯(949.0mg,2.8mmol,1.5equiv)。随后将反应液在室温条件下搅拌2小时。反应完毕减压蒸馏除去溶剂吡啶，剩余体系用饱和碳酸氢钠溶液和乙酸乙酯萃取反应液3次，将有机相合并旋干，得到的粗产品用硅胶柱层析(石油醚/乙酸乙酯4:1至0:1)进行分离纯化，得到白色固体化合物3-4(0.7g，反应收率70％)。化合物3-4的1H NMR数据:(400MHz,DMSO-d6)δ11.3(s,1H),7.37(d,J＝8.0Hz,1H),7.33-7.29(m,4H),7.20-7.17(m,5H),6.86-6.83(m,4H),5.40(d,J＝8.0Hz,1H),4.61(d,J＝4.8Hz,2H),3.83-3.79(m,2H),3.72(s,6H),3.71-3.68(m,1H),3.08-2.90(m,2H),1.90(s,1H),1.01(d,J＝6.4Hz,3H)。化合物3-4的质谱鉴定(C30H32N2O6，分子量516.1，[M+H]＝517.4).
(5)在室温氮气条件下将化合物3-4(1.2g,2.32mmol)溶解于二氯甲烷(12.0mL)中，分别向反应体系中加入4,5-二氰基咪唑(357.0mg,3.02mmol,1.3equiv)和双(二异丙基氨基)(2-氰基乙氧基)膦(1.05g,3.48mmol,1.5equiv)。随后将反应液在室温条件下搅拌2小时。反应完毕将反应液溶解于甲基叔丁基醚(10.0mL)和1％氢氧化钠溶液(10.0mL)，室温条件下继续搅拌0.5小时。剩余体系用乙酸乙酯萃取反应液3次，将有机相合并旋干，得到的粗产品用C18反相柱制备得到白色固体化合物SA000016(1.2g，反应收率71％)。化合物SA000016的1H NMR数据:(400MHz,CD3CN)δ9.39(s,1H),7.39-7.24(m,10H),6.85-6.81(m,4H),5.43-5.39(m,1H),4.21-4.18(m,1H),3.86-3.77(m,1H),3.75(s,6H),3.56-3.53(m,5H),3.28-3.05(m,2H),2.62-2.61(m,2H),2.22-2.19(m,2H),1.22-1.07(m,15H).31P NMR数据:(400MHz,CD3CN)δ147.6,146.5。化合物SA000016的质谱鉴定(C39H49N4O7P，分子量716.1，[M+H]＝717.5).
3.4修饰siRNA的制备
反义链的5’区域的第6或7位中的核苷酸位置处包含Ago、Cgo、Ugo所示的化学修饰，该修饰的siRNA参照实施例2的固相合成获得。
发明人通过前期设计和合成了大量PD-L1siRNA，并经过细胞活性验证，排除了一些效果不佳的PD-L1siRNA，保留部分活性较好的PD-L1siRNA进行进一步实验，具体序列参见下表2至4。
下表2为PD-L1siRNA的未修饰的正义链及反义链，下表3为PD-L1siRNA的经过修饰的正义链和反义链(该表从上至下分别为表1中各siRNA对应的修饰序列)，下表4为siRNA缀合物序列。其中，大写字母A、C、G、U分别表示腺苷-3'-磷酸、胞苷-3'-磷酸、鸟苷-3'-磷酸、尿苷-3'-磷酸；小写字母m表示该字母m左侧相邻的一个核苷酸为2’-甲氧基修饰的核苷酸；小写字母f表示左侧相邻的一个核苷酸为2’-氟代修饰的核苷酸；小写字母s在大写字母中间表示s左右相邻的两个核苷酸之间为硫代磷酸酯基链接； s在3’端第一个时表示与该字母s左侧相邻的一个核苷酸末端为硫代磷酸酯基。SA51的合成与缀合物合成见实施例2。
表2
表3


表4

实施例4IC50体外筛选：双萤光素酶测定法
在体外利用双萤光素酶测定法测试靶向PD-L1的siRNA的活性。siRNA初步转染的终浓度为10nM，对10nM浓度下活性剩余百分比小于35％的化合物进行两浓度1nM和0.2nM活性筛选，从经过两浓度筛选出的优选化合物进行IC50筛选。
1)细胞培养和质粒/siRNA共转染
HEK293细胞培养于10％胎牛血清的DMEM高糖培养基(Gibco，C11995500BT)中，在37℃，5％CO2条件下，生长至汇合度90％左右，然后通过胰蛋白酶(Thermo，25200072)消化，将细胞重悬。将完整的人PD-L1参考序列(NM_014143.4)克隆到双荧光素酶psiCHECK2TM载体多克隆位点XhoI和NotI之中，构建hs-psiCHECK质粒。使用LipofectamineTM2000Transfection Reagent(Thermo，11668500)将双荧光素酶质粒与siRNA共转染进1x104个细胞中。采用96孔板，每孔将0.3μL的LipofectamineTM2000加入到含有20ng质粒载体和siRNA的19.7μLOpti-MEM(Gibco，31985070)中，并使其在室温下孵育15分钟，将混合物加入96板中。然后加入重悬于80μL新鲜含10％胎牛血清的DMEM高糖培养基(Gibco，C11995500BT)的细胞。将细胞孵育24小时，检测荧光素酶(翊圣生物，11405ES80)。
2)双萤光素酶测定
在siRNA转染24小时后，测量双荧光素酶即萤火虫荧光素酶(内参对照)和海肾荧光素酶(PolyA后插入PD-L1靶序列，融合成一条mRNA)。参照产品说明手册(翊圣生物，11405ES80)，首先在多功能酶标仪(Bioteck，Synergy LX)上检测萤火虫萤光素酶发光信号，然后检测肾萤光素酶发光信号。海肾萤光素酶在微孔板上的检测顺序应与萤火虫萤光素酶的检测顺序相同。
将各孔的海肾荧光素酶信号读值对萤火虫荧光素酶(对照)信号进行比值均一化，然后与转染有相同质粒但未经siRNA处理的细胞比较，评估各siRNA的活性。所有转染设置复孔。
结果以相对于未加siRNA的对照组(对照组为100％)的剩余百分比表示，数据见表5。从表5可见，除了SD003798和SD003955以外，其他各组的实验结果较佳，对效果较好的siRNA进行进一步研究。
表5
实施例5 siRNA在SNU-387细胞中的活性，IC50体外筛选
在SNU-387细胞中采用8个浓度(10nM开始3倍稀释，共8个浓度点)开展IC50体外筛选。
SNU-387细胞培养于10％胎牛血清的RPMI-1640培养基(Thermo，11875119)中，培养条件37℃，5％CO2，然后通过胰蛋白酶消化，将细胞重悬。使用RNAiMAX(Thermo，13778150)将siRNA共转染进2x104个细胞中。采用96孔板，每孔将0.3μL的RNAiMAX加入到含有不同浓度的siRNA的19.7μL Opti-MEM(Gibco，31985070)中，并使其在室温下孵育15分钟，将混合物加入96孔板中，然后加入重悬于80μL新鲜含10％胎牛血清的RPMI-1640培养基(Thermo，11875119)中的细胞。将细胞孵育24小时，利用组织细胞提取试剂盒(志昂生物，MNTR/FX96)提取RNA，进行反转录cDNA(Takara，6210B)，通过SYBR Green qPCR(Vazyme,Q711)测量PD-L1基因的表达水平，具体操作方法见相应说明书。
目的基因PD-L1引物和探针：
正向引物：TGCAGGGCATTCCAGAAAGAT(SEQ ID NO.29)；
反向引物：CCTTGGGAACCGTGACAGTA(SEQ ID NO.30)；
内参基因β-Actin引物：
正向引物：TGCACCACCAACTGCTTAGC(SEQ ID NO.31)；
反向引物：ACTGTGGTCATGAGTCCTTCCA(SEQ ID NO.32)；
与未经siRNA处理的细胞比较计算不同浓度下的相对水平，并计算IC50值，结果见表6和图1。结果显示，SD003141、SD003143、SD003187、SD003842、SD003844、SD003887活性相对较好。
表6
实施例6包含不同化学修饰的siRNA体外在靶和脱靶评估
利用双荧光素酶报告基因检测不同化学修饰的siRNA在靶活性和脱靶活性。
HEK293细胞培养于含10％胎牛血清的DMEM高糖培养基中，在37℃，5％CO2条件下培养。参照产品说明手册，利用Lipofectamine 2000(ThermoFisher，11668019)将报告基因质粒和不同浓度(终浓度90nM开始,3倍稀释，共11个浓度)的siRNA共同转染至细胞中。转染24小时后利用双荧光素酶检测试剂盒(翊圣生物，11405ES80)进行检测。在报告子质粒的海肾荧光素酶的3’非翻译区分别插入与反义链互补配对的序列(为在靶报告质粒)和含有与正义链5’端12-19位点一致的五个串联的序列(为脱靶报告质粒)。报告子质粒中都含有萤火虫荧光素酶，作为内参。只转染质粒的组作为对照组。将各孔的海肾荧光素酶信号读值对萤火虫荧光素酶(对照)信号进行比值均一化，然后与转染有相同质粒但未经siRNA处理的细胞比较计算不同浓度下的相对水平，并计算IC50值，结果见表7。结果显示，部分序列在反义链的5’区域的第6或7位中的核苷酸进行修饰不影响活性的同时改善脱靶作用，提高了siRNA的安全性。
表7

实施例7包含不同化学修饰的siRNA在HDI小鼠体内活性
在本实施例中，综合体外在靶和脱靶评估结果，选取siRNA进行缀合合成，在HDI小鼠模型中测评体内活性。siRNA缀合物合成为实施例2通过固相合成获得。
在缀合物给药前至少14天，对六至八周龄雌性Balb/C小鼠(浙江维通利华)通过高压尾静脉注射构建人源化表达PD-L1的小鼠，在5-7秒内通过尾静脉将2μg含有PD-L1mRNA序列的质粒注射至小鼠中，以产生PD-L1-SEAP模型小鼠(Zhang G等人,“High levels of foreign gene expression inhepatocytes after tail vein injection of naked plasmid DNA.”Human GeneTherapy 1999Vol.10,p1735-1737.)。PD-L1siRNA缀合物对PD-L1表达的抑制导致分泌型碱性磷酸酶(SEAP)表达的抑制。在给药前1天，通过眼眶采血，收集血清，根据产品说明书，用Phospha-Light SEAP报导基因分析系统(Invitrogen)来测量血清中的SEAP表达水平，且根据平均SEAP水平对小鼠进行分组。其中实验组小鼠给予缀合物，溶媒组小鼠给予磷酸盐 缓冲盐水(PBS)，按照每只小鼠给予3mg/kg缀合物的剂量进行皮下给药。给药后7天、28天再次采血，收集血清，并根据产品说明书，用Phospha-Light SEAP报导基因分析系统(Invitrogen)来测量血清中的SEAP表达水平。将各动物给药后的SEAP水平除以该动物给药前的SEAP水平，以确定“针对处理前归一化”的表达比率。
结果以各组给药前后血清SEAP的剩余表达水平表示(溶媒组为100％)，相对剩余表达水平结果如表8和图2所示，在给药后7天缀合物SD004177、SD004178、SD004179、SD004240、SD004180、SD004241、SD004242、SD004181、SD004182活性较好，抑制率大于80％，给药后28天，SD004178和SD004180化合物仍然保持大于80％的活性，体现了长效性。
表8
本实施例利用HDI模型，可在体内检测基于人PD-L1基因水平，从而能够更好的预测人源PD-L1mRNA体内活性，为药物的临床试验以及药物上市提供更多且更可靠的实验数据。
实施例8 siRNA在hPD-1/PD-L1人源化小鼠AAV-HBV(乙型肝炎病毒)模型中体内药效
在本实例中，评估siRNA在hPD-1/PD-L1人源化小鼠AAV-HBV模型中的体内药效，测试抗乙肝病毒活性。siRNA缀合物合成为实施例2通过固相合成获得。
缀合物给药前6周，构建hPD-1/PD-L1人源化小鼠AAV-HBV模型。通过向6-8周龄B6-hPD-1/PD-L1雌性小鼠尾部静脉注射rAAV8-1.3HBV，制备持续性HBV感染的小鼠动物模型。AAV病毒注射剂量为1E+11vg/只，注射病毒6周后对动物进行下颌下取血，取血后离心，离心条件为5000rpm，4℃，离心10分钟。用PBS稀释血清，需按照实际情况确定稀释倍数。然后将稀释后的样品送至北京迪安医学检验实验室有限公司进行检测HBsAg(乙型肝炎表面抗原)，其测定结果按照稀释倍数进行回算。HBsAg定量方法为直接电化学发光法。根据HBsAg水平分组，在第0天对动物分别皮下注射，注射剂量为7.5mg/kg体重，每周给药1次，在实验进展至第49天取血检测HBsAg水平，评估siRNA抗乙肝病毒的活性。
给药后49天HBsAg降低见表9，结果表明SD004178和SD004180均能显著降低HBsAg水平。
表9

以上对本发明做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明的精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (19)

  1. 一种siRNA，其包括正义链和反义链，其特征在于：所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，所述正义链包括选自SEQ ID NO.1至12所示核苷酸序列中的一种或上述序列不超过3个碱基突变的核苷酸序列，所述反义链包括选自SEQ ID NO.13至24所示核苷酸序列中的一种或上述序列不超过5个碱基突变的核苷酸序列。
  2. 根据权利要求1所述的siRNA，其特征在于：按照5’到3’的方向，所述正义链的碱基突变在其核苷酸序列的3’末端，所述反义链的碱基突变在其核苷酸序列的5’末端、第2～8位、3’端最后两位中的任意一位或多位。
  3. 根据权利要求1所述的siRNA，其特征在于：所述修饰的核苷酸为2’-甲氧基修饰的核苷酸、2’-氟代修饰的核苷酸、2’-O-CH2-CH2-O-CH3修饰的核苷酸、2’-O-CH2-CH＝CH2修饰的核苷酸、2’-CH2-CH2-CH＝CH2修饰的核苷酸、2’-脱氧基核苷酸、2’‐甲氧基乙基修饰的核苷酸、硫代磷酸酯键修饰的核苷酸、VP修饰的核苷酸、LNA、ENA、cET BNA、UNA、GNA和中的一种或多种的组合，其中，R1为H、OH或CH3，Base为天然核碱基、修饰的核碱基、通用碱基或H原子。
  4. 根据权利要求1至3中任一项所述的siRNA，其特征在于：所述正义链或所述反义链中的一个及以上的核苷酸为修饰的核苷酸。
  5. 根据权利要求1至3中任一项所述的siRNA，其特征在于：按照5’到3’的方向，所述正义链的第7、8和9位的核苷酸为2’-氟代修饰的核苷酸，所述正义链的其他位置的核苷酸为2’-甲氧基修饰的核苷酸；按照5’到3’的方向，所述反义链的第2、14和16位的核苷酸为2’-氟代修饰的核苷酸，所述反义链的除第2、6、7、14和16位以外的位置的核苷酸为2’-甲氧基修饰的核苷酸；所述siRNA中以下核苷酸之间的连接中至少一个为硫代磷酸酯基连接：
    所述正义链的5’端第1个核苷酸和第2个核苷酸之间的连接；
    所述正义链的5’端第2个核苷酸和第3个核苷酸之间的连接；
    所述正义链的3’端第1个核苷酸和第2个核苷酸之间的连接；
    所述正义链的3’端第2个核苷酸和第3个核苷酸之间的连接；
    所述反义链的5’端第1个核苷酸和第2个核苷酸之间的连接；
    所述反义链的5’端第2个核苷酸和第3个核苷酸之间的连接；
    所述反义链的3’端第1个核苷酸和第2个核苷酸之间的连接；
    所述反义链的3’端第2个核苷酸和第3个核苷酸之间的连接；
    所述正义链的3’末端为硫代磷酸酯基，所述反义链的5’末端为VP修饰的核苷酸。
  6. 根据权利要求1至3中任一项所述的siRNA，其特征在于：按照5’到3’的方向，所述反义链的第6位为2’-氟代修饰的核苷酸或所述反义链的第7位为2’-甲氧基修饰的核苷酸或 其中，R1为H、OH或CH3，Base为天然核碱基、修饰的核碱基、通用碱基或H原子。
  7. 根据权利要求6所述的siRNA，其特征在于：所述反义链的第6位或第7位的核苷酸为
  8. 根据权利要求1所述的siRNA，其特征在于：所述siRNA为如下表所示序列中的任一条：
  9. 根据权利要求1所述的siRNA，其特征在于：所述siRNA为如下表所示序列中的任一条：

  10. 根据权利要求1所述的siRNA，其特征在于：所述siRNA为如下表所示序列中的任一条：

  11. 一种siRNA缀合物，其特征在于：其包括权利要求1至10中任一项所述的siRNA中的一条或多条，以及缀合至所述siRNA的缀合基团。
  12. 根据权利要求11所述的siRNA缀合物，其特征在于：所述缀合基团来自化合物SA51，所述缀合基团中的化合物SA51的个数为2～4个，2～4个所述化合物SA51通过化学键依次连接；所述化合物SA51的结构式为：
  13. 根据权利要求11或12所述的siRNA缀合物，其特征在于：所述缀合基团连接在所述正义链的3’末端和/或5’末端。
  14. 根据权利要求11所述的siRNA缀合物，其特征在于：所述siRNA缀合物的结构式如下：
    其中，表示本公开的siRNA形成的双链体，X为O或S。
  15. 一种药物组合物，其特征在于：其包括权利要求1至10中任一项所述的siRNA或权利要求11至14中任一项所述的siRNA缀合物，以及药学上可接受的载体或辅料。
  16. 根据权利要求15所述的药物组合物，其特征在于：所述药物组合物用于抑制细胞程序性死亡-配体1基因表达。
  17. 如权利要求1至10中任一项所述的siRNA，或权利要求11至14中任一项所述的siRNA缀合物，或权利要求15或16所述的药物组合物用于制备治疗和/或预防PD-L1基因表达相关的疾病的药剂的用途。
  18. 根据权利要求17所述的用途，其特征在于：所述疾病为病毒性肝炎或癌症。
  19. 根据权利要求18所述的用途，其特征在于：所述病毒性肝炎为乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒感染所导致的。
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