[go: up one dir, main page]

WO2024169770A1 - Arnsi inhibant l'expression du gène scn9a, composition pharmaceutique et son utilisation - Google Patents

Arnsi inhibant l'expression du gène scn9a, composition pharmaceutique et son utilisation Download PDF

Info

Publication number
WO2024169770A1
WO2024169770A1 PCT/CN2024/076201 CN2024076201W WO2024169770A1 WO 2024169770 A1 WO2024169770 A1 WO 2024169770A1 CN 2024076201 W CN2024076201 W CN 2024076201W WO 2024169770 A1 WO2024169770 A1 WO 2024169770A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
nucleotide
sirna
sense strand
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2024/076201
Other languages
English (en)
Chinese (zh)
Inventor
刘楠
张红丽
陈平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Siran Biotechnology Co Ltd
Original Assignee
Suzhou Siran Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Siran Biotechnology Co Ltd filed Critical Suzhou Siran Biotechnology Co Ltd
Priority to CN202480000252.6A priority Critical patent/CN118202055A/zh
Publication of WO2024169770A1 publication Critical patent/WO2024169770A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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 an siRNA for inhibiting SCN9A gene expression, a pharmaceutical composition thereof and uses thereof.
  • the generation of pain originates from the widely distributed nerve endings throughout the body, which convert the mechanical, chemical or thermal stimulation they feel into nerve impulses (animal potentials) and transmit them to the dorsal root ganglia through afferent nerve fibers, and finally to higher nerve centers, thereby causing pain.
  • the generation and conduction of action potentials depend on the activation and inactivation of voltage-gated sodium channels (Nav.s) on the cell membrane.
  • Voltage-gated sodium channels are a class of transmembrane glycoproteins composed of an ⁇ subunit and several ⁇ subunits.
  • 9 subtypes Nav1.1 to Nav1.9 have been identified in mammals. It is generally believed (Dib-Hajj SD et al., Annu Rev Neurosci. 2010; 33: 325-47) that Nav1.3, Nav1.7, Nav1.8 and Nav1.9 are related to pain, among which Na(v)1.7 has special significance and is related to a series of hereditary human pain disorders.
  • Voltage-gated sodium channel Nav1.7 is mainly expressed in DRG neurons, sympathetic neurons, and neuroendocrine cells.
  • IEM hereditary erythromelalgia
  • DIB-HAJJ S D et al. Brain: a Journal of Neurology, 2005, 128(8): 1847-1854; DRENTH J P et al., The Journal of Investigative Dermatology, 2005, 124(6): 1333-1338
  • PPD Encephalopathy
  • CIP congenital analgesia
  • opioid analgesics and non-steroidal anti-inflammatory drugs occupy the mainstream position in analgesics, but NSAIDs cause a variety of side effects including gastrointestinal bleeding, myocardial infarction and stroke. They are not suitable for people with cardiovascular diseases, diabetes, etc.
  • Opioid analgesics generally have safety and addiction problems, but people's demand for pain relief is increasing day by day.
  • the drugs in the prior art have many defects, mainly including: a) mainstream pain drugs, opioid analgesics and non-steroidal anti-inflammatory drugs, have many side effects; b) there are no marketed drugs for the pain target Nav1.7, and most of the drugs in the clinical stage for the same target are small molecule drugs.
  • RNA interference is a method of sequence-specific degradation of RNA and post-transcriptional regulation of gene expression levels.
  • RNAi is a highly efficient and sequence-specific technology that can silence or knock down genes and is developing rapidly in multiple therapeutic areas.
  • the object of the present invention is to provide an siRNA, a conjugate and a pharmaceutical composition thereof for inhibiting the mRNA transcript of the SCN9A gene, for use in, for example, preventing or treating pain.
  • the present invention provides an siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the sense strand comprises the following sequence or a nucleotide sequence with no more than 3 base mutations from the following sequence, and the antisense strand comprises the following sequence or a nucleotide sequence with no more than 5 base mutations from the following sequence.
  • the sequence of the sense chain is such as SEQ ID NO:15
  • the sequence of the antisense chain is such as SEQ ID NO:16.
  • sequence of the sense chain is such as SEQ ID NO:7
  • sequence of the antisense chain is such as SEQ ID NO:8.
  • the sequence of the sense strand is SEQ ID NO:9
  • the sequence of the antisense strand is SEQ ID NO:10.
  • sequence of the sense chain is such as SEQ ID NO: 1
  • sequence of the antisense chain is such as SEQ ID NO: 2.
  • sequence of the sense chain is such as SEQ ID NO:3
  • sequence of the antisense chain is such as SEQ ID NO:4.
  • sequence of the sense chain is such as SEQ ID NO:5
  • sequence of the antisense chain is such as SEQ ID NO:6.
  • sequence of the sense chain is such as SEQ ID NO:11
  • sequence of the antisense chain is such as SEQ ID NO:12.
  • sequence of the sense chain is such as SEQ ID NO:13
  • sequence of the antisense chain is such as SEQ ID NO:14.
  • sequence of the sense chain is SEQ ID NO: 17, and the sequence of the antisense chain is SEQ ID NO: 18.
  • the sequence of the sense chain is such as SEQ ID NO:19
  • the sequence of the antisense chain is such as SEQ ID NO:20.
  • the sequence of the sense chain is SEQ ID NO:21
  • the sequence of the antisense chain is SEQ ID NO:22.
  • the sequence of the sense chain is SEQ ID NO: 23
  • the sequence of the antisense chain is SEQ ID NO: 24.
  • the sequence of the sense chain is SEQ ID NO:25
  • the sequence of the antisense chain is SEQ ID NO:26.
  • the sequence of the sense chain is SEQ ID NO:27
  • the sequence of the antisense chain is SEQ ID NO:28.
  • the sequence of the sense chain is SEQ ID NO:29
  • the sequence of the antisense chain is SEQ ID NO:30.
  • the sequence of the sense chain is SEQ ID NO:31
  • the sequence of the antisense chain is SEQ ID NO:32.
  • the positive strand includes a nucleotide sequence with no more than 2 base mutations and no more than 1 base mutation compared to any of the above nucleotide sequences.
  • 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 strand 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 of the above-mentioned nucleotide sequences.
  • 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.
  • 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 2'-fluoro-modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 8 and 9 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides.
  • At least one of the following connections between nucleotides in the siRNA is a phosphorothioate connection:
  • the sense strand comprises phosphorothioate groups located at the following positions:
  • the sense strand contains phosphorothioate groups located at the positions shown below:
  • the antisense strand comprises phosphorothioate groups located at the positions shown below:
  • the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.
  • non-fluorinated modified nucleotides of the sense strand and the antisense strand are 2'-methoxy modified nucleotides.
  • the nucleotide at the 6th position of the sense chain is a 2'-O-hexadecyl-modified nucleotide
  • the phosphate group at the 5' end of the antisense chain is replaced by VP
  • the non-fluorinated modified nucleotides at other positions of the sense chain and the antisense chain are 2'-methoxy-modified nucleotides.
  • the siRNA is selected from the siRNAs in Table 1 or Table 2.
  • 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 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 SCN9A gene expression.
  • the fourth aspect of the present invention provides use of the above-mentioned siRNA, or the above-mentioned siRNA conjugate, or the above-mentioned pharmaceutical composition for preparing a medicament for treating and/or preventing diseases related to SCN9A gene expression.
  • the disease is selected from neuropathic pain, nociceptive pain, organ-related pain, and mixed pain.
  • the neuropathic pain is selected from postherpetic neuralgia, migraine and diabetic neuropathy.
  • the nociceptive pain is selected from osteoporosis and rheumatoid arthritis.
  • the organ-related pain is selected from visceral-related pain and pancreatitis.
  • the mixed pain is selected from low back pain, cancer pain and fibromyalgia.
  • the disease is selected from hereditary erythromelalgia (EIM), paroxysmal extreme pain disorder (PEPD), congenital analgesia (CIP), chronic pain syndrome, and small fiber neuropathy.
  • EIM hereditary erythromelalgia
  • PEPD paroxysmal extreme pain disorder
  • CIP congenital analgesia
  • chronic pain syndrome chronic pain syndrome
  • small fiber neuropathy small fiber neuropathy
  • the fifth aspect of the present invention also provides a method for inhibiting SCN9A gene expression, comprising contacting a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention with cells expressing the SCN9A gene or administering it to a subject in need.
  • the present invention also provides a method for treating and/or preventing diseases related to SCN9A gene expression, comprising administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to a subject in need.
  • the siRNA of the present invention targets SCN9A gene, which is mainly expressed in DRG neurons, sympathetic neurons, and neuroendocrine cells.
  • the activity of the siRNA of the present invention is high, and it is possible to achieve an activity far below nM, and the siRNA of the present invention has a highly selective sequence for Nav1.7, which eliminates the potential toxic and side effects caused by the inhibition of other sodium ion channels from the root.
  • the main method of using the siRNA drug of the present invention is ganglion injection, which can directly act on the DRG and spinal cord related to pain afferent by selecting the position of the ganglion, and the RNA transcript of the SCN9A gene mediated by the RNA-induced silencing complex (RISC) is cut, so as to achieve the expression of SCN9A of local targeted organs or tissues, and the effect is lasting, so as to reduce the frequency of administration, improve safety, so as to effectively control the site of pain relief, and prevent the effects on smell and acid feeling, etc.
  • the cell body of nerve cells including organelles such as nucleus and ribosomes, is mainly present in ganglia.
  • siRNA drugs can effectively inhibit the protein expression of Nav1.7, and can reduce the Nav1.7 protein in peripheral nerves and central nerves at the same time.
  • appropriate delivery technology such as CNS delivery technology
  • effective inhibition of target proteins in the central nervous system can also be achieved through intrathecal injection.
  • the present invention has the following advantages compared with the prior art:
  • the siRNA, siRNA conjugate and pharmaceutical composition provided by the present invention show excellent SCN9A gene expression inhibition activity in cell experiments and animal experiments, and have good potential for treating diseases related to SCN9A gene expression.
  • the siRNA, siRNA conjugate and pharmaceutical composition of the present invention have good tissue specificity and high safety; long duration of efficacy, subcutaneous administration, and high medication compliance; compared with existing drugs of the same type, both efficacy and safety are greatly improved.
  • FIG1 is a graph showing the in vitro on-target activity and off-target activity test results of SA00011;
  • FIG2 is a graph showing the in vitro on-target activity and off-target activity test results of SA00013;
  • FIG3 is a graph showing the in vitro on-target activity and off-target activity test results of SA00045;
  • FIG. 4 is a graph showing the test results of the SCN9A silencing level of the siRNA of Example 5 in rats.
  • 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 siRNA of the invention contains deoxynucleotides, which may be dA, dT, dC, or dG.
  • 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 connection between two or more chemical moieties each having a specific function in a covalently linked manner; accordingly, “conjugate” refers to a compound formed by covalently linking the chemical moieties.
  • siRNA conjugate means a compound formed by covalently linking one or more chemical moieties having a specific function to siRNA. siRNA conjugates should be understood as a general term for multiple siRNA conjugates or a siRNA conjugate shown in a chemical formula, depending on the context.
  • conjugated 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.
  • the 2'-O-hexadecyl group on the siRNA sense chain can be considered as a conjugated molecule, such as Uhd, Ahd, etc., which are nucleotides modified or conjugated with 2'-O-hexadecyl.
  • the VP (vinyl phosphate) at the 5' end of the siRNA antisense chain can be considered as a conjugated molecule.
  • 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, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic/glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate) and poly (2-aminoethyl ethylene phosphate).
  • the excipient may be one or more of various preparations or compounds conventionally used in the art.
  • the other pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic
  • 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.
  • siRNA 19/21nt siRNA was designed to meet the general rules of active siRNA, with human SCN9A (NM_002977.3) and monkey SCN9A (XM_005573366.3) mRNA as target genes.
  • SCN9A NM_002977.3
  • XM_005573366.3 XM_005573366.3
  • the SCN9A 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 (0.6M acetonitrile solution)
  • 0.22M diphenylacetyl disulfide PADS dissolved in a 1:1 volume ratio of acetonitrile and trimethylpyridine (Suzhou Kelema) solution was used as a sulfurization reagent, the sulfurization reaction time was 3 minutes (min), and iodine pyridine/water solution (Kelema) was used as an oxidant, and 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 0.1M triethylamine acetate TEAA and acetonitrile, and DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotides were collected and freeze-dried, and 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.
  • the capital letters A, C, G, and U represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, and uridine-3'-phosphate, respectively;
  • the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide;
  • the lowercase letter f indicates that the nucleoside adjacent to the left
  • the lowercase letter s in the middle of the uppercase letter indicates that the two adjacent nucleotides to the left and right of s are linked by a phosphorothioate group; when s is the first at the 3' end, it indicates that the end of the nucleotide adjacent to the left of the letter s is a phosphorothioate group.
  • Ahd represents 2'-O-hexadecyl adenosine-3'-phosphate
  • Chd represents 2'-O-hexadecyl cytosine-3'-phosphate
  • Ghd represents 2'-O-hexadecyl-guanosine-3'-phosphate.
  • Uhd stands for 2'-O-hexadecyl-uridine-3'-phosphate
  • VP stands for vinyl phosphate
  • dA stands for deoxyadenosine-3'-phosphate
  • HEK293 cells were cultured in DMEM high glucose medium with 10% fetal bovine serum, grown to a confluence of about 90% at 37°C, 5% CO2 , and then digested with trypsin to resuspend the cells.
  • 1-3000bp in the human SCN9A reference sequence (NM_002977.3) was cloned into the dual luciferase psiCHECK2TM vector to construct the psiCHECK-SCN9A-L plasmid
  • 2951-6099 and 9395-9410bp were cloned into the dual luciferase psiCHECK2TM vector to construct the psiCHECK-SCN9A-M plasmid.
  • Dual luciferase plasmids and siRNA were co-transfected into 1x104 cells using Lipofectamine TM 3000 (Thermo, L3000015).
  • Lipofectamine TM 3000 Thermo, L3000015
  • 0.3 ⁇ l of Lipofectamine TM 3000 was added to 19.7 ⁇ l Opti-MEM containing 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-well plate. Then, cells resuspended in 80 ⁇ l of fresh complete medium 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 siRNA. All transfections were set up in duplicate.
  • the inhibitory activity of the siRNA of the present application is significantly better than that of the positive control group SA00195, wherein, in the 5'-3' direction, the sequence of the sense chain of SA00195 is AmsAmsAmAmCmAmAmUmCfUfUfCmCmGmUmUmUmCmAmAmAm (SEQ ID NO: 33), and the sequence of the antisense chain is UmsUfsUmGmAmAfAmCmGmGmAmAmGmAfUmUfGmUmUmUmUmUmUmsCmsCm (SEQ ID NO: 34).
  • AVG means average
  • SD means standard deviation
  • SK-N-AS cells were cultured in DMEM high glucose medium with 10% fetal bovine serum at 37°C and 5% CO 2 , and then digested with trypsin and the cells were resuspended. 1 ⁇ 10 4 cells were co-transfected with siRNA using RNAiMAX (Thermo, 13778150). Using a 96-well plate, 0.3 ⁇ l of RNAiMAX was added to 19.7 ⁇ l Opti-MEM containing 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 complete medium were then added.
  • RNAiMAX Thermo, 13778150
  • Target gene primers F: GTATTTTGTGTCCCCTACCCTGTT (SEQ ID NO: 35); R: GTGCGGATCCCCTTTGCT (SEQ ID NO: 36); Probe: ATTGGCCGAATCCTACGT (SEQ ID NO: 37);
  • the results were expressed as the residual expression level of the siRNA-administered group compared to the vehicle group (the vehicle group was 100%).
  • the relative expression level of SCN9A mRNA is shown in Table 5.
  • AVG means average
  • SD means standard deviation
  • the on-target and off-target activities of compounds in different sequences were detected using dual luciferase reporter genes.
  • 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 different concentrations of siRNA were co-transfected into the cells using Lipofectamine 2000 (ThermoFisher, 11668019). After 24 hours of transfection, the dual luciferase detection kit (Yishen Bio, 11405ES80) was used for detection.
  • siRNAs with good in vitro activity or good on-target activity and off-target activity evaluated in vitro were conjugated and modified to further evaluate the SCN9A silencing level of siRNAs in rats.
  • Sprague Dawley rats 6-8 weeks old, male, were provided.
  • vehicle artificial cerebrospinal fluid, aCSF.
  • the rats were killed and dissected on day 7 after administration, and the lumbar cord, thoracic cord, cervical cord and dorsal root ganglion were respectively taken and placed in RNALater (Invitrogen, AM7021M) for subsequent RNA extraction.
  • RNALater Invitrogen, AM7021M
  • RNA reverse transcribed cDNA
  • Takara 6210B
  • SCN9A SCN9A gene was measured by probe-based qPCR (Applied Biosystems, 4444964).
  • Target gene primers F: GTCGTGTCGCTTGTTGATGG (SEQ ID NO:41); R: AGATGCGGGTCATTCAGCAT (SEQ ID NO:42); Probe: CCGACGACAGCGGCACGACT (SEQ ID NO:43);
  • FIG. 4 shows that the silencing effect of SD003625, SD003626, and SD003627 on SCN9A mRNA in the lumbar cord, thoracic cord, cervical cord, and dorsal root ganglion is significantly better than that of SD003628, SD003630, and SD003631. Among them, the effect of SD003627 targeting the dorsal root ganglion is more excellent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un ARNsi inhibant l'expression du gène SCN9A, une composition pharmaceutique et son utilisation. L'ARNsi contient un brin sens et un brin antisens. Chaque nucléotide de l'ARNsi est, indépendamment, un nucléotide modifié ou non modifié. Le brin sensé comprend une séquence nucléotidique de la séquence suivante ou ne présente pas plus de 3 mutations de bases par rapport à la séquence suivante. Le brin antisens comprend une séquence nucléotidique de la séquence suivante ou ne présente pas plus de 5 mutations de bases par rapport à la séquence suivante. La séquence de l'ARNsi est représentée dans les SEQ ID NO : 1 à SEQ ID NO : 32. L'ARNsi, un conjugué et une composition pharmaceutique fournis peuvent inhiber efficacement l'expression du gène SCN9A.
PCT/CN2024/076201 2023-02-17 2024-02-06 Arnsi inhibant l'expression du gène scn9a, composition pharmaceutique et son utilisation Ceased WO2024169770A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202480000252.6A CN118202055A (zh) 2023-02-17 2024-02-06 一种抑制SCN9A基因表达的siRNA、其药物组合物及用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310133396.6 2023-02-17
CN202310133396 2023-02-17

Publications (1)

Publication Number Publication Date
WO2024169770A1 true WO2024169770A1 (fr) 2024-08-22

Family

ID=92422222

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/076201 Ceased WO2024169770A1 (fr) 2023-02-17 2024-02-06 Arnsi inhibant l'expression du gène scn9a, composition pharmaceutique et son utilisation

Country Status (1)

Country Link
WO (1) WO2024169770A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100273857A1 (en) * 2007-09-05 2010-10-28 Medtronic, Inc. Suppression of scn9a gene expression and/or function for the treatment of pain
WO2021123814A1 (fr) * 2019-12-19 2021-06-24 Ucl Business Ltd Traitement d'une douleur chronique
US20210238608A1 (en) * 2018-06-22 2021-08-05 Hoffmann-La Roche Inc. Oligonucleotides for modulating scn9a expression
WO2021247995A2 (fr) * 2020-06-04 2021-12-09 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de la douleur neuropathique
WO2022147541A1 (fr) * 2021-01-04 2022-07-07 Exicure Operating Company Composés pour moduler l'expression de scn9a
WO2022204430A1 (fr) * 2021-03-24 2022-09-29 Atalanta Therapeutics, Inc. Arnsi double brin ayant des modifications chimiques à motifs
WO2022256565A2 (fr) * 2021-06-02 2022-12-08 Atalanta Therapeutics, Inc Compositions et méthodes d'administration d'oligonucléotides thérapeutiques au système nerveux central

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100273857A1 (en) * 2007-09-05 2010-10-28 Medtronic, Inc. Suppression of scn9a gene expression and/or function for the treatment of pain
US20210238608A1 (en) * 2018-06-22 2021-08-05 Hoffmann-La Roche Inc. Oligonucleotides for modulating scn9a expression
WO2021123814A1 (fr) * 2019-12-19 2021-06-24 Ucl Business Ltd Traitement d'une douleur chronique
WO2021247995A2 (fr) * 2020-06-04 2021-12-09 Voyager Therapeutics, Inc. Compositions et méthodes de traitement de la douleur neuropathique
WO2022147541A1 (fr) * 2021-01-04 2022-07-07 Exicure Operating Company Composés pour moduler l'expression de scn9a
WO2022204430A1 (fr) * 2021-03-24 2022-09-29 Atalanta Therapeutics, Inc. Arnsi double brin ayant des modifications chimiques à motifs
WO2022256565A2 (fr) * 2021-06-02 2022-12-08 Atalanta Therapeutics, Inc Compositions et méthodes d'administration d'oligonucléotides thérapeutiques au système nerveux central

Similar Documents

Publication Publication Date Title
AU2020257111B2 (en) RNA modulating oligonucleotides with improved characteristics for the treatment of Duchenne and Becker muscular dystrophy
AU2014284836B2 (en) Respiratory disease-related gene specific siRNA, double-helical oligo RNA structure containing siRNA, compositon containing same for preventing or treating respiratory disease
KR101718534B1 (ko) Ｈｓｐ４７ 발현의 조절
CN110799647A (zh) 双尾自递送sirna及相关的方法
JP6944942B2 (ja) ＩＬ４Ｒα、ＴＲＰＡ１、またはＦ２ＲＬ１を標的とするＲＮＡ複合体を用いたアトピー性皮膚炎および喘息の治療
CN108699556B (zh) 使用靶向myd88或tlr3的rna复合物治疗老年性黄斑变性
CA3020487A1 (fr) Traitement de la fibrose pulmonaire idiopathique a l'aide de complexes d'arn qui ciblent le facteur de croissance du tissu conjonctif
TWI857290B (zh) 用於調節ｐｎｐｌａ３表現之組合物及方法
CN102985544A (zh) 治疗β-ENaC-相关疾病的有机组合物
WO2017007886A2 (fr) Compositions pour inhiber l'expression du gène dux4 et leurs utilisations
CN120752341A (zh) 靶向sod1的寡核苷酸
KR102701681B1 (ko) 이중가닥 올리고뉴클레오티드 및 이를 포함하는 코로나바이러스감염증-19〔covid-19〕치료용 조성물
JP2022527529A (ja) Ａｔｘｎ２発現を調節するためのオリゴヌクレオチド
CN118202055A (zh) 一种抑制SCN9A基因表达的siRNA、其药物组合物及用途
WO2024169770A1 (fr) Arnsi inhibant l'expression du gène scn9a, composition pharmaceutique et son utilisation
CN116670276A (zh) 用于通过多个靶标治疗疼痛的治疗组合物
CN118480552B (zh) 一种靶向CEBPA基因表达的saRNA及其用途
JP2019134706A (ja) デュシェンヌ型及びベッカー型筋ジストロフィーの治療のための改善された特徴を有するｒｎａ調節オリゴヌクレオチド
WO2025045130A1 (fr) Molécule d'arndb pour inhiber l'expression du gène sod1 et son utilisation
WO2024169907A1 (fr) Arnsi pour la régulation de l'expression du complément c3, son conjugué, sa composition pharmaceutique et son utilisation
WO2025190276A1 (fr) Molécule polynucléotidique pour la suppression de l'expression de dmpk et son utilisation
JP2025501269A (ja) アンチセンスオリゴヌクレオチド及びその使用
TW202444904A (zh) 用於抑制粒線體醯胺肟還原組分1(MARC1)之表現之RNAi藥劑、其醫藥組合物及使用方法
TW202440921A (zh) 經修飾之寡核苷酸
CN118202053A (zh) 一种调节补体C3表达的siRNA、其缀合物和药物组合物及用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24756144

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE