WO2025201467A1 - Conjugué oligonucléotidique polypeptidique pour inhiber l'expression d'un gène cible dans le système nerveux central et son utilisation - Google Patents

Conjugué oligonucléotidique polypeptidique pour inhiber l'expression d'un gène cible dans le système nerveux central et son utilisation

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WO2025201467A1
WO2025201467A1 PCT/CN2025/085394 CN2025085394W WO2025201467A1 WO 2025201467 A1 WO2025201467 A1 WO 2025201467A1 CN 2025085394 W CN2025085394 W CN 2025085394W WO 2025201467 A1 WO2025201467 A1 WO 2025201467A1
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polypeptide
pharmaceutically acceptable
stereoisomer
oligonucleotide conjugate
acceptable salt
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Chinese (zh)
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范福顺
刘新建
傅天成
林莹莹
梁豪
翁运幄
钱长庚
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Bebetter Med Inc
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Bebetter Med Inc
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Priority to CN202580000534.0A priority Critical patent/CN121127266A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • 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 claims priority to the Chinese patent application with application number 2024103688430, filed with the Patent Office of China on March 28, 2024, and entitled “Polypeptide oligonucleotide conjugates for inhibiting the expression of target genes in the central nervous system and their applications”, and the Chinese patent application with application number 2024113531318, filed with the Patent Office of China on September 26, 2024, and entitled “Polypeptide oligonucleotide conjugates for inhibiting the expression of target genes in the central nervous system and their applications”.
  • the entire contents of the two patent applications are incorporated into the present invention by reference.
  • the present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide oligonucleotide conjugate for delivering oligonucleic acid drugs for inhibiting the expression of target genes in the central nervous system and its application.
  • peptide-drug conjugates have great prospects for biomedical applications.
  • PDCs not only focus on antibody drugs and small molecule drugs, but also use specific chemical reactions such as click chemistry to couple peptides with nucleic acid drugs (such as small interfering RNA, siRNA). Due to the targeting of peptides, targeted delivery of siRNA can be achieved, which greatly expands the application prospects of extrahepatic delivery of nucleic acid drugs (Rizvi SFA et al. ACS Pharmacol Transl Sci. 2024; 7(2): 309-334).
  • amino acid residues in the amino acid sequence are replaced by methylated amino acid residues
  • the linking group is preferably:
  • the LA in P-LA is linked to the C-terminus of the polypeptide ligand.
  • the LA in P-LA is linked to the seventh amino acid residue from the N-terminus of the polypeptide ligand.
  • the LA in P-LA is linked to the 15th amino acid residue from the N-terminus of the polypeptide ligand.
  • the unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGKRNNFKTEEY, and the LA in P-LA is linked to the 15th amino acid (i.e., lysine) residue from the N-terminus of the polypeptide ligand.
  • the unmodified amino acid sequence of the polypeptide ligand is TFFYGGKRGKRNNFKTEEY, and the LA in P-LA is linked to the 7th, 10th, or 15th amino acid (i.e., lysine) residue in the polypeptide ligand from the N-terminus.
  • the unmodified amino acid sequence of the polypeptide ligand is TFFYGGCRGKRNNFKTEEY, and the LA in P-LA is linked to the 10th amino acid (i.e., lysine) residue from the N-terminus of the polypeptide ligand.
  • the LA in P-LA is linked to a norleucine residue in the polypeptide ligand.
  • the unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGZRNNFKTEEY, and the LA in P-LA is linked to the 10th amino acid (norleucine) residue from the N-terminus of the polypeptide ligand.
  • LA in P-LA is linked to an amino acid residue containing a side chain carboxyl group in the polypeptide ligand.
  • the LA in P-LA is linked to a glutamic acid residue or an aspartic acid residue in the polypeptide ligand.
  • LE is selected from the following structures:
  • Each R is independently selected from: H or a hydroxyl protecting group
  • n 2 is independently selected from an integer between 0-10.
  • LE is selected from:
  • LA is selected from the following structures:
  • each i, j, h, q, s, and e are independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
  • c is selected from: 0, 1;
  • X is selected from: O, S, CH 2 ;
  • LB in formula (I) is selected from the following structures:
  • Each i, j, and e are independently selected from: 0, 1, 2, 3, and 4;
  • c is selected from: 0, 1;
  • k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • X is selected from: CH 2 , O.
  • LB in formula (I) is selected from the following structures:
  • LB in formula (II) has the structure shown below:
  • X is selected from: O, S, CH 2 ;
  • Each i and e are independently selected from: 0, 1, 2, 3, 4;
  • LC in formula (II) is selected from the following structures:
  • polypeptide oligonucleotide conjugate is selected from the following compounds:
  • c is selected from: 0, 1;
  • k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • X is selected from: CH 2 , O;
  • n1 is independently selected from the group consisting of: 0, 1, 2, and 3.
  • the oligonucleic acid drug is selected from the group consisting of small interfering RNA (siRNA), antisense oligonucleotide (ASO) and peptide nucleic acid (PNA).
  • siRNA small interfering RNA
  • ASO antisense oligonucleotide
  • PNA peptide nucleic acid
  • the oligonucleic acid drug is a small interfering RNA (siRNA), and the LE is linked to the phosphate group or the thiophosphate group at the 3' and/or 5' end of the sense strand of the siRNA.
  • siRNA small interfering RNA
  • the polypeptide-oligonucleotide conjugate of the present invention is prepared by the following method: a click chemistry reaction is performed between an alkyne-containing linker-nucleic acid sense chain molecule and an azide-modified polypeptide to obtain a polypeptide-sense chain conjugate, and the polypeptide-sense chain conjugate is then annealed with a corresponding nucleic acid antisense chain to obtain the polypeptide-oligonucleotide conjugate; the polypeptide is a polypeptide ligand for the LRP-1 receptor;
  • the alkyne-containing linker-nucleic acid sense chain molecule has a structure as shown in the following formula (III) or formula (IV):
  • LA, LB, LC, LE, n, a, and b are as described above;
  • the alkyne-containing linker-nucleic acid sense strand molecule is selected from the following structures:
  • k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • X is selected from: CH 2 , O.
  • the alkyne-containing linker-nucleic acid molecule sense strand is selected from the following structures:
  • the azide-modified polypeptide is selected from the following structures:
  • the present invention provides applications of the polypeptide-oligonucleotide conjugate, including the following:
  • polypeptide oligonucleotide conjugate of the present invention or its pharmaceutically acceptable salt or stereoisomer is used as an active ingredient in the preparation of a drug for inhibiting the expression of a target gene in the central nervous system.
  • the central nervous system target gene is selected from one or more of APP, MC4R, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and NLRP3.
  • polypeptide oligonucleotide conjugate of the present invention or its pharmaceutically acceptable salt or stereoisomer is used as an active ingredient in the preparation of a drug for treating diseases related to the central nervous system (CNS).
  • CNS central nervous system
  • the target of the disease related to the central nervous system is selected from APP, MC4R, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, and NLRP3.
  • the disease associated with the central nervous system is Alzheimer's disease, cachexia, cerebral amyloid angiopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, spinocerebellar disorders, prion diseases and Lafora disease.
  • the present invention provides a method for inhibiting the expression of a target gene in the central nervous system, comprising: administering a safe and effective amount of the polypeptide oligonucleotide conjugate of the present invention or a pharmaceutically acceptable salt or a stereoisomer thereof; or administering a safe and effective amount of the drug of the present invention for inhibiting the expression of a target gene in the central nervous system.
  • the present invention provides a method for treating diseases related to the central nervous system, comprising: administering a safe and effective amount of the polypeptide oligonucleotide conjugate of the present invention or a pharmaceutically acceptable salt or a stereoisomer thereof; or administering a safe and effective amount of a drug for treating diseases related to the central nervous system.
  • the present invention couples the polypeptide ligand of the LRP-1 receptor with an oligonucleic acid drug via a linker group to obtain a polypeptide oligonucleotide conjugate based on a novel LRP-1 targeting peptide.
  • the polypeptide oligonucleotide conjugate can be used to deliver nucleic acid drugs such as small interfering RNA (siRNA) for the treatment of central nervous system diseases, and is used to inhibit the expression of one or more target genes in central nervous system tissue cells. It has the advantages of good in vivo effect, long duration of action and good safety.
  • siRNA small interfering RNA
  • the attachment of the heterocyclic substituent can be achieved through a carbon atom or through a heteroatom.
  • safe and effective amount means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.
  • “Pharmaceutically acceptable excipients” refers to: one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficiently low toxicity.
  • “Compatibility” here means that the components of the composition can be mixed with the active ingredient of the present invention (polypeptide oligonucleotide conjugate or its pharmaceutically acceptable salt or stereoisomer) and with each other without significantly reducing the efficacy of the active ingredient.
  • Precipitation and purification Add pre-cooled anhydrous ether to the filtrate and centrifuge to obtain a white precipitate. Wash the precipitate three times with anhydrous ether and dry under reduced pressure at room temperature to obtain a crude polypeptide product as an off-white powder. Dissolve the crude polypeptide in acetonitrile/water and purify it by high performance liquid chromatography.
  • the Mtt protecting group on the amino group of the lysine side chain is first selectively removed using a DCM solution containing 2% TFA, and the peptide is then reacted with 2-(2-(2-aminoethoxy)ethoxy)acetic acid according to the conventional amino acid condensation and Fmoc removal conditions described above, and then reacted with 6-azidohexanoic acid before the peptide is cleaved.
  • Ang2(K10Mtt)-Wang Resin was synthesized on a 0.2 mmol scale on a PurePep Chorus 4-channel peptide synthesizer according to the conditions described in Example 1. 20 mL of DCM and 0.4 mL of TFA were added to Ang2(K10Mtt)-Wang Resin, and after shaking at room temperature for 15 minutes, the resin was washed three times with DCM to obtain Ang2(K10NH 2 )-Wang Resin.
  • Ang2(K10NH 2 )-Wang Resin was placed in 15 mL of DMF, and 6-azidohexanoic acid (157 mg, 1 mmol), HCTU (414 mg, 1 mmol), and DIEA (129 mg, 1 mmol) were added sequentially. The mixture was shaken at room temperature for 1 hour, washed with DMF and DCM, and cleaved with TFA at room temperature for 2 hours.
  • the mixture was precipitated with diethyl ether and purified by HPLC to obtain the target polypeptide Ang2(K10N 3 ) (150 mg, white powdery solid) (theoretical molecular weight: 2482.70, measured molecular weight: 1242.30 [M+2H] 2+ ).
  • Ang2-Wang resin was synthesized on a 0.2 mmol scale on a PurePep Chorus 4-channel peptide synthesizer according to the conditions described in Example 1.
  • Ang2-Wang resin was mixed with 1.5 equivalents of 3-maleimidopropionic acid hydroxysuccinimide ester (BMPS) for 1 h to obtain Maleimido-Ang2-Wang resin.
  • BMPS 3-maleimidopropionic acid hydroxysuccinimide ester
  • Example 1-3 According to the method of Example 1-3, the azide-modified polypeptides shown in Table 1-1 were synthesized, and their theoretical molecular weights and measured molecular weights are shown in Table 1-1 below.
  • Step 1 Preparation of N6-(tert-Butoxycarbonyl)-N2-(6-((tert-Butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (Compound A3): A mixture of N6-(tert-Butoxycarbonyl)-L-lysine methyl ester (5.0 g, 19.2 mmol, 1.0 eq), tert-Butoxycarbonyl 6-aminohexanoic acid (4.89 g, 21.1 mmol, 1.1 eq), and N,N-diisopropylethylamine (5.46 g, 42.3 mmol, 2.2 eq) in dichloromethane was stirred at room temperature.
  • Step 2 Preparation of (6-aminohexanoyl)-L-lysine methyl ester (Compound A4): N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (1.0 g, 2.11 mmol, 1.0 equiv) and a solution of hydrogen chloride in dioxane (10 mL, 2 mol/L) were mixed and reacted for 1.5 hours at room temperature. The reaction was complete as monitored by LCMS. The reaction solution was concentrated under reduced pressure, and water (30 mL) was added. The pH was adjusted to approximately 7-8 by adding sodium hydroxide solution.
  • Step 3 Preparation of N6-((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine methyl ester (Compound A5): Under nitrogen protection, (6-aminohexanoyl)-L-lysine methyl ester (450 mg) was added.
  • Step 4 Preparation of N6-((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine (Compound A6): To a mixture of tetrahydrofuran, methanol, and water (6 ml) was added (280 mg, 0.448 mmol, 1.0 equiv) of (1R, 8S, 9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine methyl ester (280 mg, 0.448 mmol, 1.0 equiv).
  • a lithium hydroxide (13 mg, 0.538 mmol, 1.2 equiv) aqueous solution (1 ml) was then added, and the mixture was stirred at room temperature for 1 hour. Following completion of the reaction as monitored by LCMS, the reaction solution was concentrated under reduced pressure, water (30 ml) was added, and the pH was adjusted to approximately 3-4 by adding 1 mol/L dilute hydrochloric acid. The mixture was diluted with water (20 ml), and extracted with dichloromethane (30 ml). The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate.
  • Step 1 Preparation of (S)-22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oic acid methyl ester
  • Compound B1 (6-aminohexanoyl)-L-lysine methyl ester (3.0 g, 10.9 mmol, 1.0 eq), 2,2-dimethyl-4-oxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oic acid methyl ester were added at room temperature.
  • the reaction solution was diluted with dichloromethane and extracted. The organic layer was washed twice with saturated sodium bicarbonate and twice with saturated ammonium chloride, and dried over anhydrous sodium sulfate.
  • a dioxane solution (40 mL, 2 mol/L) was mixed and reacted for 1.5 hours. The reaction was complete as monitored by LCMS. The reaction solution was concentrated under reduced pressure, and water (30 mL) was added. Sodium hydroxide solution was added to adjust the pH to approximately 7-8. The aqueous phase was concentrated under reduced pressure using an oil pump. The resulting solid was washed with a 10/1 dichloromethane solution and stirred.
  • Step 4 Preparation of (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25ynoic acid (Compound B4): Methyl 1-hydroxy-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoate (310 mg, 0.398 mmol, 1.0 eq) was added to a mixture of tetrahydrofuran, methanol, and water (6 mL).
  • Step 9 Synthesis of solid support DT-Linker-CPG
  • the mixture was filtered, and the filter cake was washed three times with dichloromethane, acetonitrile, and dichloromethane, followed by vacuum drying for one hour.
  • the filter cake was then placed in a mixture of pyridine (45 mL) and acetic anhydride (15 mL) and shaken on a shaker for two hours.
  • the mixture was filtered again, and the filter cake was washed three times with dichloromethane, acetonitrile, and dichloromethane in sequence.
  • the filter cake was dried under vacuum for one hour to obtain the product DT-Linker-CPG (6.43 g) as a yellow solid.
  • oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis method. Aminolysis reagents were added to the synthesized oligonucleotides and incubated at 45-80°C to separate the oligonucleotides from the solid phase carrier and free the oligonucleotides. The crude oligonucleotides were then precipitated with ethanol, and the supernatant was discarded by high-speed centrifugation. This process was repeated twice to obtain the crude oligonucleotides, and the precipitate was resuspended in DEPC water. The crude oligonucleotides were purified by ion-pairing HPLC, and the collected products were dried to powder in a vacuum centrifugal dryer. The purified product was dissolved in DEPC water and analyzed by TOF LC-MS.
  • the nucleotide concentration was determined by dissolving it in DEPC water. The volumes required for equimolar amounts of the sense and antisense strands were calculated. The equimolar amounts of the peptide-sense and antisense strands were then mixed and heated at 75°C for 5 minutes, followed by annealing and cooling to room temperature to prepare the peptide-siRNA duplex. After cooling to room temperature, the annealed product was diluted with 10 volumes of DEPC water and transferred to a Millipore 15mL ultrafiltration centrifuge tube (molecular cutoff 3 kDa) for ultrafiltration.
  • Ang2(K10N 3 )-B04-12006SM2 and 12006AM2 were mixed and annealed according to the method in Example 13 to obtain Ang2(K10N 3 )-12006.2-2.
  • Ang2(K10N 3 )-B12-12006SM1 and 12006AM1 were mixed and annealed according to the method in Example 13 to obtain Ang2(K10N 3 )-12006.1-1.
  • 12006SM1-B08 160 mg, 22 ⁇ mol was dissolved in 20 mL of phosphate buffer (0.1 M, pH 7.4), and BPL-2 (569 mg, 660 ⁇ mol) was dissolved in 10 mL of DMF.
  • the DMF solution of BPL-2 and the phosphate buffer of 12006SM1-B08 were mixed and shaken at 40°C for 60 min. After the reaction was complete as monitored by LC-MS, 3 mL of 3 M NaCl solution and 90 mL of pre-cooled ethanol were added to the reaction solution. The mixture was shaken thoroughly and centrifuged for 3 min (4°C, 15,000 rpm). The supernatant was discarded, and the precipitate was dissolved in approximately 5 mL of DEPC water.
  • the precipitate was purified by HPLC and lyophilized to obtain 12006SM1-B08-BPL-2 (120 mg, 14.9 ⁇ mol, yield: 68%) as a white powder (molecular weight: 8028.56, measured: 8027.78).
  • Example 22 Effect of Linking Single or Double Peptides on the In Vivo Activity of Peptide-siRNA in Wild-Type Mice by Intrathecal Injection
  • This example tested the effects of Ang2 conjugation, tandem dipeptides, and parallel dipeptides on the in vivo activity of peptide-siRNA.
  • Four-week-old female C57 wild-type mice were administered intrathecally according to the method of Example 21. The mice were sacrificed on day 11, and mAPP expression was measured in various brain tissues. The results are shown in Table 5.
  • the results in Table 10 show that under low-dose conditions, the activity of the dipeptide BiAng2(K10N 3 )-12006.1-1 was significantly improved at 14 days compared to C16(6-G)-12006.1-1 delivered by C16. This indicates that the siRNA delivered by the polypeptide Ang2 has more significant specificity and efficiency than the siRNA delivered by C16, and low-dose injection can also reduce toxic side effects. Therefore, the polypeptide Ang2 of the present invention is an ideal carrier for delivering siRNA to the central nervous system.
  • Example 29 Testing the in vivo activity of different peptide-siRNA forms by injecting them into the cisterna magna in wild-type rats
  • This example compares the siRNA delivery capabilities of the LRP-1 ligands Ang1 and Ang2.
  • the corresponding peptide-siRNA conjugates, BiAng2( K10N3 )-12006.1-1 and BiAng1( K10N3 )-12006.1-1 both utilize lysine 10 as the siRNA attachment site and possess a dipeptide structure.
  • the drug was administered to rats via cisterna magna injection, with each rat receiving a 0.3 mg dose.
  • Other experimental procedures were the same as in Example 21. Rats were sacrificed on day 28, and rAPP expression was measured in various brain tissues. The results are shown in Table 13.
  • Example 30 Testing the in vivo activity of peptide-siRNA containing different linkers by injection into the cisterna magna of wild-type mice
  • This example compares the in vivo activity of a peptide-siRNA (BiAng2( K10N3 )-12006.1-1) from the present invention with that of a peptide-siRNA (BiAng2( K10N3 )-Q22-12006.3-5) using a Q22 linker in mice.
  • the drugs were injected into the mice via cisterna magna injection, with the dose reduced to 0.1 mg per mouse.
  • Other experimental procedures were the same as in Example 21.
  • the mice were sacrificed, and different brain tissues were collected to measure mAPP expression. The results are shown in Table 14.
  • This example compares the in vivo activity of the polypeptide-siRNA (BiAng2( K10N3 )-12006.3-5) of the present invention with that of the hexadecyl chain-modified oligonucleotide C16(6-G)-12006.3-5 in mice.
  • C16(6-G)-12006.3-5 is modified with a hexadecyl fatty chain at the 6th nucleotide residue of the sense strand. Hexadecyl modification of the 6th nucleotide residue is an effective strategy for improving nucleic acid delivery efficiency.
  • the drug was injected into mice using different injection methods: intrathecal injection (IT) and cisterna magna injection (ICM). Each mouse received a 0.25 mg injection dose. Other experimental methods were the same as in Example 21. On day 7, the mice were sacrificed, and different brain tissues were collected to measure mAPP expression. The results are shown in Table 15.
  • AD1747580 (WO2023192977A2), a known siRNA targeting ⁇ -synuclein (SNCA) for the treatment of Parkinson's disease
  • SNCA ⁇ -synuclein
  • the activity of this peptide-siRNA was tested in mice.
  • This example used cisterna magna injection to deliver the peptide-siRNA to mice, with a dose of 0.25 mg per mouse.
  • Other experimental procedures were the same as in Example 21. On day 14, the mice were sacrificed, and different brain tissues were collected to measure mSNCA (mouse SNCA) expression. The results are shown in Table 16.
  • the criteria for FOB were (1) the mouse was intelligent, alert, and responsive; (2) the mouse stood or arched its back without stimulation; (3) the mouse showed any movement without stimulation; (4) the mouse exhibited forward movement after being lifted; (5) the mouse showed any movement after being lifted; (6) the mouse responded to a tail pinch; and (7) the mouse breathed evenly.
  • the mouse was given a score of 0 if it met the criteria and 1 if it did not. After evaluating all seven criteria, the scores for each mouse were summed and averaged within each treatment group. The results are presented in Table 18.

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Abstract

La présente invention concerne un conjugué oligonucléotidique polypeptidique ayant une structure telle que représentée dans la formule suivante (I) ou (II), ou un sel pharmaceutiquement acceptable de celui-ci ou un stéréoisomère de celui-ci, et son utilisation. Le conjugué oligonucléotidique polypeptidique de la présente invention peut être utilisé pour inhiber l'expression d'un ou de plusieurs gènes cibles dans une cellule tissulaire du système nerveux central, et présente les avantages d'avoir un bon effet in vivo, un temps d'action long et une bonne sécurité.
PCT/CN2025/085394 2024-03-28 2025-03-27 Conjugué oligonucléotidique polypeptidique pour inhiber l'expression d'un gène cible dans le système nerveux central et son utilisation Pending WO2025201467A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220048992A1 (en) * 2020-08-13 2022-02-17 Bioasis Technologies, Inc. Combination therapies for delivery across the blood brain barrier
WO2022204293A1 (fr) * 2021-03-23 2022-09-29 Arizona Board Of Regents On Behalf Of Arizona State University CIBLAGE SÉLECTIF DE β-AMYLOIDE OLIGOMÈRE
WO2023010134A1 (fr) * 2021-07-30 2023-02-02 Alnylam Pharmaceuticals, Inc. Ligands peptidiques pour l'administration de composés d'arni au snc et à l'oeil

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220048992A1 (en) * 2020-08-13 2022-02-17 Bioasis Technologies, Inc. Combination therapies for delivery across the blood brain barrier
WO2022204293A1 (fr) * 2021-03-23 2022-09-29 Arizona Board Of Regents On Behalf Of Arizona State University CIBLAGE SÉLECTIF DE β-AMYLOIDE OLIGOMÈRE
WO2023010134A1 (fr) * 2021-07-30 2023-02-02 Alnylam Pharmaceuticals, Inc. Ligands peptidiques pour l'administration de composés d'arni au snc et à l'oeil

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