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WO2024060649A1 - 用于抑制TMPRSS6基因表达的siRNA或其盐、药物及其应用 - Google Patents

用于抑制TMPRSS6基因表达的siRNA或其盐、药物及其应用 Download PDF

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Publication number
WO2024060649A1
WO2024060649A1 PCT/CN2023/095104 CN2023095104W WO2024060649A1 WO 2024060649 A1 WO2024060649 A1 WO 2024060649A1 CN 2023095104 W CN2023095104 W CN 2023095104W WO 2024060649 A1 WO2024060649 A1 WO 2024060649A1
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Prior art keywords
bbd
antisense strand
sirna
justice chain
tmprss6
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French (fr)
Inventor
刘新建
范福顺
钱长庚
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Bebetter Med Inc
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Bebetter Med Inc
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Priority to CN202380009674.5A priority Critical patent/CN117136236B/zh
Priority to EP23866946.9A priority patent/EP4596693A1/en
Publication of WO2024060649A1 publication Critical patent/WO2024060649A1/zh
Priority to US19/093,270 priority patent/US20250250574A1/en
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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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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 the priority of Chinese patent application CN2022112033373 titled “siRNA, drugs and applications for inhibiting TMPRSS6 gene expression” submitted on September 29, 2022.
  • the disclosure of this invention is incorporated herein by reference; the present invention It also requests priority from the Chinese patent application 2023100881658 titled “siRNA, drugs and applications for inhibiting TMPRSS6 gene expression” submitted on January 19, 2023. Part of the content of this invention is incorporated herein by reference.
  • the invention belongs to the field of biomedicine, and specifically relates to siRNA or salts thereof, medicines and applications thereof for inhibiting the expression of TMPRSS6 gene.
  • RNA interference is the process by which messenger RNA (mRNA) is cleaved by small interfering RNA (siRNA), and is widely used to study the regulation of gene expression in various cells. Delivery of siRNA to cells in vitro can significantly inhibit gene expression (Jackson et al. 2006, RNA 12: 1179-1187.). In the past few years, RNA interference (RNAi) has become the most widely used gene knockout technology (Hu et al. 2020, Signal Transduct Target Ther 5: 101.).
  • RNAi Small interfering RNA
  • miRNA microRNA
  • RISC RNA-induced silencing complex
  • RISC RNA-induced silencing complex
  • ⁇ -thalassemia is one of the most common genetic diseases, usually caused by mutations in the ⁇ -globin gene or its promoter, resulting in reduced or missing ⁇ -globin synthesis (Cao and Kan 2013, Cold Spring Harb Perspect Med 3 :a011775.Rivella 2015, Haematologica 100:418-430.). More than 180 different ⁇ -globin gene mutations have been found in patients with ⁇ -thalassemia, and they may occur at any level between transcription, processing of primary messenger ribonucleic acid transcripts, translation, or post-translational stability of the gene product Affect gene function.
  • Beta-thalassemia typically presents with ineffective erythropoiesis and consequent extramedullary hematopoiesis, splenomegaly, and systemic iron loading. Affected individuals show extreme clinical heterogeneity, ranging from being almost asymptomatic to exhibiting life-threatening severe anemia. Beta-thalassemia can be divided into thalassemia major and relatively less severe anemia (non-transfusion-dependent thalassemia). or thalassemia intermedia) (Musallam et al. 2021, Am J Hematol 96:1518-1531.).
  • iron homeostasis In patients with thalassemia, hepcidin, the main regulator of iron homeostasis, is chronically suppressed. Leading to uncontrolled absorption of dietary iron and insufficient iron retention by the reticuloendothelial system, patients absorb abnormally high levels of iron, exacerbating ineffective erythropoiesis and anemia, requiring iron chelation to prevent clinical sequelae associated with iron overload (Richard et al .2020, Am J Hematol 95:68-77). Iron homeostasis in patients needs to be regulated to avoid toxicity due to its excess (Nualkaew et al. 2021, Mol Ther 29:2841-2853.). If left untreated, iron overload can lead to organ failure and death.
  • TMPRSS6 inhibits hepcidin activation by cleaving membrane heme, thereby playing a role in iron homeostasis. Inhibiting the expression of TMPRSS6 can regulate iron homeostasis, reduce iron overload, and thereby alleviate the symptoms of thalassemia (Ginzburg and Rivella 2011, Blood 118: 4321-4330.).
  • TMPRSS6-HCM-9 is an siRNA targeting TMRPSS6 and is in phase I clinical trials for the treatment of thalassemia.
  • Data from a previously conducted Phase I clinical trial of SLN124 in healthy volunteers showed that SLN124 is safe and effective in reducing plasma iron levels, and has a long-lasting effect.
  • the SLN124 sequence is published in patent US_11174483_B2. There are currently few drugs for treating thalassemia on the market, and siRNA therapy has good market prospects.
  • Polycythemia can be divided into two types: secondary polycythemia and vera. Secondary polycythemia can be alleviated by improving the inducing cause.
  • Polycythemia vera PV
  • MPDS myeloproliferative disorder
  • Hct hematocrit
  • Hb hemoglobin
  • the loss of function of the TMPRSS6 gene leads to excessive production of hepcidin, which in turn improves abnormal erythropoiesis and prevents or limits iron overload.
  • Oligonucleotides targeting TMPRSS6 provide a solution for the prevention or treatment of polycythemia.
  • an antisense oligonucleotide drug IONIS-TMPRSS6-LRx (ISIS 702843) targeting TMPRSS6 is in Phase II clinical trials for the treatment of polycythemia vera.
  • IONIS-TMPRSS6-LRx ISIS 702843
  • siRNAs will be more helpful in the treatment of the above diseases and provide more options for clinical treatment.
  • the purpose of the present invention is to provide a double-stranded siRNA or a pharmaceutically acceptable salt thereof for inhibiting the expression of TMPRSS6 and its application, which has the advantages of good in vitro and in vivo test results and weak non-target activity.
  • a first aspect of the present invention provides an siRNA or a pharmaceutically acceptable salt thereof for inhibiting the expression of TMPRSS6, which consists of a sense strand and an antisense strand.
  • the sense strand and the antisense strand form a double strand through base pairing.
  • TMPRSS6 which consists of a sense strand and an antisense strand.
  • the sense strand and the antisense strand form a double strand through base pairing. , whose base sequence is selected from any group shown in a-e or a sequence that differs from it by one, two or three nucleotides:
  • Double-stranded siRNA SEQ ID NO:BBD-2051 in which the sense strand sequence is as shown in UGGGAACUUACUACAACUCCA (SEQ ID NO:103), and the antisense strand sequence is as shown in UGGAGUUGUAGUAAGUUCCCAGG (SEQ ID NO:104).
  • Double-stranded siRNA SEQ ID NO:BBD-2083 in which the sense strand sequence is as shown in UGCUACUCUGGUAUUUCCUAA (SEQ ID NO:167), and the antisense strand sequence is as shown in UUAGGAAAUACCAGAGUAGCACC (SEQ ID NO:168);
  • Double-stranded siRNA SEQ ID NO: BBD-2047 in which the sense strand sequence is as shown in CGCCUGGGAACUUACUACAAU (SEQ ID NO:95), and the antisense strand sequence is as shown in AUUGUAGUAAGUUCCCAGGCGGG (SEQ ID NO:96);
  • siRNA SEQ ID NO: BBD-2086 double-stranded siRNA SEQ ID NO: BBD-2086, wherein the sense strand sequence is shown as GGGUGCUACUCUGGUAUUUCA (SEQ ID NO: 173), and the antisense strand sequence is shown as UGAAAUACCAGAGUAGCACCCCC (SEQ ID NO: 174);
  • Double-stranded siRNA SEQ ID NO: BBD-2087 in which the sense strand sequence is as shown in GGGGUGCUACUCUGGUAUUUA (SEQ ID NO:175), and the antisense strand sequence is as shown in UAAAUACCAGAGUAGCACCCCCG (SEQ ID NO:176).
  • the sense strand includes no more than 3, 2, 1, or 0 unmodified nucleotides, and the modified nucleotides in the sense strand include 2'-O-methyl Modified nucleotides, 2'-deoxynucleotides, 2'-fluoro modified nucleotides, the sense strand contains 1-3 phosphorothioate bonds at the 5'-end.
  • the antisense strand includes no more than 3, 2, 1, or 0 unmodified nucleotides
  • the modified nucleotides in the antisense strand include 2'-O -Methyl-modified nucleotides, 2'-deoxynucleotides, 2'-fluoro-modified nucleotides, 5'-(E)-vinylphosphonic acid nucleotides and isoglycerol nucleotides (isoGNA )
  • the antisense strand contains 1-3 phosphorothioate bonds at each of the 5'-end and 3'-end; a double-stranded RNAi reagent, wherein at least one strand contains 3 having at least 1-3 nucleotides 'Protruding end.
  • the isoglycerol nucleotide is located on the antisense strand, preferably at at least 1 of the 4-8 positions from the 5' end, so that the siRNA or its pharmaceutical
  • the above acceptable salt has low off-target activity and good safety profile.
  • positions 11-13 at the 5' end of the complementary paired siRNA antisense strand or positions 9-11 at the 5' end of the sense strand contain a total of 1-6 2'-deoxynucleotides.
  • isoglycerol nucleotide is at least one of the following structures:
  • the 2'-deoxynucleotide is at least one of the following structures:
  • the names of the modified duplexes are: BBD-2051.210, BBD-2051.211, BBD-2051.28, BBD-2083.313, BBD-2083.413, BBD-2051.25, BBD-2051.26, BBD-2051.27, BBD-2051.28, BBD-2051.29, BBD-2051.213, BBD-2051.214, BBD-2051.21 5.
  • the modified siRNA is:
  • Antisense strand VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmA*mG*mG,
  • Antisense strand VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmA*mG*mG,
  • Antisense strand VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmA*mG*mG,
  • Antisense strand VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC,
  • Antisense strand VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC,
  • a second aspect of the present invention provides pharmaceutical preparations for inhibiting TMPRSS6 expression, including any of the above-mentioned siRNA or a pharmaceutically acceptable salt thereof, and a targeted delivery ligand connected or packaged with the siRNA. or delivery vehicle.
  • the structure of the ligand is as follows:
  • the ligand is linked to the 3' end of the sense strand of the siRNA.
  • the ligand is connected to the 3' end of the sense strand of siRNA in the following manner:
  • the fourth aspect of the present invention is to provide the application of the biological preparation or pharmaceutical preparation in the preparation of drugs for the prevention and treatment of diseases related to TMPRSS6 or iron overload or diseases associated with iron overload.
  • the fifth aspect of the present invention is to provide a method for inhibiting the expression of TMPRSS6 in cells, which method includes:
  • step (b) Maintaining the cells generated in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting the expression of the TMPRSS6 gene in the cell.
  • a sixth aspect of the present invention provides a method for treating a subject suffering from TMPRSS6-related disorders or iron overload or iron overload-related conditions, comprising administering to the subject a therapeutically effective amount of any of the above.
  • siRNA or a pharmaceutically acceptable salt thereof or any of the above-mentioned biological agents or pharmaceutical agents thereby treating the subject.
  • the cells are within the subject.
  • the subject is a human.
  • TMPRSS6 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.
  • the subject's serum hepcidin concentration is increased by at least about 10%; and/or wherein the subject's serum iron concentration is decreased by at least about 20%; and/or wherein the subject is The percent transferrin saturation in the subject was reduced by at least about 20%.
  • the disease associated with TMPRSS6 or iron overload or a disease associated with iron overload is polycythemia, thalassemia, hemochromatosis, myelodysplastic syndrome, porphyria cutanea tarda , aplastic anemia, sideroblastic anemia, iron-refractory iron deficiency anemia, hereditary anemia, severe chronic hemolysis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia or Microcytic anemia, long-term blood transfusion leading to iron overload, and non-alcoholic fatty liver disease (NAFLD)-related iron overload.
  • NAFLD non-alcoholic fatty liver disease
  • the thalassemia is selected from ⁇ -thalassemia, ⁇ -thalassemia and ⁇ -thalassemia.
  • the polycythemia is polycythemia vera.
  • the hereditary anemia is selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, Day-Bruch anemia, Schuh-Day syndrome, red blood cell membrane disorders, glucose-6 -Phosphate dehydrogenase deficiency and hereditary hemorrhagic telangiectasia.
  • the inventor of this application has studied siRNA sequences and through a large number of experiments, screened out a number of siRNA sequences that can be used to inhibit the expression of TMPRSS6, and based on this, further made appropriate modifications to improve the silencing ability of the target and Reduce non-target activity.
  • siRNA sequence with good biological activity in inhibiting the expression of TMPRSS6 was obtained. It is more active than TMPRSS6-HCM-9, which is the fastest progressing clinically, and is expected to be used in clinical trials. Treatment of diseases associated with TMPRSS6 expression or iron overload or iron overload-related diseases.
  • Figure 7 evaluates the off-target activity of different modified BBD-2083 sequences and AD-1554911 sequences.
  • Iron overload is the excessive deposition of iron in the body and leads to structural damage and dysfunction of important organs (especially the heart, liver, pituitary gland, pancreas and joints).
  • Polycythemia refers to the number of red blood cells and hemoglobin per unit volume of blood being higher than the upper limit of the reference value.
  • Hemochromatosis is a disease caused by iron metabolism disorders caused by excessive iron storage in the body due to a high-iron diet, massive blood transfusions, or systemic diseases.
  • Thalassemia (alphathalassaemia), also known as thalassemia (thalassemia), should be called "globin dysgenesis anemia” according to the National Medical Terminology Approval Committee. It is due to the blockage or complete inhibition of the synthesis of one or more globin peptide chains, resulting in abnormal composition of Hb components and causing chronic hemolytic anemia.
  • TMPRSS6 gene can be evaluated based on the level of any variable related to TMPRSS6 gene expression, such as TMPRSS6 mRNA level, TMPRSS6 protein level, hepcidin mRNA level, hepcidin protein level, transferrin saturation in tissue or serum levels, or iron levels. Inhibition can be assessed by a decrease in absolute or relative levels of one or more of these variables compared to control levels.
  • the control level may be any type of control level used in the art, such as pre-dose baseline levels, or levels measured from similar subjects, cells or samples, untreated or control treated, and known populations. level.
  • the disease associated with TMPRSS6 expression or iron overload or a disease associated with iron overload is polycythemia, thalassemia, hemochromatosis, myelodysplastic syndrome, porphyria cutanea tarda, Aplastic anemia, sideroblastic anemia, iron-refractory iron deficiency anemia, hereditary anemia, severe chronic hemolysis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia or minor Cellular anemia, long-term blood transfusion leading to iron overload and non-alcoholic fatty liver disease (NAFLD)-related iron overload.
  • NAFLD non-alcoholic fatty liver disease
  • siRNA agents of the invention to cells, such as cells within a subject (eg, a human subject, such as a subject suffering from a TMPRSS6-related disorder, such as hemochromatosis) can be accomplished through a variety of different pathways.
  • delivery can be performed by contacting cells with the siRNA of the invention either in vitro or in vivo.
  • In vivo delivery may also be by administering a composition comprising siRNA or a salt thereof to a subject.
  • any method of delivering nucleic acid molecules can refer to existing delivery technologies.
  • factors to be considered for delivering siRNA molecules include, for example, the biological stability of the delivered molecule. , prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue.
  • the formulation can be administered locally (for example, by direct injection or implantation into the tissue or topical administration). Topical administration to The therapeutic site is to maximize the local concentration of the drug agent, limit the exposure of the agent to systemic tissues that can be damaged by the agent or degrade the agent, and allow for a lower total dose of the iRNA molecule to be administered.
  • the RNA can be modified or delivered using a drug delivery system; both methods work to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modifications to the RNA or pharmaceutical carrier may also allow the siRNA composition to be targeted to the target tissue and avoid off-target effects.
  • siRNA molecules can be modified, such as lipid particles, by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.
  • the present invention also includes pharmaceutical compositions and preparations, which include any of the siRNAs of the present invention or salts thereof.
  • the pharmaceutical preparation contains a pharmaceutical composition of siRNA or a salt thereof described herein and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions containing the iRNA can be used to treat diseases or conditions related to TMPRSS6, such as hemochromatosis. These pharmaceutical compositions are formulated based on the mode of delivery.
  • compositions are formulated for systemic administration via parenteral delivery, such as intravenous (IV) delivery or subcutaneous (SC) delivery.
  • parenteral delivery such as intravenous (IV) delivery or subcutaneous (SC) delivery.
  • the pharmaceutical formulation of siRNA or a salt thereof may be administered in solution, preferably in a sterile solution, for example by injection.
  • a "therapeutically effective amount” includes, when administered to a patient for the treatment of a TMPRSS6-related disease, an amount of siRNA or a salt thereof sufficient to effect treatment of the disease (e.g., by attenuating, alleviating or maintaining the existing disease). , or one or more symptoms of a disease).
  • the "therapeutically effective amount” may depend on the pharmaceutical formulation of the siRNA or salt thereof, how the formulation is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, expression of TMPRSS6 of the patient to be treated vary depending on the stage of the mediated pathological process, the type of prior treatment or combination of treatments (if any), and other independent characteristics.
  • the "prophylactically effective amount” includes an amount of a pharmaceutical preparation containing siRNA or a salt thereof that is sufficient to prevent or alleviate symptoms when administered to a subject who has not yet experienced or exhibited symptoms of a TMPRSS6-related disease but may be susceptible to the disease.
  • the “preventatively effective amount” may depend on how the siRNA or its salt is administered, the risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, and expression of TMPRSS6 mediated by the patient to be treated. The stage of the pathological process, the type of previous treatment or combination of treatments (if any), and other independent characteristics vary.
  • “Therapeutically effective amount” or “prophylactically effective amount” also includes an amount of siRNA that provides a reasonable benefit-risk ratio acceptable for any treatment.
  • the ligand is preferably an N-acetylgalactosamine derivative (Galnac carrier), other types of delivery carriers such as lipids specifically delivered to liver cells Any body that can achieve delivery of siRNA can be used in the present invention.
  • Galnac carrier N-acetylgalactosamine derivative
  • GalNAc-nucleic acid is a monoconjugate formed by a carbohydrate compound and a nucleic acid.
  • N-acetylated galactosamine is covalently conjugated to the 3′ and/or 5′ end of the sense chain of RNA of different sequences in a trivalent state to form GalNAc-siRNA drugs, thereby achieving specific delivery to hepatocytes, and allowing the drug to enter the cells and exert its function through endocytosis.
  • Galnac vector and siRNA are as follows:
  • nucleotide monomers used in nucleic acid sequence representation are as follows:
  • the pharmaceutically acceptable salt of siRNA can be a sodium salt or a potassium salt.
  • a sodium salt of siRNA is produced.
  • a solid-phase oligonucleotide synthesis protocol is used to synthesize 0.2-1 ⁇ mol oligonucleotides.
  • the Galnac-containing siRNA sequence needs to be pre-packed in a Galnac-coupled CPG column. synthesized on. Add an aminolysis reagent to the synthesized oligonucleotide and incubate it at 45-80°C to separate the oligonucleotide from the solid-phase carrier and free the oligonucleotide.
  • the crude oligonucleotide was purified by ion-pairing HPLC method, and the collected product was dried in a vacuum centrifugal dryer to powder. The purified product was dissolved in DEPC water and analyzed by TOF LC-MS.
  • TMPRSS6-hcm-9 is the sequence entered clinical stage by Silence Therapeutics (publication number: CN113164768A), which is used for positive comparison sequences.
  • Unmodified siRNA was used in the screening assay at a final concentration of 1 nM.
  • 96-well plate RNA extraction and reverse transcription Use Dynabeads mRNA DIRECT kit (Ambion) to extract 96-well plate cell mRNA, aspirate the culture medium in the 96-well plate, wash it once with DPBS, and add 50-300 ⁇ l cell lysis solution to each well , then add 20-100 ⁇ l beads, shake on the oscillator, place the 96-well plate on the magnetic separation rack, suck away the lysate in the wells, add 50-300 ⁇ l washing buffer A to each well, pipette and place it on the magnetic separation rack, suck away the washing buffer A, then blow up the beads with washing buffer B, transfer to a new 96-well plate, place it on the magnetic separation rack, suck away the washing buffer B, and then blow up the beads with buffer B.
  • Real-time fluorescence quantitative PCR After reverse transcription, place the 96-well plate on the magnetic separation rack until the beads are adsorbed on the bottom, suck away the reversal reagent, add the prepared QPCR system to the 96-well PCR plate, and seal the plate with membrane The plate was sealed and PCR was performed on the StepOnePlus real-time PCR system (applied biosystems).
  • the negative control AD-1955 sequence is: CUUACGCUGAGUACUUCGAdTdT (SEQ ID NO.187)
  • Table 2 shows the experimental results of screening unmodified siRNA in Hep3B cells at a transfection concentration of 1 nM.
  • Table 3 describes multiple siRNA modified sense strand sequences.
  • Table 4 describes multiple siRNA modified antisense strand sequences.
  • Table 5 describes the modified siRNA double-stranded sequence
  • Table 6 shows the experimental results of screening modified siRNA double strands in Hep3B cells at a transfection concentration of 1 nM.
  • Table 7 shows the experimental results of screening modified siRNA double strands in Hep3B cells at a concentration of 10nM during transfection.
  • Table 8 describes multiple BBD-2051 modified sense strand sequences.
  • Table 9 describes multiple BBD-2051 modified antisense strand sequences.
  • Table 10 describes multiple BBD-2051 modified siRNA double-stranded sequences.
  • Example 3 Integrate the hTMPRSS6 (human TMPRSS6) gene into the liver-targeted AVV8 expression vector, prepare the virus [VectorBuilder, AAV8LP] and infect mice to obtain transgenic mice stably expressing hTMPRSS6.
  • Mouse hepatocytes were digested and extracted with collagenase through inferior vena cava perfusion. Filter through tissue cell strainer (BIOLOGIX, 15-1070) to obtain mouse primary liver cells with good activity, resuspended in DMEM culture medium + 10% FBS + 1X penicillin and streptomycin, and use Scepter automatic cell counter ( Millipore) to measure cell density.
  • tissue cell strainer BIOLOGIX, 15-1070
  • 96-well plate transfection Conduct in vitro experiments in mouse primary hepatocytes. Mix siRNA, Opti-MEM and INTERFERin (Polyplus transfection) in a 96-well plate and incubate at room temperature for 10 minutes (free uptake siRNA) No need to add INTERFERin), then add complete medium containing mouse primary hepatocytes to each well. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours.
  • 96-well plate RNA extraction and reversal Use Dynabeads mRNA DIRECT kit (Ambion) to extract 96-well plate cell mRNA, aspirate the culture medium in the 96-well plate, wash it once with DPBS, and add 50-300 ⁇ l cell lysis solution to each well.
  • Real-time fluorescence quantitative PCR After the inversion, place the 96-well plate on the magnetic separation rack until the beads are adsorbed on the bottom, suck away the inversion reagent, add the prepared QPCR system to the 96-well PCR plate, and seal the plate with a sealing film plate, and PCR was performed on the StepOnePlus real-time PCR system (applied biosystems). Data were analyzed using the ⁇ Ct method and assays were normalized using cells transfected with 1 nM AD-1955.
  • Table 11 shows the experimental results of screening multiple BBD-2051 modified siRNA double-stranded sequences delivered by Galnac at a concentration of 1 nM in mouse primary hepatocytes expressing hTMPRSS6.
  • Table 12 shows the experimental results of screening the double-stranded sequences of BBD-2051 modified siRNA in Hep3B cells at a transfection concentration of 1 nM.
  • Table 13 shows the experimental results of screening the BBD-2051 modified siRNA double-stranded sequence at multiple concentrations via Galnac delivery in mouse primary hepatocytes expressing hTMPRSS6.
  • Table 14 describes multiple BBD-2083 modified sense strand sequences.
  • Table 15 describes multiple BBD-2083 modified antisense strand sequences.
  • Table 16 describes multiple BBD-2083 modified siRNA double-stranded sequences.
  • Table 17 shows the experimental results of screening multiple BBD-2083 modified siRNA double-stranded sequences and AD-1554911 delivered by Galnac at 1 nM concentration in mouse primary hepatocytes expressing hTMPRSS6
  • Table 18 shows the experimental results of BBD-2083 modified siRNA double-stranded sequence and AD-1554911 screening at a transfection concentration of 1 nM in Hep3B cells.
  • Table 19 shows the experimental results of BBD-2083 modified siRNA double-stranded sequence and AD-1554911 screening at a transfection concentration of 0.3nM in Hep3B cells.
  • Table 20 describes multiple BBD-2047 modified sense strand sequences.
  • Table 21 describes multiple BBD-2047 modified antisense strand sequences.
  • Table 22 describes multiple BBD-2047 modified siRNA double-stranded sequences.
  • Table 23 shows the experimental results of screening multiple BBD-2047 modified siRNA double-stranded sequences delivered by Galnac at a concentration of 10 nM in mouse primary hepatocytes expressing hTMPRSS6.
  • Table 24 shows the experimental results of screening multiple BBD-2047 modified siRNA double-stranded sequences delivered by Galnac at a concentration of 5 nM in mouse primary liver cells expressing hTMPRSS6
  • Table 25 shows the experimental results of screening multiple BBD-2047 modified siRNA double-stranded sequences in primary mouse hepatocytes expressing hTMPRSS6 via Galnac delivery at a concentration of 1 nM.
  • Table 26 shows the experimental results of screening multiple BBD-2047 modified siRNA double-stranded sequences delivered by Galnac at a concentration of 0.5 nM in mouse primary hepatocytes expressing hTMPRSS6.
  • Table 27 shows the experimental results of BBD-2047 modified siRNA double-stranded sequence and TMPRSS6-HCM-9 screening at a concentration of 10 nM in Hep3B cells.
  • Example 4 evaluates the IC50 of different sequences in inhibiting hTMPRSS6 expression in Hep3B cells.
  • siRNA was tested with 100nM as the highest concentration and 3-fold gradient dilution at 8 concentration points.
  • 96-well plate RNA extraction and reversal Use Dynabeads mRNA DIRECT kit (Ambion, Cat.61021) to extract 96-well plate cell mRNA, aspirate the culture medium in the 96-well plate, wash it once with DPBS, and add 50-300 ⁇ l to each well.
  • Real-time fluorescence quantitative PCR After reverse transcription, place the 96-well plate on the magnetic separation rack until the beads are adsorbed on the bottom, suck away the reversal reagent, add the prepared QPCR system to the 96-well PCR plate, and seal the plate with membrane The plate was sealed and PCR was performed on the StepOnePlus real-time PCR system (applied biosystems).
  • Table 28 shows the activity results of different modified double-stranded sequences in Hep3B cells
  • Adenovirus integrates hTMPRSS6
  • Transgenic mice stably expressing hTMPRSS6 were obtained by infecting mice with 1-10x10 11 purified recombinant AAV8 virus particles.
  • mice Fourteen days after the mice were injected with the virus, the mice were evenly divided into groups, with 4 mice in each group. siRNA was dissolved in physiological saline and injected subcutaneously at doses of 1 mg/kg and 3 mg/kg.
  • the liver was harvested at different time points after siRNA injection, and the RNA in the liver tissue was extracted using TRI REAGENT (MRC, Cat. No.: TR118).
  • the extracted RNA was reverse transcribed into cDNA using PrimeScript RT regent Kit (Takara, Cat. No.: RR047A).
  • the prepared QPCR system was added to the 96-well PCR plate, sealed with a sealing film, and QPCR was performed on the StepOnePlus real-time PCR system (applied biosystems) to detect the expression of hTMPRSS6.
  • the liver was removed 14 days after siRNA injection and the expression of hTMPRSS6 was detected.
  • the results are shown in Figure 1.
  • the dose was 1 mg/kg, the active expression of different modified sequences in the body was different. Multiple sequences showed significant effects on degrading target genes in mice. , its activity is higher than TMPRSS6-hcm-9-Galnac.
  • the liver was removed at different time points after siRNA injection to detect the expression of hTMPRSS6.
  • the results are shown in Figure 2.
  • the dose of 3mg/kg, the duration of the drug effect of BBD-2083.31-Galnac and BBD-2083.313-Galnac is longer than that of TMPRSS6-hcm-9-Galnac.
  • AD-1554911-Galnac has a long drug effect, and we found that adding VP modification to the BBD-2083 sequence has higher in vivo activity (BBD-2083.313-GalnacVS BBD-2083.31-Galnac).
  • the liver was taken at different time points after siRNA injection to detect the expression of hTMPRSS6.
  • the results are shown in Figure 3.
  • the dosage of 3 mg/kg was used.
  • the activity of different modified BBD-2051 was very good, and the drug effect lasted longer than TMPRSS6-hcm-9-Galnac.
  • the drug has a long duration of effect.
  • the subcutaneous dose of wild-type mice is 10 mg/kg.
  • the livers are harvested on days 7 and 21.
  • RNA in the liver tissue is extracted using TRI REAGENT (MRC, Cat. No.: TR118).
  • the extracted RNA is extracted using PrimeScript RT regent Kit (Takara, Cat. No.: RR047A). Reverse transcribed into cDNA.
  • the prepared QPCR system was added to the 96-well PCR plate, sealed with a sealing film, and QPCR was performed on the StepOnePlus real-time PCR system (applied biosystems) to detect mTMPRSS6 expression.
  • the results are shown in Figure 4.
  • the results showed that BBD-2051.210 and BBD-2051.211 could reduce endogenous TMPRSS6 expression in wild-type mice.
  • the subcutaneous dose of wild-type mice is 10 mg/kg.
  • a small amount of blood is taken from the tail of the mouse. Let it stand at room temperature for 30 minutes. Centrifuge at 1000xg for 10 minutes. Take the supernatant serum and follow the Iron Assay Kit-Colorimetric detection kit to dilute the serum and add Reducer. solution, continue to add 100 ⁇ l Probe Solution after incubating at 37°C for 15 minutes, and continue incubating at 37°C for 1 hour. Detect the absorbance at 593nm and calculate the iron content of the serum sample based on the standard curve. The results are shown in Figure 5. The results showed that BBD-2051.210 and BBD-2051.211 could reduce the iron content in the serum of wild-type mice.
  • the psiCheck2-Off-target plasmid was co-transfected with siRNA, and the experiment was performed in a 24-well plate. 6 to 8 hours before transfection, cells were inoculated into 0.5 mL of cell growth medium per well. Transfect 50ng psiCheck2-Off-target plasmid and 0.0001nM to 100nM siRNA through transfection reagent, and culture at 37°C for 24h.
  • Dual-Luciferase Reporter Assay System (Promega, cat#E1980) was used to detect the fluorescence signal values of Renilla fluorescence and firefly luciferase. Calculate the normalized value.
  • Normalized value Renilla luciferase reading enzyme/firefly luciferase reading enzyme.
  • Relative expression fold normalized value of experimental group/normalized value of control group.
  • mice, rat, dog, monkey and human serum or liver S9 were incubated with BBD-2051.210-Galnac at 37°C for 24 hours in vitro to evaluate the metabolic stability of BBD-2051.210-Galnac.
  • the results are shown in Table 29, BBD-2051.210-Galnac double chain in the serum of different species, the serum stability of the antisense chain is mouse > monkey > rat > human > dog, and the serum stability of the sense chain is monkey > rat > mouse > Human >Dog; the results are shown in Table 30.
  • BBD-2051.210-Galnac double strands are in liver S9 of different species.
  • the stability of the antisense chain in liver S9 is dog > human > monkey > mouse > rat.
  • the sense chain is in liver S9.
  • the stability of S9 is dog>mouse>human>monkey>rat.
  • Rat liver homogenate was incubated with BBD-2051.210-Galnac at 37°C for 72 hours in vitro to evaluate the metabolic stability of BBD-2051.210-Galnac. After incubation of BBD-2051.210-Galnac in liver homogenate for 72 hours, the percentage of antisense strand remaining was 94.98 and the percentage of sense strand remaining was 54.24. It can be seen from the data that the BBD-2051.210-Galnac chain is stable in liver homogenate.

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Abstract

本发明提供了用于抑制人体细胞中TMPRSS6表达的siRNA或者药学上可接受的盐，以及对所述siRNA进行了合适的修饰，以提高靶标的沉默能力并降低非靶点活性。本发明还提供了含有上述siRNA的用于抑制TMPRSS6表达的生物制剂或者药物制剂。本发明通过一系列体外和体内试验，获得了具有很好的抑制TMPRSS6表达生物活性的siRNA序列，且比临床上进展最快的TMPRSS6-HCM-9活性更高，有望临床应用于与TMPRSS6表达异常相关的疾病、或者铁超负荷或铁过载相关联疾病的治疗，具有很好的应用前景。

Description

用于抑制TMPRSS6基因表达的siRNA或其盐、药物及其应用
本发明要求2022年9月29日提交的名称为《用于抑制TMPRSS6基因表达的siRNA、药物及其应用》的中国专利申请CN2022112033373的优先权，该发明公开的内容通过援引合并于此；本发明还要求2023年01月19日提交的名称为《用于抑制TMPRSS6基因表达的siRNA、药物及其应用》的2023100881658的中国专利申请的优先权，该发明的部分内容通过援引合并于此。
技术领域
本发明属于生物医药领域，具体是涉及用于抑制TMPRSS6基因表达的siRNA或其盐、药物及其应用。
背景技术
RNA干扰(RNAi)是信使RNA(mRNA)被小干扰RNA(siRNA)切割的过程，广泛用于研究各种细胞中基因表达的调节。在体外将siRNA递送到细胞中可明显抑制基因表达(Jackson et al.2006,RNA 12:1179-1187.)。在过去的几年里，RNA干扰(RNAi)已成为最广泛使用的基因敲除技术(Hu et al.2020,Signal Transduct Target Ther 5:101.)。小干扰RNA(siRNA)或短单链RNA如microRNA(miRNA)与RNA诱导的沉默复合物(RISC)形成复合物，并进一步与互补的mRNA结合，mRNA在RNA酶的帮助下被切割以抑制特定基因的表达，RNAi的机制可以保护细胞免受病毒和转座子的侵害(Weingartner et al.2020,Mol Ther Nucleic Acids 21:242-250.)。
β-地中海贫血是最常见的遗传性疾病之一，通常是由β珠蛋白基因或其启动子的突变引起β-珠蛋白合成减少或缺失而导致(Cao and Kan 2013,Cold Spring Harb Perspect Med 3:a011775.Rivella 2015,Haematologica 100:418-430.)。在β-地中海贫血患者中发现了180多种不同的β-珠蛋白基因突变，它们可能在转录、初级信使核糖核酸转录物的加工、翻译或基因产物的翻译后稳定性之间的任何水平上影响基因功能。β-地中海贫血通常表现为无效的红细胞生成和随之而来的髓外造血、脾肿大和系统性铁负载。受影响的个体表现出极端的临床异质性，从几乎无症状到表现出危及生命的严重贫血，β-地中海贫血可以分为重型地中海贫血，相对不太严重的贫血(非输血依赖性地中海贫血或中间型地中海贫血)(Musallam et al.2021,Am J Hematol 96:1518-1531.)。
在地中海贫血患者中，铁稳态的主要调节剂铁调素长期受到抑制。导致膳食铁的吸收失控和网状内皮系统的铁保留不足，患者吸收异常高水平的铁，加剧了无效的红细胞生成和贫血，需要铁螯合以防止与铁过载相关的临床后遗症(Richard et al.2020,Am J Hematol 95:68-77)。需要调节患者体内的铁稳态，以避免由于其过量而产生毒性(Nualkaew et al.2021,Mol Ther 29:2841-2853.)。如果不加以治疗，铁过载会导致器官衰竭和死亡。由此，在β-地中海贫血和其他与铁相关的疾病中，铁过载的管理已成为主要焦点(Beliveau et al.2019,Cell Chem Biol 26:1559-1572e1559.)。铁调素的产生主要受一种由TMPRSS6基因编码的跨膜丝氨酸蛋白酶调节。TMPRSS6(跨膜蛋白酶，丝氨酸6)也称为蛋白裂解酶-2，这是一种II型丝氨酸蛋白酶。它主要在肝脏中表达，但在肾脏中也发现了高水平的TMPRSS6 mRNA， TMPRSS6通过切割膜血红素抑制铁调素活化，从而在铁稳态中起作用，抑制TMPRSS6的表达可以调节铁稳态，降低铁过载，从而缓解地中海贫血的病症(Ginzburg and Rivella 2011,Blood 118:4321-4330.)。
TMPRSS6-HCM-9是一种靶向TMRPSS6的siRNA，正在开展用于治疗地中海贫血的一期临床试验。之前开展的SLN124在健康志愿者中的一期临床试验数据显示SLN124在降低血浆铁水平，安全有效的，并且作用持续时间长。SLN124序列公布于专利US_11174483_B2中。目前市场上治疗地中海贫血药物少，siRNA疗法有很好的市场前景。
红细胞生成失败和慢性贫血导致铁代谢异常和全身铁过载，进而导致心脏、肝脏和内分泌功能障碍。地中海贫血症的治疗选择有限，主要的治疗手段是红细胞输血(Longo et al.2021,Int J Mol Sci 22.)。当前急需新的用于有效治疗与铁负载相关联的病症的方法。
红细胞增多症分为继发性红细胞增多症和真性红细胞增多症两种情况。继发性红细胞增多症改善诱发原因即可得到缓解。真性红细胞增多症(PV)是一种骨髓增生性疾病(MPDS)，常常导致异常高的红细胞压积(Hct)和血红蛋白(Hb)浓度，进而导致血栓栓塞并发症。TMPRSS6基因的功能丧失导致铁调素的过度产生，进而改善红细胞生成异常，防止或限制铁超载。靶向TMPRSS6的寡核苷酸为预防或治疗红细胞增多症提供了一种方案。目前一项靶向TMPRSS6的反义寡核苷酸药物IONIS-TMPRSS6-LRx(ISIS 702843)治疗真性红细胞增多症二期临床。开发更多的siRNA，更有助于上述疾病的治疗，也为临床治疗提供更多的选择。
发明内容
基于此，本发明的目的是提供一种用于抑制TMPRSS6表达的双链siRNA或其药学上可接受的盐及其应用，其具有体外、体内试验效果好、非靶点活性弱的优势。
本发明的第一个方面，提供了一种用于抑制TMPRSS6表达的siRNA或其药学上可接受的盐，由正义链及反义链组成，正义链和反义链通过碱基配对形成双链，其碱基序列选自a-e所示中的任意一组或与其相差一个、两个或三个核苷酸的序列：
a)双链siRNA SEQ ID NO:BBD-2051，其中正义链序列如UGGGAACUUACUACAACUCCA(SEQ ID NO:103)所示，所述反义链序列如UGGAGUUGUAGUAAGUUCCCAGG(SEQ ID NO:104)所示。
b)双链siRNA SEQ ID NO:BBD-2083，其中正义链序列如UGCUACUCUGGUAUUUCCUAA所示(SEQ ID NO:167)，所述反义链序列如UUAGGAAAUACCAGAGUAGCACC所示(SEQ ID NO:168)；
c)双链siRNA SEQ ID NO:BBD-2047，其中正义链序列如CGCCUGGGAACUUACUACAAU所示(SEQ ID NO:95)，所述反义链序列如AUUGUAGUAAGUUCCCAGGCGGG所示(SEQ ID NO:96)；
d)双链siRNA SEQ ID NO:BBD-2086，其中正义链序列如GGGUGCUACUCUGGUAUUUCA所示(SEQ ID NO:173)，所述反义链序列如UGAAAUACCAGAGUAGCACCCCC所示(SEQ ID NO:174)；
e)双链siRNA SEQ ID NO:BBD-2087，其中正义链序列如GGGGUGCUACUCUGGUAUUUA所示(SEQ ID NO:175)，所述反义链序列如UAAAUACCAGAGUAGCACCCCCG所示(SEQ ID NO:176)。
在其中的一些实施例中，所述正义链包括不多于3、2、1或0个未修饰的核苷酸，正义链中修饰的核苷酸，包含选自2'-O-甲基修饰的核苷酸、2'-脱氧核苷酸、2'-氟修饰的核苷酸，所述正义链在5'-末端含有1-3个硫代磷酸酯键。
在其中的一些实施例中，所述反义链包括不多于3、2、1或0个未修饰的核苷酸，所述反义链中修饰的核苷酸包含选自2'-O-甲基修饰的核苷酸、2'-脱氧核苷酸、2'-氟修饰的核苷酸、5’端-(E)-乙烯基膦酸核苷酸和异甘油核苷酸(isoGNA)，所述反义链在5'-末端和3'-末端各含有1-3个硫代磷酸酯键；双链RNAi试剂，其中至少一条链包含具有至少1-3个核苷酸的3'突出端。
在其中的一些优选的实施例中，所述异甘油核苷酸(isoGNA)位于反义链，优选位于5'端起的4-8位中的至少1位，以使所述siRNA或其药学上可接受的盐非靶点活性低，具有良好的安全性。
在其中的一些实施例中，互补配对的siRNA反义链5'端第11-13位或正义链5'端第9-11位共含有1-6个2'-脱氧核苷酸。
其中，所述异甘油核苷酸(isoGNA)为如下结构中的至少一种：
其中，所述2'-脱氧核苷酸为如下结构中的至少一种：
其中，所述尿苷-5’端-(E)-乙烯基膦酸核苷酸的结构如下：
在一些优选的实施例中，所述修饰双链体的名称为：BBD-2051.210、BBD-2051.211、 BBD-2051.28、BBD-2083.313、BBD-2083.413、BBD-2051.25、BBD-2051.26、BBD-2051.27、BBD-2051.28、BBD-2051.29、BBD-2051.213、BBD-2051.214、BBD-2051.215、BBD-2051.217、BBD-2051.218、BBD-2051.219；BBD-2083.41、BBD-2083.410、BBD-2083.411、BBD-2083.412、BBD-2083.414、BBD-2083.415、BBD-2083.416、BBD-2083.418、BBD-2083.419、BBD-2083.420、BBD-2083.422、BBD-2083.423、BBD-2083.424。
在一些更优选的实施例中，所述修饰的siRNA为：
BBD-2051.210：
正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
反义链：VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
BBD-2051.211：
正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
反义链：VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
BBD-2051.28：
正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
反义链：VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
BBD-2083.313：
正义链：mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA，
反义链：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
BBD-2083.413：
正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
反义链：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
本发明的第二个方面，提供了用于抑制TMPRSS6表达的药物制剂，包括上述任一所述siRNA或其药学上可接受的盐，以及与所述siRNA连接或包裹的靶向性递送配体或递送载体。
在其中一些实施例中，所述配体为N-乙酰基半乳糖胺(Galnac)衍生物。
在其中一些实施例中，所述配体的结构如下：

在其中一些实施例中，所述配体与siRNA的正义链的3’端连接。
在其中一些实施例中，所述配体与siRNA的正义链的3’端连接方式如下：

本发明的第三个方面，提供了上述siRNA或其药学上可接受的盐在制备抑制TMPRSS6表达的生物制剂或者药物制剂中的应用。
本发明的第四个方面，是提供所述生物制剂或者药物制剂在制备预防和治疗与TMPRSS6相关的疾病或铁超负荷或铁超载相关联的疾病药物中的应用。
本发明的第五个方面，是提供一种抑制TMPRSS6在细胞内表达的方法,该方法包括:
(a)将细胞与上述任一种的siRNA或其药学上可接受的盐或上述任一种的生物制剂或者药物制剂接触；和
(b)将步骤(a)中产生的细胞维持足以获得TMPRSS6基因的mRNA转录本的降解的时间,从而抑制TMPRSS6基因在细胞中的表达。
本发明的第六个方面，是提供一种治疗患有TMPRSS6相关病症或铁超负荷或铁超载相关联的受验者的方法，包含对该受验者给药治疗有效量的上述任一种siRNA或其药学上可接受的盐或上述任一种所述的生物制剂或者药物制剂，从而治疗该受验者。
在其中一些实施例中，其中所述细胞在受试者内。
在其中一些实施例中，其中对象是人。
在其中一些实施例中，其中TMPRSS6表达被抑制至少约30％、约40％、约50％、约60％、约70％、约80％、约90％、约95％、约98％或约100％。
在其中一些实施例中，所述受试者的血清铁调素浓度增加至少约10％；和/或其中所述受试者的血清铁浓度降低至少约20％；和/或其中所述受试者中的转铁蛋白饱和百分比降低至少约20％。
在其中一些实施例中，所述与TMPRSS6相关的疾病或铁超负荷或铁超载相关联的疾病为红细胞增多症、地中海贫血、血色素沉着症、骨髓增生异常综合征、迟发性皮肤卟啉症、再生障碍性贫血、铁粒幼细胞性贫血、铁难治性缺铁性贫血、遗传性贫血、严重慢性溶血、帕金森病、阿尔兹海默症、弗里德赖希氏共济失调或小细胞贫血、长期输血导致铁过载及非酒精性脂肪性肝病(NAFLD)相关的铁过载。
在其中一些实施例中，所述地中海贫血选自α-地中海贫血、β-地中海贫血和δ-地中海贫血。
在其中一些实施例中，所述红细胞增多症是真性红细胞增多症。
在其中一些实施例中，所述遗传性贫血选自镰状细胞性贫血、地中海贫血、范可尼贫血、戴-布二氏贫血、舒-戴二氏综合征、红细胞膜障碍、葡萄糖-6-磷酸脱氢酶缺乏和遗传性出血性毛细血管扩张症。
本申请的发明人经过对siRNA序列研究，并通过大量的实验，筛选到多条能够用于抑制TMPRSS6表达的siRNA序列，并在此基础上，进一步进行了合适的修饰以提高靶标的沉默能力并降低非靶点活性。其中，通过一系列体外和体内试验中，获得了具有很好的抑制TMPRSS6表达生物活性的siRNA序列，且比临床上进展最快的TMPRSS6-HCM-9活性更高，有望应用于临床上进行与TMPRSS6表达相关的疾病或铁超负荷或铁过载相关联疾病的治疗。
附图说明
图1在表达hTMPRSS6的小鼠中评估不同修饰的BBD-2047序列、BBD-2051序列、BBD-2083序列和TMPRSS6-HCM-9序列对肝中hTMPRSS6表达影响(1mg/kg)。
图2在表达hTMPRSS6的小鼠中评估不同修饰的BBD-2083序列和和TMPRSS6-HCM-9序列对肝中hTMPRSS6表达影响(3mg/kg)。
图3在表达hTMPRSS6的小鼠中评估不同修饰的BBD-2051序列和TMPRSS6-HCM-9序列对肝中hTMPRSS6表达影响(3mg/kg)。
图4在野生型小鼠中评估不同修饰的BBD-2051序列对肝中mTMPRSS6表达影响(10mg/kg)。
图5在野生型小鼠中评估不同修饰的BBD-2051序列对血中铁含量的表达影响(10mg/kg)。
图6评估不同修饰的BBD-2051序列非靶点活性。
图7评估不同修饰的BBD-2083序列和AD-1554911序列非靶点活性。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
定义 为了便于理解本技术，下面定义了一些术语和短语。
铁过载是铁在体内过度沉积，并导致重要脏器(尤其是心脏、肝脏、垂体、胰腺和关节)的结构损害和功能障碍。
红细胞增多症是指单位容积血液中红细胞数量及血红蛋白量高于参考值高限。
血色素沉着症是由于高铁饮食、大量输血或全身疾病造成体内铁质储积过多，而发生的铁质代谢障碍所致的疾病。
地中海贫血(alphathalassaemia)又称海洋性贫血(thalassemia)，据全国医学名词审定委员会规定应称为“珠蛋白生成障碍贫血”。是由于一种或多种珠蛋白肽链合成受阻或完全抑制，导致Hb成分组成异常，引起慢性溶血性贫血。
TMPRSS6基因的表达可基于与TMPRSS6基因表达相关的任何变量的水平而评价，如组织或血清中的TMPRSS6 mRNA水平、TMPRSS6蛋白质水平、铁调素mRNA水平、铁调素蛋白质水平、运铁蛋白饱和度水平、或铁水平。抑制可通过此等变量之一者或多者的绝对水平或相对水平相较于对照水平的下降而评价。该对照水平可为该技术领域中采用的任何类型的对照水平，如给药前基线水平、或自未治疗或经对照治疗的类似受验者、细胞或样本测得的水平、以及已知族群水平。
在其中一些实施例中，与TMPRSS6表达相关的疾病或铁超负荷或铁超载相关联的疾病为红细胞增多症、地中海贫血、血色素沉着症、骨髓增生异常综合征、迟发性皮肤卟啉症、再生障碍性贫血、铁粒幼细胞性贫血、铁难治性缺铁性贫血、遗传性贫血、严重慢性溶血、帕金森病、阿尔兹海默症、弗里德赖希氏共济失调或小细胞贫血、长期输血导致铁过载及非酒精性脂肪性肝病(NAFLD)相关的铁过载。
将本发明的siRNA剂递送至细胞，如受验者(如人受验者，如患有TMPRSS6相关病症如血色素沉着症的受验者)内的细胞，可通过多种不同路径达成。举例而言，可通过令细胞与本发明的siRNA或于活体外或于活体内接触而施行递送。活体内递送亦可通过将包含siRNA或其盐的组成物给药至受验者。
通常，任一递送核酸分子的方法(活体外或活体内可参考已有的递送技术。对于活体内递送，为了递送siRNA分子考虑的因素系包括，举例而言，所递送的分子的生物学安定性、非专一性效果的预防、以及所递送的分子到达目标组织内的蓄积。可通过局部给药(举例而言，通过直接注射或植入组织或外用给药)制剂。局部给药至治疗位点是最大化该药物制剂的局部浓度，限制该剂曝露至可被该剂伤害或可令该剂降解的全身性组织，以及容许待给药的iRNA分子的较低总剂量。
对于全身性给药iRNA而治疗疾病，该RNA可经修饰或使用药物递送系统递送；两种方法皆作用以防止通过活体内的核酸内切酶及核酸外切酶造成的dsRNA的快速降解。对RNA或药学载剂的修饰亦可容许该siRNA组成物靶向至目标组织，并避免脱靶效果。siRNA分子可通过化学接合至亲脂性基如胆固醇而修饰，例如脂质颗粒，以提升细胞摄取并防止降解。
本发明也包括药物组成物及制剂，其包括本发明的任一所述siRNA或其盐。在其中一些实施例中，所述药物制剂含有本文中描述的siRNA或其盐以及药学可接受的载体的药物组成物。含有该iRNA之药物组成物有用于治疗与TMPRSS6相关的疾病或病症，如血色素沉着症。此等药物组成物基于递送模式而配制。
在其中一些实施例中，配制为用于经由不经肠道递送如静脉(IV)递送或皮下(SC)递送而全身给药的组成物。于本发明的方法中，该siRNA或其盐的药物制剂可于溶液中给药，优选在无菌溶液中给药，例如通过注射给药。
本发明中，“治疗有效量”包括，当对患者给药用于治疗TMPRSS6相关疾病时，siRNA或其盐的量系足以造成该疾病的治疗(如，透过削弱、减轻或维持现有疾病、或疾病的一种或多种症候)。该“治疗有效量”可依据该siRNA或其盐的药物制剂、该制剂如何给药、疾病及其严重性、及待治疗的患者的病史、年龄、体重、家族病史、基因组成、通过TMPRSS6表现介导的病理过程的阶段、先前治疗或联合治疗的类型(若有)、及其他独立特征而改变。
本发明中，“预防有效量”包括，当对尚未经历或显现TMPRSS6相关疾病的症候但可能易患该疾病的受验者给药时，包含siRNA或其盐的药物制剂的量足以预防或减轻该疾病或该疾病的一种或多种症候。减轻该疾病包括延缓该疾病的进程或降低后续发展的疾病的严重性。该“预防有效量”可依据该siRNA或其盐的药物制的如何给药、疾病的风险程度、及待治疗的患者的病史、年龄、体重、家族病史、基因组成、通过TMPRSS6表现介导的病理过程的阶段、先前治疗或联合治疗的类型(若有)、及其他独立特征而改变。
“治疗有效量”或“预防有效量”也包括一种siRNA的量，以任何治疗可接受的合理的效益、风险比。
本发明所述靶向性递送配体或其他类型递送载体，所述配体优选为N-乙酰基半乳糖胺衍生物(Galnac载体)，其他类型递送载体如特异性递送到肝脏细胞的脂质体只要能实现siRNA的递送都可用于本发明。
近年来，Galnac载体目前已经有比较深入研究，GalNAc-核酸是糖类化合物与核酸形成的单缀合物，将N-乙酰化的半乳糖胺以三价态的方式共价缀合到不同序列的RNA的正义链3′和/或5′末端，形成GalNAc-siRNA药物，从而实现向肝细胞的特异性递送，并通过细胞内吞作用使药物进入细胞和发挥功能。
以下实施例中，Galnac载体及连接siRNA后的结构如下：
核酸序列表示中使用的核苷酸单体的缩写及结构如下：
具体结构如下：

本领域技术人员根据已有的技术，所述siRNA的药学上可接受的盐可为钠盐或钾盐，例如，在纯化的时候，产生siRNA的钠盐。
以下结合具体实施例对本发明作进一步详细的说明。
实施例1 siRNA的合成
在北京擎科生物科技有限公司12通道核酸合成仪上，使用固相寡核苷酸合成方案进行0.2-1μmol的寡核苷酸合成，含Galnac的siRNA序列需要在偶联Galnac的CPG预填充柱上进行合成。在合成的寡核苷酸中加入氨解试剂，并在45-80℃下孵育，将寡核苷酸与固相载体分离使寡核苷酸游离下来。然后用乙醇沉淀粗制寡核苷酸，高速离心弃去上清液，重复两遍获得粗制寡核苷酸，并将沉淀重悬于DEPC水中。通过离子配对HPLC方法对粗制寡核苷酸进行纯化，将收集到的产物在真空离心干燥器中干燥至粉末状。用DEPC水溶解纯化后得到的产物，并通过TOF LC-MS进行分析。测定寡核苷酸浓度，计算等摩尔量的正义链、反义链所需体积，将等摩尔量的正义链、反义链混合均匀，通过95℃加热5分钟，然后自然冷却至室温的退火方法制备双链。
表1：未修饰siRNA的正义链和反义链序列。TMPRSS6-hcm-9为Silence Therapeutics公司进入临床阶段的序列(公开号：CN113164768A)，用于阳性比对序列。





实施例2在Hep3B细胞中采用脂质体转染方法进行siRNA体外筛选
细胞培养和96孔板转染：在Hep3B细胞中进行体外实验，采用MEM+10％FBS+1X青霉素链霉素+1X非必需氨基酸培养基，细胞平铺面积达80％时用胰蛋白酶消化，并用Scepter自动细胞计数仪(Millipore，#PHCC00000)测定细胞密度，同时在96孔板中将siRNA、Opti-MEM和INTERFERin(Polyplus transfection)混合并置于室温孵育10分钟，然后每孔加入含有Hep3B细胞的完全培养基，将96孔板放于37℃，5％CO2的培养箱中孵育24小时。
未修饰的siRNA以1nM最终浓度进行筛选试验。
96孔板RNA提取、反转录：使用Dynabeads mRNA DIRECT试剂盒(Ambion)提取96孔板细胞mRNA，将96孔板中培养基吸走，用DPBS清洗一次，每孔加入50-300μl细胞裂解液，再加入20-100μl beads,在振荡器上震荡，将96孔板置于磁分离架上，吸走孔内裂解液，每孔加入50-300μl的清洗缓冲液A，吹打后置于磁分离架上，吸走清洗缓冲液A，然后用清洗缓冲液B将beads吹起，转移到新的96孔板，置于磁分离架，吸走清洗缓冲液B，再用缓冲液B将beads吹起转移到96孔PCR板中，同时配制反转试剂，将96孔PCR板置于磁分离架上，吸走清洗缓冲液B，每孔加入20μl的反转试剂，用封板膜封板后在PCR仪上进行25℃孵育10分钟，然后37℃孵育两小时后85℃，5分钟，降温至4℃，反转录结束。
实时荧光定量PCR：反转录结束后将96孔板置于磁分离架上直到beads都吸附在底部，吸走反转试剂，将配好的QPCR体系加入到96孔PCR板中，封板膜封板，在StepOnePlus的实时PCR系统(applied biosystems)上进行PCR。
采用ΔΔCt方法分析数据，并采用1nM阴性对照序列转染的细胞进行测试标准化。
阴性对照AD-1955序列为：CUUACGCUGAGUACUUCGAdTdT(SEQ ID NO.187)
UCGAAGUACUCAGCGUAAGdTdT(SEQ ID NO.188)。
表2为Hep3B细胞中未修饰siRNA在转染1nM浓度下筛选的实验结果


所得实验结果显示，表1中的siRNA在1nM浓度下对Hep3B细胞中TMPRSS6的表达产生了不同程度的抑制。经过比较和综合考虑后，我们对其中部分活性好的序列进行修饰，以提高其体内的稳定性及活性。
表3描述了多条siRNA修饰正义链序列。


表4描述了多条siRNA修饰反义链序列。

表5描述了修饰siRNA双链序列



表6为Hep3B细胞中修饰siRNA双链在转染1nM浓度下筛选的实验结果
表7为Hep3B细胞中修饰siRNA双链在转染10nM浓度下筛选的实验结果

接下来对上述活性比较好的BBD-2051.11、BBD-2083.11、BBD-2047.11三条序列分别进行进一步的修饰，以找到最适合的修饰方法。
表8描述了多条BBD-2051修饰正义链序列。

表9描述了多条BBD-2051修饰反义链序列。
表10描述了多条BBD-2051修饰siRNA双链序列


实施例3：将hTMPRSS6(人源TMPRSS6)基因整合到肝靶向的AVV8表达载体，制备病毒【VectorBuilder，AAV8LP】后感染小鼠，获得稳定表达hTMPRSS6的转基因小鼠。
小鼠原代肝细胞提取：通过下腔静脉灌注法，用胶原酶消化提取小鼠肝细胞。经组织细胞滤网(BIOLOGIX，15-1070)过滤，得到活性良好的小鼠原代肝细胞，重悬于DMEM培养基+10％FBS+1X青霉素链霉素中，用Scepter自动细胞计数仪(Millipore)测定细胞密度。
96孔板转染：在小鼠原代肝细胞中进行体外实验，在96孔板中分别将siRNA、Opti-MEM和INTERFERin(Polyplus transfection)混合并置于室温孵育10分钟(自由摄取方式siRNA则无需添加INTERFERin)，然后每孔加入含有小鼠原代肝细胞的完全培养基。将96孔板放于37℃，5％CO2的培养箱中孵育24小时。
96孔板RNA提取、反转：使用Dynabeads mRNA DIRECT试剂盒(Ambion)提取96孔板细胞mRNA，将96孔板中培养基吸走，用DPBS清洗一次，每孔加入50-300μl细胞裂解液，再加入20-100μl beads,在振荡器上震荡后，将96孔板置于磁分离架上，吸走孔内裂解液，每孔加入50-300μl的清洗缓冲液A，吹打后置于磁分离架上，吸走清洗缓冲液A，然后用清洗缓冲液B将beads吹起，转移到新的96孔板，置于磁分离架上，吸走清洗缓冲液B，再用缓冲液B将beads吹起转移到96孔PCR板中，同时配好反转录试剂，将96孔PCR板置于磁分离架上，吸走清洗缓冲液B，每孔加入20μl的反转试剂，用封板膜封板后在PCR仪上进行25℃孵育10分钟，然后37℃孵育两小时后85℃，5分钟，降温至4℃，反转录结束。
实时荧光定量PCR：反转结束后将96孔板置于磁分离架上直到beads都吸附在底部，吸走反转试剂，将配好的QPCR体系加入到96孔PCR板中，封板膜封板，在StepOnePlus的实时PCR系统(applied biosystems)上进行PCR。采用ΔΔCt方法分析数据，并采用1nM AD-1955转染的细胞进行测试标准化。
表11为在表达hTMPRSS6的小鼠原代肝细胞中多条BBD-2051修饰siRNA双链序列通过Galnac递送在1nM浓度下筛选的实验结果。
表12为Hep3B细胞中BBD-2051修饰siRNA双链序列在转染1nM浓度下筛选的实验结果。
表13为在表达hTMPRSS6的小鼠原代肝细胞中BBD-2051修饰siRNA双链序列通过Galnac递送在多个浓度下筛选的实验结果。
表14描述了多条BBD-2083修饰正义链序列。

表15描述了多条BBD-2083修饰反义链序列。
表16描述了多条BBD-2083修饰siRNA双链序列。



表17为在表达hTMPRSS6的小鼠原代肝细胞中多条BBD-2083修饰siRNA双链序列和AD-1554911通过Galnac递送在1nM浓度下筛选的实验结果

表18为Hep3B细胞中BBD-2083修饰siRNA双链序列和AD-1554911在转染1nM浓度下筛选的实验结果
表19为Hep3B细胞中BBD-2083修饰siRNA双链序列和AD-1554911在转染0.3nM浓度下筛选的实验结果
通过上述实验我们发现，2083这条序列，通过加入VP修饰后，体外活性得到了大幅加强。
表20描述了多条BBD-2047修饰正义链序列。
表21描述了多条BBD-2047修饰反义链序列。

表22描述了多条BBD-2047修饰siRNA双链序列。



表23为在表达hTMPRSS6的小鼠原代肝细胞中多条BBD-2047修饰siRNA双链序列通过Galnac递送在10nM浓度下筛选的实验结果
表24为在表达hTMPRSS6的小鼠原代肝细胞中多条BBD-2047修饰siRNA双链序列通过Galnac递送在5nM浓度下筛选的实验结果

表25为在表达hTMPRSS6的小鼠原代肝细胞中多条BBD-2047修饰siRNA双链序列通过Galnac递送在1nM浓度下筛选的实验结果

表26为在表达hTMPRSS6的小鼠原代肝细胞中多条BBD-2047修饰siRNA双链序列通过Galnac递送在0.5nM浓度下筛选的实验结果
表27为Hep3B细胞中BBD-2047修饰siRNA双链序列和TMPRSS6-HCM-9在10nM浓度下筛选的实验结果
实施例4评估不同序列在Hep3B细胞中抑制hTMPRSS6表达的IC50。
细胞培养和96孔板转染：在Hep3B细胞中进行体外实验，采用MEM+10％FBS+1X青霉素链霉素(Gibco,Cat.15070-063)+1X非必需氨基酸(cell cook,Cat.CM1008L)培养基，细胞平铺面积达80％时用胰蛋白酶消化，并用Scepter自动细胞计数仪(Millipore，#PHCC00000)测定细胞密度，同时在96孔板中将siRNA与Opti-MEM和INTERFERin(Polyplus transfection)混合并置于室温孵育10分钟，然后每孔加入含有Hep3B细胞的完全培养基，将96孔板放于37℃，5％CO2的培养箱中孵育24小时。
siRNA以100nM为最高浓度，3倍梯度稀释8个浓度点进行试验。
96孔板RNA提取、反转：使用Dynabeads mRNA DIRECT试剂盒(Ambion,Cat.61021)提取96孔板细胞mRNA，将96孔板中培养基吸走，用DPBS清洗一次，每孔加入50-300μl细胞裂解液，再加入20-100μl beads,在振荡器上震荡，将96孔板置于磁分离架上，吸走孔内裂解液，每孔加入50-300μl的清洗缓冲液A，吹打后置于磁分离架上，吸走清洗缓冲液A，然后用清洗缓冲液B将beads吹起，转移到新的96孔板，置于磁分离架，吸走清洗缓冲液B，再用缓冲液B将beads吹起转移到96孔PCR板中，同时配制反转试剂，将96孔PCR板置于磁分离架上，吸走清洗缓冲液B，每孔加入20ul的反转试剂，用封板膜封板后在PCR仪上进行25℃孵育10分钟，然后37℃孵育两小时后85℃，5分钟，降温至4℃，反转录结束。
实时荧光定量PCR：反转录结束后将96孔板置于磁分离架上直到beads都吸附在底部，吸走反转试剂，将配好的QPCR体系加入到96孔PCR板中，封板膜封板，在StepOnePlus的实时PCR系统(applied biosystems)上进行PCR。
采用ΔΔCt方法分析数据，并采用1nM阴性对照序列转染的细胞进行测试标准化。计算各浓度点对阴性对照的降低百分比，并通过S形剂量-反应曲线拟合计算IC50值。
表28为Hep3B细胞中不同修饰双链序列的活性结果
实施例5在表达hTMPRSS6的小鼠中评估不同修饰的BBD-2047序列、BBD-2051序列和BBD-2083序列对肝中hTMPRSS6表达影响
1.腺病毒整合hTMPRSS6
通过1-10x1011个纯化重组AAV8病毒颗粒感染小鼠，获得稳定表达hTMPRSS6的转基因小鼠。
2.给药
小鼠注射病毒14天后，将小鼠平均分组，每组4只。siRNA用生理盐水溶解，皮下注射，给药剂量1mg/kg和3mg/kg。
3.取肝检测
siRNA注射后不同时间点取肝，通过TRI REAGENT(MRC，货号：TR118)提取肝组织中的RNA，用PrimeScript RT regent Kit(Takara,货号：RR047A)将提取的RNA反转录为cDNA。配好的QPCR体系加入到96孔PCR板中，封板膜封板，在StepOnePlus的实时PCR系统(applied biosystems)上进行QPCR，检测hTMPRSS6表达量。
siRNA注射后14天取肝，检测hTMPRSS6表达量，结果参见图1，给药剂量1mg/kg时不同修饰的序列在体内的活性表达不同，多条序列显示出显著的小鼠体内降解靶基因效果，其活性比TMPRSS6-hcm-9-Galnac更高。
siRNA注射后不同时间点取肝，检测hTMPRSS6表达量，结果参见图2，给药剂量3mg/kg，BBD-2083.31-Galnac和BBD-2083.313-Galnac的药效时间持续比TMPRSS6-hcm-9-Galnac和AD-1554911-Galnac药效时间长，且我们发现在BBD-2083这条序列中加入VP修饰后，其体内活性更高(BBD-2083.313-GalnacVS BBD-2083.31-Galnac)。
siRNA注射后不同时间点取肝，检测hTMPRSS6表达量，结果参见图3，给药剂量3mg/kg,不同修饰BBD-2051的活性均很好，且药效时间持续比TMPRSS6-hcm-9-Galnac药效时间长。
实施例6在野生型小鼠中评估不同修饰BBD-2051序列对肝中mTMPRSS6和mHepcidin1表达以及血清中铁含量的影响
野生型小鼠皮下剂量10mg/kg，7、21天取肝，通过TRI REAGENT(MRC，货号：TR118)提取肝组织中的RNA，用PrimeScript RT regent Kit(Takara,货号：RR047A)将提取的RNA反转录为cDNA。配好的QPCR体系加入到96孔PCR板中，封板膜封板，在StepOnePlus的实时PCR系统(applied biosystems)上进行QPCR，检测mTMPRSS6表达量，结果如图4所示。结果表明，BBD-2051.210和BBD-2051.211可以降低野生型小鼠内源TMPRSS6表达。
野生型小鼠皮下剂量10mg/kg，第7天断尾取少量血，室温静置30分钟，1000xg离心10分钟，取上清血清，按照Iron Assay Kit-Colorimetric检测试剂盒，稀释血清后加入Reducer solution，37℃培养15min后继续加入100μl Probe Solution，继续37℃培养1h。检测593nm处吸光度，根据标准品曲线计算得到血清样本的铁含量，结果参考图5。结果表明，BBD-2051.210和BBD-2051.211可以降低野生型小鼠血清中铁的含量。
实施例7评估不同修饰的BBD-2051序列和BBD-2083序列非靶点活性
设计siRNA对应的Off-Target序列，将Off-target序列合成并连接到psiCheck2载体上。psiCheck2-Off-target质粒与siRNA共转染，24孔板中进行实验，在转染前6～8小时，每孔0.5mL细胞生长培养基中接种细胞。通过转染试剂转染50ng psiCheck2-Off-target质粒和0.0001nM至100nM siRNA，37℃培养24h。用Dual-Luciferase Reporter Assay System(Promega,cat#E1980)检测海肾荧光和萤火虫荧光素酶的荧光信号值。计算归一化值。归一化值＝海肾荧光素酶读数酶/萤火虫荧光素酶读数。相对表达倍数＝实验组归一化值/对照组归一化值。
通过实验可知，参见图6，通过在反义链的第5、6和7位引入ISOGNA，体外实验结果显示BBD-2051修饰序列(特别是BBD-2051.29，BBD-2051.210)非靶点活性很低，具有良好的安全性。
参见图7，体外实验结果显示BBD-2083修饰序列非靶点活性低，具有良好的安全性。实施例8评估BBD-2051.210在血清和肝脏S9中的体外代谢稳定性
将小鼠、大鼠、犬、猴和人血清或肝S9与BBD-2051.210-Galnac在体外37℃下孵育24小时，评估BBD-2051.210-Galnac的代谢稳定性。通过实验数据，结果参见表29， BBD-2051.210-Galnac双链在不同种属的血清中，反义链在血清稳定性为小鼠>猴子>大鼠>人>犬，正义链在血清稳定性为猴子>大鼠>小鼠>人>犬；结果参见表30，BBD-2051.210-Galnac双链在不同种属的肝S9中，反义链在肝S9稳定性为犬>人>猴子>小鼠>大鼠，正义链在肝S9稳定性为犬>小鼠>人>猴子>大鼠。
表29 BBD-2051.210-Galnac血清稳定性结果
表30 BBD-2051.210-Galnac肝S9稳定性结果
实施例9评估BBD-2051.210在肝匀浆中的体外代谢稳定性
将大鼠肝匀浆与BBD-2051.210-Galnac在体外37℃下孵育72小时，评估BBD-2051.210-Galnac的代谢稳定性。BBD-2051.210-Galnac在肝匀浆中孵育72小时后，反义链剩余的百分比为94.98，正义链剩余的百分比54.24。由数据可知，BBD-2051.210-Galnac链在肝匀浆中是稳定的。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (19)

  1. 用于抑制细胞中TMPRSS6表达的siRNA或其药学上可接受的盐，其由正义链及反义链组成，其特征在于，其碱基序列选自a-e所示中的任意一组：
    a)BBD-2051,其正义链如SEQ ID NO.103所示，反义链如SEQ ID NO.104所示；
    b)BBD-2083,其正义链如SEQ ID NO.167所示，反义链如SEQ ID NO.168所示；
    c)BBD-2047,其正义链如SEQ ID NO.95所示，反义链如SEQ ID NO.96所示；
    d)BBD-2086,其正义链如SEQ ID NO.173所示，反义链如SEQ ID NO.174所示；
    e)BBD-2087,其正义链如SEQ ID NO.175所示，反义链如SEQ ID NO.176所示。
  2. 根据权利要求1所述的siRNA或其药学上可接受的盐，其特征在于，所述正义链包括不多于3、2、1或0个未修饰的核苷酸，所述正义链中的修饰的核苷酸包含分别选自2'-O-甲基修饰的核苷酸、2'-脱氧核苷酸、2'-氟修饰的核苷酸，所述正义链在5'-末端含有1，或2，或3个硫代磷酸酯键，所述2'-脱氧核苷酸为如下结构中的至少一种：
    所述反义链包括不多于3、2、1或0个未修饰的核苷酸，所述反义链中的修饰的核苷酸包括分别选自2'-O-甲基修饰的核苷酸、2'-脱氧核苷酸、2'-氟修饰的核苷酸、5’端-(E)-乙烯基膦酸核苷酸和异甘油核苷酸，所述反义链在5'-末端和3'-末端各含有1-3个硫代磷酸酯键；所述异甘油核苷酸(isoGNA)选自如下结构一种：
    所述尿苷-5’端-(E)-乙烯基膦酸核苷酸的结构如下：
  3. 根据权利要求2所述的siRNA或其药学上可接受的盐，其特征在于，所述异甘油核苷酸位于反义链，且位于5'端起的4-8位中的至少1位。
  4. 根据权利要求1-3任一项所述的siRNA或其药学上可接受的盐，其特征在于，所述siRNA的至少一条链包含具有至少1-3个核苷酸的3'突出端；且互补配对的siRNA反义链5'端第11-13位或正义链5'端第9-11位共含有1-6个2'-脱氧核苷酸。
  5. 根据权利要求1-3任一项所述的siRNA或其药学上可接受的盐，其中，经过修饰的siRNA选自以下任意一种：
    BBD-2051.210：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.211：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.28：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*fG*mGmAmGmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2083.313：
    正义链：mU*mG*mCmUmAmCfUmCfUfGfGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.413：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2051.25：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：mU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.26：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：mU*fG*mGmAmGisoGNA-TmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.27：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：mU*fG*mGmAmGmUisoGNA-TmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.29：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*fG*mGmAisoGNA-GmUmUmGmUfAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.213：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：mU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG；
    BBD-2051.214：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：mU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.215：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：mU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.217：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*dG*mGmAisoGNA-GmUmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.218：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*dG*mGmAmGisoGNA-TmUmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2051.219：
    正义链：mU*mG*mGmGmAmAfCmUfUfAfCmUmAmCmAmAmCmUmCmCmA，
    反义链：VPU*dG*mGmAmGmUisoGNA-TmGmUmAmGdTmAfAmGfUmUmCmCmCmA*mG*mG、
    BBD-2083.41：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*fU*mAmGmGmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.410：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.411：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.412：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.414：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*fU*mAmGisoGNA-GmAmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.415：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*fU*mAmGmGisoGNA-AmAmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.416：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*fU*mAmGmGmAisoGNA-AmAmUfAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.418：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.419：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.420：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：mU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.422：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*dT*mAmGisoGNA-GmAmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.423：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*dT*mAmGmGisoGNA-AmAmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC、
    BBD-2083.424：
    正义链：mU*mG*mCmUmAmCfUmCfUfGdGmUmAmUmUmUmCmCmUmAmA，
    反义链：VPU*dT*mAmGmGmAisoGNA-AmAmUmAmCdCmAfGmAfGmUmAmGmCmA*mC*mC。
  6. 根据权利要求1-5任一项所述的siRNA或其药学上可接受的盐，其特征在于，所述药学上可接受的盐为钠盐或钾盐。
  7. 权利要求1-6任一项所述的siRNA或其药学上可接受的盐在制备用于抑制TMPRSS6表达的生物制剂或者药物制剂中的应用。
  8. 一种用于抑制TMPRSS6表达的生物制剂或者药物制剂，其特征在于，包括权利要求1-6任一项所述的siRNA或其药学上可接受的盐，以及与所述siRNA连接或包裹的靶向性递送配体或递送载体。
  9. 根据权利要求8所述的生物制剂或者药物制剂，其特征在于，所述配体为N-乙酰基半乳糖胺衍生物；或递送载体为特异性递送到肝脏细胞的脂质体、多肽或抗体。
  10. 根据权利要求9所述的生物制剂或者药物制剂，其特征在于，所述N-乙酰基半乳糖胺衍生物的结构如下：
  11. 根据权利要求10所述的生物制剂或者药物制剂，其特征在于，所述N-乙酰基半乳糖胺衍生物与siRNA的正义链的3'端连接方式如下：
  12. 权利要求8-11任一项所述的生物制剂或者药物制剂在制备用于抑制细胞中TMPRSS6表达的药物中的应用。
  13. 权利要求8-11任一项所述的生物制剂或者药物制剂在制备预防或治疗与铁超负荷或铁过载相关联的病症的药物中的应用。
  14. 根据权利要求12或13所述应用，其特征在于，所述与TMPRSS6表达相关的疾病或铁超负荷或铁过载相关联的疾病为红细胞增多症、地中海贫血、血色素沉着症、骨髓增生异常综合征、迟发性皮肤卟啉症、再生障碍性贫血、铁粒幼细胞性贫血、铁难治性缺铁性贫血、遗传性贫血、严重慢性溶血、帕金森病、阿尔兹海默症、弗里德赖希氏共济失调或小细胞贫血、长期输血导致铁过载及非酒精性脂肪性肝病(NAFLD)相关的铁过载。
  15. 根据权利要求14所述应用，其特征在于，所述地中海贫血为α-地中海贫血、β-地中海贫血或δ-地中海贫血。
  16. 根据权利要求14所述应用，其特征在于，所述红细胞增多症是真性红细胞增多症。
  17. 根据权利要求14所述应用，其特征在于，所述遗传性贫血选自镰状细胞性贫血、地中海贫血、范可尼贫血、戴-布二氏贫血、舒-戴二氏综合征、红细胞膜障碍、葡萄糖-6-磷酸脱氢酶缺乏和遗传性出血性毛细血管扩张症。
  18. 一种抑制TMPRSS6在细胞内表达的方法,其特征在于，该方法包括:
    (a)将细胞与权利要求1所述的siRNA或其药学上可接受的盐或权利要求8所述的生物制剂或者药物制剂接触；和
    (b)将步骤(a)中产生的细胞维持足以获得TMPRSS6基因的mRNA转录本的降解的时间,从而抑制TMPRSS6基因在细胞中的表达。
  19. 一种治疗患有TMPRSS6相关病症或铁超负荷或铁超载相关联的受验者的方法，其特征在于，包含对该受验者给药治疗有效量的权利要求1所述的siRNA或其药学上可接受的盐或权利要求所述8的生物制剂或者药物制剂，从而治疗该受验者。
PCT/CN2023/095104 2022-09-29 2023-05-18 用于抑制TMPRSS6基因表达的siRNA或其盐、药物及其应用 Ceased WO2024060649A1 (zh)

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