WO2022100706A1 - Réactif d'arni et composition pour inhiber l'expression du gène surf4 - Google Patents

Réactif d'arni et composition pour inhiber l'expression du gène surf4 Download PDF

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WO2022100706A1
WO2022100706A1 PCT/CN2021/130426 CN2021130426W WO2022100706A1 WO 2022100706 A1 WO2022100706 A1 WO 2022100706A1 CN 2021130426 W CN2021130426 W CN 2021130426W WO 2022100706 A1 WO2022100706 A1 WO 2022100706A1
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surf4
mice
short antisense
antisense oligonucleotide
liver
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陈晓伟
王潇
许柏林
方欢
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北京大学
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    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • 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
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Definitions

  • the present invention relates to short antisense oligonucleotides and RNAi agents for inhibiting SURF4 gene expression, and compositions comprising one or more of the short antisense oligonucleotides or RNAi agents.
  • the present invention also relates to methods and uses of the short antisense oligonucleotides, RNAi agents or compositions for lowering blood lipids in a subject, or for treating or preventing diseases associated with dyslipidemia.
  • SURF4 is a transmembrane cargo receptor on the endoplasmic reticulum.
  • Transmembrane cargo receptors are generally believed to mediate the recognition of selected cargoes (such as proteins or lipids) by the envelope complex (Dancourt, J., and Barlowe, C. (2010). Annual review of biochemistry 79, 777-802).
  • the yeast homolog of SURF4, Erv29p mediates the transport of ⁇ -factors, wherein ER export of these cargoes is accelerated by concentrating the proteins to be transported into COPII vesicles (Belden, W.J., and Barlowe, C. (2001). Science 294, 1528-1531; and Otte, S., and Barlowe, C. (2004).
  • Lipids are important energy sources and structural components, and also undertake physiological functions such as signal transduction. Lipids differ from other biomolecules in that their bulk transport requires specialized lipoproteins because of their hydrophobicity. The mechanism by which lipid-carrying lipoproteins are differentiated from general proteins and selectively enter the secretory pathway is unclear. Dyslipidemia can lead to a variety of cardiovascular and metabolic diseases. For example, low-density lipoprotein cholesterol (LDL-C) is thought to be closely related to atherosclerosis. Arteriosclerosis can further cause stroke, coronary heart disease and other diseases with high mortality and disability rates, and is also related to hypertension, fatty liver, diabetes and other diseases. With the improvement of living standards, the incidence of diseases related to dyslipidemia in the population tends to increase.
  • LDL-C low-density lipoprotein cholesterol
  • LDL low density lipoprotein
  • MTP microsomal triglyceride transfer protein
  • ASO antisense oligonucleotides
  • PCSK9 inhibitors are new blood lipid-lowering drugs that have attracted much attention in recent years.
  • PCSK9-inhibitor lipid-lowering drugs including Amgen's Evolocumab, Sanofi's Alirocumab, and Novartis' small interfering RNA lipid-lowering drug Leqvio (inclisiran) High hopes.
  • PCSK9 proprotein convertase subtilisin Kexin type 9
  • LDLR low density lipoprotein receptor
  • PCSK9 inhibitors increase the clearance of LDL, the "bad blood lipid", by preventing the degradation of LDLR by PSCK9, thereby achieving the effect of lowering blood lipids.
  • PCSK9 inhibitors cannot achieve the desired LDL-lowering effect.
  • the inventors of the present invention have revealed a lipid carrier-specific transport program that can efficiently and precisely supply lipids in response to physiological demands, and whose entry is defined by the molecular switch SAR1B and the cargo receptor SURF4.
  • the inventors of the present invention discovered and confirmed for the first time that the inactivation of the cargo receptor-encoding gene SURF4 in the liver completely prevented pathogenic lipid abnormalities and atherosclerosis, even in the absence of LDL receptor (LDLR) In cases where lipid clearance is defective, it does not cause significant damage or inflammation of the liver.
  • LDLR LDL receptor
  • the inventors constructed an antisense oligonucleotide molecule capable of inhibiting the expression of the SURF4 gene for regulating lipid abnormalities (reducing blood lipids) and then treating and preventing diseases related to lipid abnormalities. this invention.
  • the present invention provides short antisense oligonucleotides of 8-30 nucleotides in length that target nucleotides encoding SURF4.
  • the short antisense oligonucleotides comprise 15-25 nucleotides, more preferably 19-23 nucleotides, eg, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24 or 25 nucleotides.
  • the short antisense oligonucleotide comprises a sequence selected from the antisense strand of Table 1 or Table 2 or a sequence that differs from it by 1 or 2 nucleotides, or consists of a sequence selected from Table 1 Or the sequence of the antisense strand of Table 2 or the sequence that differs from it by 1 or 2 nucleotides.
  • the present invention provides an RNAi agent for inhibiting the expression of the SURF4 gene, comprising the short antisense oligonucleotide of the first aspect, and a sense strand at least partially complementary to the short antisense oligonucleotide.
  • the sense strand is at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to the short antisense oligonucleotide over the length of the short antisense oligonucleotide .
  • the RNAi agent comprises a sense and/or antisense strand selected from Table 1 or Table 2. More preferably, the RNAi agent is selected from the combination of sense and antisense strands listed in Table 1 or Table 2.
  • the present invention provides a composition for inhibiting the expression of the SURF4 gene, comprising (a) the short antisense oligonucleotide of the first aspect or the RNAi agent of the second aspect, and (b) a pharmaceutically acceptable excipients.
  • the present invention provides the use of the short antisense oligonucleotide of the first aspect or the RNAi reagent of the second aspect in the preparation of a medicament for the treatment or prevention of dyslipidemia-related diseases such as cardiovascular and cerebrovascular diseases Disease or metabolic disease associated with dyslipidemia.
  • the disease is selected from the group consisting of hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, abnormal cholesterol metabolism, abnormal lipid metabolism, hyperLDL-C hyperlipidemia, atherosclerosis Sclerosing cardiovascular and cerebrovascular diseases such as myocardial infarction, stroke, and atherosclerosis.
  • the present invention provides the use of the short antisense oligonucleotide of the first aspect or the RNAi reagent of the second aspect in the preparation of a medicament for reducing low-density lipoprotein (LDL) in the blood of a subject. ), very low density lipoprotein (VLDL) and one, two or all three of triglycerides.
  • the medicament is for lowering low density lipoprotein (LDL) in the blood of a subject.
  • the present invention provides a method of treating or preventing a disease associated with dyslipidemia, such as a cardiovascular metabolic disease, in a subject, comprising administering to the subject a therapeutically effective amount of the RNAi agent of the second aspect or the third aspect composition.
  • the cardiometabolic disease is selected from the group consisting of hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, abnormal cholesterol metabolism, abnormal lipid metabolism, hyperLDL-C hyperlipidemia , Atherosclerotic cardiovascular disease such as atherosclerosis.
  • the present invention provides a method of reducing one, two or all three of low density lipoprotein (LDL), very low density lipoprotein (VLDL) and triglycerides in blood in a subject, comprising administering to the subject a therapeutically effective amount of the RNAi agent of the second aspect or the composition of the third aspect.
  • the method is for reducing low density lipoprotein (LDL) in the blood of a subject.
  • the subject is deficient in LDLR expression.
  • the subject is a patient with familial hyperlipidemia with a genetic defect, such as an inherited LDLR deficiency.
  • Figure 1 is a schematic diagram showing the co-action mechanism of SURF4 and SAR1B revealed by the inventors, both of which recognize lipoproteins and mediate their packaging into transport vesicles for ER export.
  • Figure 2 is a schematic diagram showing the transmembrane structure and sequence of SURF4.
  • Figure 3 is a mass spectrogram of SURF4 peptides identified as novel COPII factors using ortho-dependent proteomic studies. Proteins biotinylated using SAR1B-BirA* were then purified with streptavidin beads and visualized with avidin-HRP after separation by SDS-PAGE. Asterisk: SAR1B-BirA* fusion protein. Three independent experiments are shown as a representative. Right: SURF4 peptide identified by mass spectrometry.
  • Figure 4 is a schematic and experimental evidence illustrating the packaging of SURF4 into reconstituted COPII vesicles in a SAR1B-dependent manner.
  • Left Schematic representation of the in vitro reconstitution assay of COPII vesicles.
  • Right Immunoblotting of vesicle marker proteins with the indicated antibodies.
  • FIG. 5 shows a schematic diagram of the process of tissue-specific knockout of Surf4 in Example 2.
  • Figure 6A-B shows the results of knockout of SURF4 by CRISPR-mediated gene editing, in which three different Surf4-targeting gRNAs and a control gRNA were used to knock out Surf4 protein and mRNA in mouse liver,
  • A Results of immunoblotting of total liver protein from mice
  • B results of analysis of total mRNA from liver extracted from mice using qPCR (***: P ⁇ 0.001, two-tailed Student's t-test).
  • Figures 7A-B show the reduction of total cholesterol (A) and total triglyceride (B) levels in mouse plasma after knockdown of SURF4 by CRISPR-mediated gene editing.
  • Time points (x-axis) represent time after AAV injection. Data are presented as mean ⁇ SEM. ***: P ⁇ 0.001 (two-tailed Student's t-test).
  • Figures 8A-B show the cholesterol and triglyceride content of different lipoproteins in the control and Surf4 CRISPR mice of Figure 7A, (A) after fractionation of lipoproteins in plasma into VLDL, LDL and HDL by FPLC Cholesterol measurements, (B) Triglyceride measurements after fractionation of lipoproteins in plasma into VLDL, LDL and HDL by FPLC.
  • Figure 10 is a graph of histological staining of mouse liver sections showing that deficiency of hepatic Surf4 results in lipid accumulation in the liver. Upper part: H&E staining; lower part: Oil red O staining.
  • Figure 15 is a schematic diagram showing Surf4 (Surf4 flox) and deleted alleles between loxP sites.
  • 16A-C are bar graphs showing the dose-dependent role of Surf4 in lipid regulation. Plasma total cholesterol ( A ), triglyceride (B) and ApoB (C) levels. Data are presented as mean ⁇ SEM. Asterisk: ***: P ⁇ 0.001; ****: P ⁇ 0.0001 (two-tailed Student's t-test).
  • Figures 17A-B are bar graphs showing that Surf4 lowers blood lipids in a dose-dependent manner when fasted for 24 hours followed by a high fructose diet for 12 hours.
  • FIGS. 19A-B are photographs showing the intracellular localization of SURF4.
  • (B) SURF4 mAb validation in immunostaining, methanol-fixed hepatocytes isolated from WT or Surf4 KO mouse liver and stained with anti-SURF4 antibody, followed by confocal microscopy, scale bar 5 ⁇ m.
  • Figure 21 is a schematic diagram of a model describing the bidirectional shuttle of SURF4 between the ER and the Golgi.
  • Figure 22 is a schematic diagram depicting the design of CRISPR-resistant SURF4 cDNA.
  • Upper part mouse Surf4 cDNA sequence (SEQ ID NO:5) and corresponding protein sequence (SEQ ID NO:6); lower part: human SURF4 cDNA sequence (SEQ ID NO:7) and corresponding protein used for complementation experiments sequence (SEQ ID NO:8).
  • PAM regions and gRNA targeting sequences are indicated in dark and light gray, respectively. Mutations in the human SURF4 cDNA eliminated the PAM motif but did not alter the protein coding.
  • Figures 23A-B are bar graphs showing the results of repletion experiments demonstrating that Surf4 defective in recycling is unable to replenish lipoprotein secretion.
  • Figure 24 is an immunoblot showing that Sar1b deficiency increases Surf4-APOB interaction.
  • FLAG-tagged Surf4 was introduced into livers of WT or Sar1b LKO (KO) mice by AAV, liver samples were lysed and subjected to anti-FLAG IP, and total liver lysates (Inputs) or immunocomplexes were detected by immunoblotting after SDS-PAGE protein present in FLAG IP. Shown are representative results from three independent experiments.
  • Figure 25 is a bar graph showing the synergistic effect of Sar1b and Surf4 haploinsufficiency in lowering blood lipids.
  • Data are presented as mean ⁇ SEM. **: P ⁇ 0.01; ****: P ⁇ 0.0001, compared to CTL.
  • Statistical methods were one-way ANOVA test and Tukey posthoc test.
  • Figure 26 is the results of H&E and Oil Red O staining of liver samples obtained from the same mice as in Figure 25 .
  • 27A-D are bar graphs showing the coordination of SAR1B and SURF4 in lipid trafficking.
  • Figure 29 is a bar graph of plasma AST and ALT levels for the mice in Figure 28 after 3 months of feeding with a high cholesterol diet, with similar data for the three mice.
  • FIGS 32A-D show the effect of hepatic inactivation of Surf4 on cholesterol and triglyceride levels in different lipoprotein fractions.
  • Plasma cholesterol in different lipoprotein populations VLDL, LDL, HDL
  • CTL FPLC in control
  • hepatic Surf4 KO Surf4 LKO, triangles
  • hepatic Surf heterozygous Surf4 LHets, squares
  • mice Levels (A) and their area under the curve (B), and plasma triglyceride levels (C) and their area under the curve (G). Numbers in B and D indicate percent reduction in hepatic Surf4 heterozygous mice compared to controls.
  • ApoB-L AopB-containing lipoprotein that combines VLDL and LDL.
  • Figures 33A-D show the results of RNAi agents S4-1, S4-2, S4-3 in in vitro cell experiments.
  • A SURF4 mRNA level
  • B Western blotting result of SURF4 protein
  • C Western blotting result of APOB protein
  • D APOB level in medium.
  • Figures 34A-B show the results of ten candidate siRNA agents targeting murine SURF4 in in vitro cell experiments.
  • Figures 35A-B show the effect of siRNA agents targeting murine SURF4 in experiments in mice overexpressing human PCSK9.
  • A Serum cholesterol level
  • B Serum triglyceride level.
  • the term “about” means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, ie, the limitations of the measurement system. For example, “about” can mean within 1 or greater than 1 standard deviation, according to the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of the given value. Alternatively, particularly for biological systems or processes, the term may represent an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a numerical value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean within an acceptable error range for the particular value.
  • nucleotide generally refers to a base-sugar-phosphate combination. Nucleotides can include analogs or derivatives of nucleotides as well as synthetic nucleotides. Nucleotides can be labeled for detection by known techniques. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels.
  • expression refers to one or more processes by which polynucleotides are transcribed from a DNA template (eg, into mRNA or other RNA transcripts) and/or the transcribed mRNA is then translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.
  • Up-regulation or “down-regulation” of expression generally refers to an increase or decrease in the level of expression of a polynucleotide (eg, RNA, eg, mRNA) and/or polypeptide sequence relative to its level of expression in the wild-type state.
  • a polynucleotide eg, RNA, eg, mRNA
  • polypeptide sequence relative to its level of expression in the wild-type state.
  • modulate in reference to expression or activity refers to altering the level of expression or activity. Regulation can occur at the transcriptional and/or translational level.
  • subject means a vertebrate, preferably a mammal such as a mouse or a human. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • treatment refers to a method for obtaining beneficial or desired results, including but not limited to therapeutic benefit and/or prophylactic benefit.
  • treatment may comprise administering a therapeutically effective amount of a short antisense oligonucleotide, RNAi agent or composition directed against SURF4 of the invention.
  • Therapeutic benefit refers to any treatment-related improvement in one or more diseases or symptoms being treated.
  • prevention refers to the administration of a SURF4-targeting agent of the present invention to a subject at risk of developing a particular disease or symptom, or to a subject in the presence of one or more physiological signs of a disease, prior to the development of a particular disease. of short antisense oligonucleotides, RNAi agents or compositions, resulting in prophylactic benefit even though the disease or symptoms may not yet manifest.
  • an effective amount refers to the amount of a composition, such as the amount of a composition comprising an antisense oligonucleotide or RNAi agent of the invention, when administered to a subject in need thereof Sufficient to produce the desired activity or effect, such as a hypolipidemic effect.
  • Lipoproteins are complexes containing proteins and lipids. Lipoproteins present in human blood are classified according to lipid content and ultracentrifugation density and mainly include chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). Among them, LDL is responsible for transporting cholesterol from the liver to human cells and is a major risk factor for atherosclerosis, also known as "bad cholesterol lipoprotein” or "bad blood lipids”.
  • VLDL very low density lipoproteins
  • LDL low density lipoproteins
  • HDL high density lipoproteins
  • RNAi agent of the present invention can reduce blood lipids, especially LDL, VLDL and triglycerides in plasma.
  • Human SURF4 is a 269 amino acid protein with approximately 99% identity to its mouse homolog. Human SURF4 has six predicted transmembrane domains and a short cytoplasmic tail containing a tri-lysine sorting motif for COPI interaction and ER recycling (Jackson, L.P. et al. (2012) Dev Cell 23 , 1255-1262), as shown in Figure 2.
  • Newly synthesized proteins and lipids enter the secretory pathway via COPII-coated vesicles and are assembled at the endoplasmic reticulum (ER) by the GTPase SAR1, but how lipid-carrying lipoproteins are distinguished from ordinary protein cargoes in the ER The mechanism by which these lipoproteins are released and selectively secreted was not clear prior to the present invention.
  • SURF4 is a highly efficient COPII cargo receptor that transmembrane multiple times.
  • Genome-wide association analysis (GWAS) and functional characterization studies also found that plasma LDL-cholesterol in humans is closely related to SURF4, and that acute inactivation of Surf4 results in almost complete disappearance of plasma lipids and lipoproteins in mice, and thus protects mice from immune in developing atherosclerosis induced by PCSK9 and dietary challenge.
  • RNA interference RNA interference
  • the present invention provides antisense oligonucleotides targeting the SURF4 gene, and double-stranded RNAi reagents targeting the SURF4 gene.
  • the antisense oligonucleotide and double-stranded RNAi agent of the present invention can induce the degradation of SURF4 mRNA by acting on SURF4 mRNA.
  • Specific examples of double-stranded RNAi reagents targeting human SURF4 are listed in detail in Table 1, and specific examples of double-stranded RNAi reagents targeting murine SURF4 are listed in Table 2, but those skilled in the art can understand that this
  • the scope of the invention is not limited to these specific RNAi agents, but encompasses any RNAi agent that targets SURF4 mRNA and is capable of acting on SURF4 mRNA to produce the effect of reducing SURF4 mRNA levels.
  • Transcript 1 (SEQ ID NO: 10) is the most classical and common transcript and is preferably used as the basis for sequence design of antisense oligonucleotides and RNAi reagents. Antisense oligonucleotides and RNAi agents of the invention can also be designed based on other transcripts.
  • RNA molecules such as modifying nucleotides or linking with other groups
  • modifying RNA molecules such as modifying nucleotides or linking with other groups
  • improving the silencing efficiency of target genes, conferring Tissue/cell specificity, etc. but as long as the modified RNA molecule still contains 8-30 nucleotides as defined herein and has the ability to target the SURF4 gene, it falls within the scope of the present invention.
  • the antisense oligonucleotides, RNAi agents and compositions against SURF4 of the present invention can be used to prevent or treat diseases or symptoms related to lipid metabolism, especially cardiovascular metabolic diseases, more particularly atherosclerotic cardiovascular diseases disease (ASCVD).
  • the disease or condition is associated with abnormal lipids, especially abnormal elevations of lipids, such as diseases or conditions caused by abnormal elevations of lipids, such as abnormal elevations of triglycerides and/or cholesterol.
  • the cardiometabolic disease is selected from the group consisting of: hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, abnormal cholesterol metabolism, abnormal lipid metabolism, hyperlipidemia, atherosclerosis Cardiovascular diseases such as atherosclerosis.
  • the antisense oligonucleotides, RNAi agents and compositions against SURF4 of the present invention are particularly suitable for hyperLDL-C hyperemia patients.
  • the antisense oligonucleotides, RNAi agents and compositions of the present invention are also particularly suitable for those with LDLR defects such as genetic defects, and thus are not significantly responsive to drugs that improve LDLR function or act on LDLR inhibitors (such as PCSK9 inhibitors) or Non-responsive patients, eg, patients with familial hyperlipidemia.
  • the LDL-C in the patient's circulation can be reduced by 30%, 40%, 50%, or even more by administering the RNAi agent of the present invention.
  • RNAi agents of the invention can be delivered to a subject in a variety of ways.
  • RNAi agents or compositions of the invention are delivered to the subject in a tissue-specific manner, preferably in a liver-specific manner.
  • Tissue-specific delivery can be achieved depending on a variety of modalities.
  • liposomes with tissue-specific antibodies can be used, or non-nucleotide groups can be attached to RNAi agents to confer tissue-specific distribution and uptake.
  • RNAi agents can confer tissue-specific distribution and uptake.
  • US9370582B2 discloses a method for coupling RNAi molecules with GalNAc (N-acetylgalactosamine) to achieve liver-targeted delivery
  • GalNAc is a highly efficient ligand for the asialoglycoprotein receptor expressed on hepatocytes, It is thus possible to confer liver targeting specificity to the molecule to which it is coupled.
  • lipid nanoparticles are used to deliver the RNAi agent or composition.
  • LNPs lipid nanoparticles
  • the RNAi agents are delivered in the form of naked siRNA molecules.
  • it is preferable to modify it including but not limited to: sugar ring modification, such as substitution of fluoro (fluoro) or oxy-methyl (Ome) at position 2 of the ribose ring Hydrogen (H); backbone modifications such as phosphorothioate modifications (s).
  • sugar ring modification such as substitution of fluoro (fluoro) or oxy-methyl (Ome) at position 2 of the ribose ring Hydrogen (H)
  • backbone modifications such as phosphorothioate modifications (s).
  • mice housing and all experimental procedures were approved by the Peking University Laboratory Animal Care and Use Committee, which is an AAALAC-accredited animal institution. All mice used were bred on a C57BL6/J background. Mice were housed at 22°C with a 12-hour light/dark cycle and had free access to normal food and water unless otherwise stated. All mice used for the experiments were from in-housing mating. Animals were monitored for overall development and health according to AAALAC guidelines, and littermates were used as controls in experiments unless otherwise stated.
  • Sar1b liver-specific knockout mice were obtained by combining Sar1b fl/fl mice (KOMP: 061774, RRID: MMRRC_061774-UCD) with albumin promoter-driven Cre recombinase transgenic mice (JAX: 016833, RRID: IMSR_JAX: 016833 ) produced by hybridization.
  • Surf4 fl/fl mice were generated by a CRISPR/Cas9-based protocol using wild-type C57BL6/J mice. Briefly, Cas9 mRNA, sgRNA and donor plasmid were injected into zygotes. Microinjection was performed by BIOCYTOGEN. Founder Surf4 fl/+ mice were backcrossed with C57BL6/J for two rounds. Surf4 fl/fl mice were generated from offspring of Surf4 fl/+ mice. Cre-dependent spCas9 knock-in (KI) mice were purchased from Jackson Lab (RRID:IMSR_JAX:026556) (Platt et al., (2014). Cell 159, 440-455).
  • KI Cre-dependent spCas9 knock-in
  • mice Male mice aged 8-16 weeks were used in all experiments unless otherwise stated. Mice were assigned to different groups according to genotype or randomly assigned. For studies on specific diets, 8-week-old mice received a high-cholesterol diet for 12 weeks, or 8-week-old mice received a high-fructose diet for 2 weeks.
  • HEK293A cells (Thermo Fisher, R70507) were obtained from Thermo Fisher.
  • 293T cells (ATCC, CRL-3216, RRID: CVCL_0063) and HepG2 (ATCC, HB-8065, RRID: CVCL_0027) were obtained from ATCC.
  • Huh7 cells (JCRB Cell Ban, JCRB0403, RRID:CVCL_0336) were obtained from JCRB. Gender information for cell lines is available on the relevant supplier's website.
  • Primary hepatocytes from mice were isolated from the livers of male mice with the indicated genotypes.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • PEI polyethyleneimine
  • mice were fasted for 16 hours, blood was drawn from the tip of the tail using a heparin capillary, and plasma was obtained by centrifugation at 6000 rpm, 4°C for 5 minutes. Triglyceride and total cholesterol levels were measured using commercially available kits (Zhongsheng Beikong and Sigma) according to the instructions.
  • FPLC Fast Protein Liquid Chromatography
  • lipidomic analysis serum lipids were extracted by the Bligh-Dyer lipid extraction method, during which a lipid internal standard mixture (SPLASHTM Lipidomix) was added. The extracted lipids were subjected to LC-MS analysis as described in previous reports. Lipid separation was performed on an AQUITY UPLC system (Waters, Milford, MA) equipped with a CSH C18 column, 100 x 2.1 mm id, 1.7 ⁇ m (Waters, Milford, MA). Gradient elution was performed at a flow rate of 0.4 ml/min and the column was thermostated at 55°C.
  • AQUITY UPLC system Waters, Milford, MA
  • CSH C18 column 100 x 2.1 mm id, 1.7 ⁇ m
  • Mobile phase A consisted of water-ACN (40:60, v/v) supplemented with 10 mM ammonium formate and 0.1% formic acid
  • mobile phase B consisted of ACN-IPA (10:90, v/v) supplemented with 10 mM ammonium formate and 0.1% formic acid v/v) composition.
  • the initial conditions started at 40% B and immediately started a linear gradient from 40% to 43% B within 2 minutes (curve 6). Over the next 0.1 minutes, the percentage of mobile phase B was increased to 50%. Over the next 9.9 minutes, the gradient was further increased to 54% B and the amount of mobile phase B was increased to 70% within 0.1 minutes. In the last part of the gradient, mobile phase B increased to 99% in 5.9 minutes.
  • the eluent composition returned to its initial state within 0.1 min, and the column was equilibrated at initial conditions for 1.9 min before the next injection, resulting in a total run time of 20 min.
  • the injection was 2 ⁇ l and the autosampler temperature was set to 4°C. For all experiments, the same chromatographic conditions were used.
  • a UPLC system was used in conjunction with an equipped SYNAPT G2-S qTOF HDMS mass spectrometer (SYNAPT G2-S HDMS, Waters Corporation) for analysis.
  • Electrospray (ESI source) positive and negative ionization modes were used. Data were acquired in full scan mode in the m/z range of 50-1,200 Da by MassLynx 4.1 software. Unfragmented ion data and fragmented ion data were collected by alternating acquisitions running at alternating collision energies. Data alignment and ion chromatogram extraction were analyzed by Progenesis QI version. Lipid identification was performed using the LIPID MAPS database. R 3.5.1 was used to generate heatmaps.
  • mice were fasted for 16 hours and injected intravenously with tyloxapol at a dose of 500 mg/kg body weight. Blood samples were collected at the indicated time points, and plasma was separated by centrifugation. Triglycerides were determined as described above.
  • liver tissue was fixed in 4% PFA and then paraffin-embedded. Hematoxylin and eosin (H&E) staining and Sirius red staining were performed on 5 ⁇ m paraffin sections.
  • H&E Hematoxylin and eosin
  • Sirius red staining were performed on 5 ⁇ m paraffin sections.
  • liver samples were embedded in OCT gel and then snap frozen. Frozen tissues were cryosectioned at 8 ⁇ m and stained with Oil Red O according to the manufacturer’s instructions. Liver macrophages were visualized by immunohistochemical staining for F4/80.
  • the protein concentration of the samples was adjusted to 3 mg/mL using 100 mM TEAB.
  • the enriched proteins were denatured in 6M urea/TEAB, reduced with 10 mM dithiothreitol (DTT, J&K Scientific) for 35 min at 35°C, and concomitant with 20 mM iodoacetamide (Sigma-Aldrich) at 35°C Stir and seal in the dark for 30 minutes.
  • the reaction mixture was diluted to 2M urea/TEAB and digested with 0.6 ⁇ g trypsin overnight at 37°C with stirring. The digested samples were subjected to reductive dimethylation.
  • Immuno(co)precipitation experiments were extracted with buffer A (50 mM Tris, pH 7.5, 150 mM sodium chloride, 1% Nonidet P-40, and 10% glycerol, supplemented with protease inhibitor tablets (Roche)) at 4°C Proteins from mouse plasma, cultured cells, or mouse liver.
  • the cell extracts were centrifuged at 13000g for 15 minutes at 4°C, the supernatant was collected and 60ul of cell lysate was taken.
  • the remaining lysates were incubated with 15 ⁇ l of M2 or anti-HA agarose beads (Sigma-Aldrich) for 3 h at 4°C, then washed 4 to 8 times with buffer A (for mass spectrometry).
  • Immune complexes were then solubilized with 1.5x SDS/PAGE sample buffer and separated on a 3-15% Tris-acetic acid SDS/PAGE gel. After SDS/PAGE, the proteins in the gel were transferred to a nitrocellulose membrane and analyzed by IB. Rabbit polyclonal and mouse monoclonal antibodies against the C-terminal epitope of SURF4 (SEQ ID NO: 9) were prepared by Proteintech Group ( www.ptglab.com ).
  • HEK293A cells were transfected with FLAG-SURF4, and proteins were extracted with buffer A, followed by centrifugation at 13000g. The supernatant was dissolved and then separated using 4%-16% Blue Native-PAGE prior to IB.
  • Semi-intact cells were freshly prepared by permeabilizing Huh7 cells with 40 ug/ml digitonin in B88 (20 mM Hepes, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate and 2 mM magnesium acetate). The final volume of each reaction was 100 ⁇ L, containing ATP regeneration system (1 mM ATP, 40 mM phosphocreatine and 0.2 mg/mL creatine phosphokinase), GTP (0.2 mM), murine liver cytosol containing 100 ⁇ g protein and 50 ⁇ g Proteins in semi-intact cells.
  • ATP regeneration system (1 mM ATP, 40 mM phosphocreatine and 0.2 mg/mL creatine phosphokinase
  • GTP 0.2 mM
  • murine liver cytosol containing 100 ⁇ g protein and 50 ⁇ g Proteins in semi-intact cells.
  • reaction mixture was incubated in siliconized 1.5 mL microcentrifuge tubes at 30°C for 60 minutes, then cooled on ice to stop the reaction. After centrifugation at 14,000g for 10 minutes to remove the medium speed precipitate (donor membrane), 75 ⁇ L of the supernatant was taken and centrifuged in a Beckman TLA100 rotor at 55,000 RPM for 30 minutes at 4°C. The pellet (vesicle fraction) was used for immunoblot analysis.
  • the AAV DNA vector for CRISPR-mediated acute KO of the liver contains a liver-specific TBG promoter, Myc-tagged Cre recombinase (for recombination with a stop codon flanked by loxP), and Human U6 promoter for noncoding sgRNA transcription.
  • the vector pX602-Cre-sgRNA was generated using pX602-AAV-TBG::NLS-SaCas9-NLS-HAOLLAS-bGHpA;U6::BsaI-sgRNA (PX602, Addgene, 61593-).
  • AAV-TBG-CRE, AAV-TBG-FLAG-hSURF4 and AAV-TBG-FLAG-hSURF4 AAAs were generated from AAV-TBG-GFP (Addgene, 105535) by replacing the GFP sequence with the corresponding cDNA.
  • AAV-TBG-GFP Additional DNA sequence
  • the PAM sequence in hSURF4(CGG) was mutated to CTT, as shown in Figure 22, and then cloned into pAAV-TBG-hSURF4 by standard mutagenesis procedures.
  • sgRNAs were designed using the Benchling platform ( https://benchling.com/ ) to maximize editing efficiency and minimize off-target effects.
  • the sgRNA sequences are shown in SEQ ID Nos: 1-4.
  • synthetic oligonucleotides were inserted into the pX602-AAV-Cre-sgRNA backbone for genome editing in murine liver, or into pLenti-CRISPR V2 Cas9 D10A for use in pLenti-CRISPR V2 Cas9 D10A according to previously published methods Genome editing in cell lines (Platt et al., 2014).
  • Genomic DNA was extracted from both cells and tissues using the TIANamp Genomic DNA Kit (Tiangen) according to the manufacturer's protocol. The isolated genomic DNA was then used as a template for PCR for sequencing and SURVEYOR nuclease assay using a high-fidelity polymerase, as described in a previous article (Platt et al., 2014).
  • AAV was packaged and produced in HEK293T cells. Briefly, polyethylenimine (PEI) was used for transfection with AAV transfer plasmid (7 ⁇ g/5 cm dish), Rep/Cap (2/8) plasmid (7 ⁇ g/15 cm dish) and helper plasmid (20 ⁇ g/15 cm dish). cell. 60 hours after transfection, cells were removed and collected. Virus purification and titer quantification were performed as previously described (Platt et al., 2014). Virus was titered by qPCR using a custom SYBR Green assay (Tiangen). AAV delivery was by tail vein injection.
  • PEI polyethylenimine
  • spCas9 KI mice were administered 2 ⁇ 10 11 viral genome copies of AAV-Cre-sgRNA.
  • 1 ⁇ 10 11 GFP-expressing or human SURF4-expressing viral copies were simultaneously injected into each spCas9 KI mouse along with 2 ⁇ 10 11 viral genome copies with targeting sgRNAs.
  • RNA samples were homogenized in TRIzol reagent (Thermo Fisher) and RNA was extracted according to the manufacturer's protocol. A total of 1 ⁇ g of RNA was used to generate complementary cDNA in a 20 ⁇ l reaction volume by the M-MLV Reverse Transcriptase Kit (invitrogen). Quantitative PCR reactions were performed on a 7500 FAST system (Applied Biosystems) using SYBR green. Genes of interest were normalized to ⁇ -actin. The design of RT-qPCR primers is carried out according to conventional methods in the art.
  • GTT Glucose tolerance test
  • ITT insulin tolerance test
  • mice were fasted overnight with free access to water.
  • Glucose (1 g/kg body weight) was injected intraperitoneally. Blood glucose was measured by tail bleed using a glucometer at 0, 15, 30, 45, 60 and 120 minutes after glucose injection.
  • mice were fasted for 4 hours and then injected intraperitoneally with insulin (0.75 U/kg body weight). Blood glucose was measured at the same time points as the GTT analysis.
  • mice were euthanized and perfused with sodium phosphate buffer (PB, 100 mM, pH 7.4) and pre-fixation solution (2.5% (vol/vol) glutaraldehyde in PB, 0.8% paraformaldehyde).
  • PB sodium phosphate buffer
  • pre-fixation solution (2.5% (vol/vol) glutaraldehyde in PB, 0.8% paraformaldehyde).
  • Liver tissue samples were cut and fixed in pre-fixation solution for 2 hours at room temperature. The liver tissue samples were then cut into small pieces (0.2 x 0.3 x 0.5 mm 3 ) and fixed overnight at 4°C in the same pre-fixing solution. After rinsing with PB, the tissue was immersed in 0.1 M imidazole in PB for 30 min and then post-fixed with 2% osmium tetroxide in PB.
  • the samples were stained with 1% uranyl acetate overnight at 4 °C. Gradient dehydration was accomplished by gradually increasing the concentration of acetone and embedding in epoxy resin (60°C, 24 hours).
  • the samples were sectioned using a Leica EM UC7 ( ⁇ 60 nm thick) and placed on a copper grid. Images were taken in a double-blind fashion on a FEI Tecnai G2 20 Twin electron microscope (FEI, USA) equipped with an Eagle TM 4k CCD digital camera.
  • Wild-type or Surf4-depleted mouse livers were homogenized in HES buffer (20 mM Hepes, pH 7.4, 1 mM EDTA, 250 mM sucrose) and centrifuged at 3,000 g for 5 min to remove nuclei or unruptured cells. Mix 1.5ml of supernatant with 1.5ml of 60% iodixanol in a centrifuge tube, then add 1.3ml of 20% iodixanol and 1.2ml of 10% iodixanol layer by layer. The gradient mixture was centrifuged at 350,000 g for 4 hours at 4°C and divided into 25 fractions starting from the top of the gradient.
  • mice For mouse experiments, “n” corresponds to the number of mice used. For experiments using cell cultures, “n” corresponds to the number of independent replicates.
  • Experiments involving the measurement of blood lipids, blood glucose and body weight in mice were performed in a blinded fashion, in which mice were randomized according to ear tag identification numbers. Imaging, histology, and analysis of atherosclerotic plaques were also performed in a blinded fashion before decoding the sample identity. No data points were excluded from the study. Data are assumed to be continuously normally distributed.
  • Example 1 Identification of the relationship between SURF4 as a SAR1B partner and changes in blood lipids in humans
  • liver-specific knockout of Sar1b in mice using the Cre-loxP recombinase system it was surprising to find that Sar1b LKO mice exhibited significantly lower blood lipid levels than littermate controls under fasting conditions, and no signs of death were observed (data not shown). Then, through secretomic studies, it was found that liver Sar1b deficiency only inhibits the secretion of proteins related to lipoprotein cycling, such as apolipoprotein B (APOB), apolipoprotein E (APOE), apolipoprotein A1 (APOA1), SAA4, and SAA1. It has an effect and has little effect on other common secreted proteins in plasma such as albumin, transferrin and alpha 1 antitrypsin.
  • APOB apolipoprotein B
  • APOE apolipoprotein E
  • APOA1 apolipoprotein A1
  • SAA4 apolipoprotein A1
  • gRNAs SEQ ID NOs: 1-3 targeting different regions of the Surf4 gene were designed to avoid potential "off-target” effects of any single gene editing event.
  • AAV was used to co-deliver hepatocyte-specific Cre and three gRNAs targeting different exons of the mouse Surf4 gene, and a gRNA targeting LacZ (SEQ ID NO: 4) was used as a control.
  • Efficient inactivation of hepatic Surf4 was confirmed by immunoblotting experiments on liver lysates (Fig. 6A) and quantitative PCR experiments for Surf4 mRNA (Fig. 6B), whereas control liver Surf4 was not inactivated.
  • partial but not complete knockdown of Surf4 (for example, half knockdown) can also be considered, so that while achieving the effect of reducing blood lipids, part of the ability of Surf4 is still retained, so that lipids will not accumulate in the liver.
  • the realization of the effect also depends on the "dose-dependent" property of Surf4 found in the present invention.
  • the inventors constructed a Surf4 allele flanked by lox sites (FIG. 15) and used AAV-mediated Cre delivery to the liver for inactivation of the gene in mice.
  • Complete deletion of the Surf4 allele in the liver clears plasma cholesterol, triglyceride or ApoB levels compared to control mice (Surf4 +/+ , TBG-Cre), comparable to the use of CRISPR-mediated Surf4 in Example 2 Consistent results were observed with inactivation ( Figures 16A-C).
  • mice heterozygous for hepatic Surf4 also exhibited a significant 20-25% reduction in blood lipids compared to control mice (data on the far right of each panel in Figures 16A-C).
  • Liver Surf4 deficiency caused an approximately 80% reduction in plasma cholesterol and total triglycerides compared to controls when measured under fasted re-fed conditions, indicating lipid uptake from the small intestine in this experimental setting
  • the lipids may contribute to circulating lipids (Figure 17).
  • Hepatic Surf4 heterozygosity still reduced lipids under refeeding conditions compared to control mice (data on the far right of the two panels in Figure 17), but did not result in a significant accumulation of lipids in the liver ( Figure 18) .
  • FLAG-Surf4 was first introduced into the livers of control or Sar1b LKO mice by AAV.
  • Sar1b deficiency caused APOB accumulation in the liver and also caused an increase in the interaction between APOB and SURF4 (as measured by anti-FLAG immunoprecipitation) ( Figure 24), thereby demonstrating that SURF4 and SAR1B are involved in recognizing lipoproteins and mediating lipids Proteins are packaged into transport vesicles for continuous cooperation in ER export ( Figure 1).
  • Example 6 The lipid-lowering effect of SURF4 is independent of LDL receptor function
  • RNAi reagents SEQ ID Nos: 11-214.
  • the RNA duplexes of the sequences in Table 1 below were synthesized as candidate SURF4 RNAi reagents by Qingke Biotechnology.
  • the RNAi Control Reagent (which does not target mRNA in any mammalian cells) provided by Chinke Biotechnology was used as a control in all RNAi evaluation experiments.
  • SURF4 RNAi In vitro evaluation of SURF4 RNAi was performed in the human liver cell line Huh7. The cells were seeded in 6-well plates, and the candidate SURF4 RNAi reagents and control RNAi reagents in Table 1 were transfected with the transfection reagent LipoFectamine RNAiMax (Thermo Fisher), respectively.
  • RT-PCR was used to compare endogenous SURF4 mRNA levels in cells transfected with different SURF RNAi reagents and cells transfected with a control RNAi reagent, and cells transfected with different SURF RNAi reagents and cells transfected with different SURF RNAi reagents were compared by immunoblotting. Endogenous SURF4 protein levels in cells transfected with control RNAi reagents to assess the effect of different SURF4 RNAi reagents on reducing SURF4.
  • the supernatants of cells transfected with different SURF RNAi reagents and cells transfected with control RNAi reagents were collected, and the amount of APOB protein in the supernatant was compared to evaluate the effect of SURF RNAi reagents on reducing lipid secretion at the cellular level.
  • RNAi reagents S4-1, S4-2 and S4-3 are shown in FIG. 33 .
  • all three SURF4 RNAi agents significantly reduced the level of SURF4 mRNA ( Figure 33A-B), and decreased the level of APOB in the medium ( Figure 33C-D).
  • AAV-mediated human SURF4 cDNA was complemented in the liver of liver-specific Surf4 knockout mice for animal-level testing of SURF4 RNAi reagents.
  • Mice expressing human SURF4 were randomly divided into groups of 5 or more, and the candidate SURF4 RNAi reagents in Table 1 encapsulated by lipid nanoparticles were injected through the tail vein respectively, and the control group was injected with control RNAi reagents.
  • serum was collected and LDL-C, HDL-C were measured.
  • the livers of mice injected with different RNAi reagents were taken, and the mRNA and protein levels of SURF4 in the livers of the mice were detected.
  • SURF4 RNAi reagent Combined with the expression level of SURF4 in liver and the levels of LDL-C and HDL-C in serum, the effect of SURF4 RNAi reagent on reducing the expression level of SURF4 and reducing blood lipids was evaluated. At the same time, the sampled livers were stained with H/E, Oil Red O and Sirius Red staining, etc., to evaluate the health status of the livers injected with different SURF4 RNAi reagents.
  • SURF4 RNAi reagent The efficacy and safety of SURF4 RNAi reagent can be further verified through animal experiments.
  • the beneficial effects of inhibiting SURF4 expression are expected to outweigh the potential burden on the liver due to lipid accumulation.
  • RNA double strands were synthesized by Qingke Biotechnology Co., Ltd. as SURF4 siRNA reagents, and GalNAc modification was added to the 3' end of the sense strand during in vitro synthesis to achieve liver-targeted delivery.
  • siRNA Control Reagent (which does not target any mRNA in mammalian cells) provided by Kinco Biotechnology was used as a control in all siRNA evaluation experiments.
  • Table 2 shows the sequences (SEQ ID Nos: 215-234) and specific modifications of all ten candidate SURF4 siRNA agents, where capital letters indicate RNA bases and lower case letters indicate modifications; m represents 2'O-methyl ( 2'O-Methyl), f stands for 2'-Fluoro (2'-Fluoro), s stands for thiomodification between two bases.
  • SURF4 siRNAs were evaluated in vitro in the murine liver cell line Hepa1-6 cells.
  • the cells were seeded in 6-well plates, and the candidate SURF4 siRNA reagent and the control siRNA reagent (NC siRNA) were transfected with the transfection reagent RNAiMax (Thermo Fisher), respectively.
  • RT-PCR was used to compare endogenous SURF4 mRNA levels in cells transfected with different SURF siRNA agents and cells transfected with a control siRNA agent.
  • FIG. 34 The results of reducing SURF4 mRNA levels and protein expression levels by SURF4 siRNA agents are shown in FIG. 34 .
  • various candidate SURF4 siRNA agents significantly reduced the level of SURF4 mRNA (Figure 34A) and reduced SURF4 expression in Hepa1-6 cells ( Figure 34B).
  • Two of the siRNAs, namely 8# and 9# (m_surf4-8 and m_surf4-9) were selected to continue the experiment.
  • a liver-specific overexpression of human PCSK9 (hPCSK9) with the D374Y mutation was constructed.
  • the D374Y mutant is a gain of function mutation found in human familial hyperlipidemia patients.
  • the degradation ability of LDLR is 5-30 times higher than that of wild-type PCSK9.
  • It is a function-enhancing mutation of PCSK9, which can lead to Elevation of plasma LDL levels.
  • Human PCSK9 with this mutation can also achieve similar effects in mice, so this mouse can be used as a hyperlipidemia model to simulate the occurrence of human familial hyperlipidemia, and to verify that SURF4 inhibition can reduce hyperlipidemia. aspect efficacy.
  • GalNAc-modified SURF4 siRNA reagents as prepared in Example 9 were injected into the mice for testing the effects of these reagents at the animal level. The specific experimental process is described as follows.
  • C57BL6/J mice were randomly divided into 7 groups.
  • An expression plasmid expressing hPCSK9 with D374Y mutation was constructed in AAV vector, packaged into AAV virus, and the mice were injected into the tail vein to express the activated form of hPCSK9.
  • SURF4 siRNA reagents siRNA 8# and siRNA 9# were injected subcutaneously, and the control group was injected with control siRNA reagent (NC siRNA).
  • NC siRNA control siRNA reagent
  • serum was collected, cholesterol and triglyceride contents were measured, and the effect of SURF4 siRNA reagent on reducing SURF4 expression level and blood lipid was evaluated.
  • FIG. 35A The results of the SURF4 siRNA reagent reducing serum cholesterol (FIG. 35A) and triglycerides (FIG. 35B) are shown in FIG. 35 .
  • FIG. 35B The results of the SURF4 siRNA reagent reducing serum cholesterol (FIG. 35A) and triglycerides (FIG. 35B) are shown in FIG. 35 .
  • overexpression of the activated form of hPCSK9 mimics the hyperlipidemia phenotype of human familial hyperlipidemia patients.
  • the hypolipidemic effect of SURF4 siRNA reagent was significant.
  • the siRNA agent targeting SURF4 can effectively reduce the mRNA level of SURF4, thereby achieving lipid-lowering function.

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Abstract

L'invention concerne un oligonucléotide antisens court pour inhiber l'expression de SURF4, et un réactif d'ARNi et une composition de celui-ci. L'oligonucléotide antisens court, le réactif d'ARNi ou la composition peuvent réduire les lipides sanguins chez un sujet, et peuvent être utilisés pour traiter ou prévenir des maladies liées à la dyslipidémie.
PCT/CN2021/130426 2020-11-12 2021-11-12 Réactif d'arni et composition pour inhiber l'expression du gène surf4 WO2022100706A1 (fr)

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CN109957565A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种修饰的siRNA分子及其应用
CN110882378A (zh) * 2018-09-10 2020-03-17 上海清流生物医药科技有限公司 一种蛋白在制备预防和治疗动脉粥样硬化及并发症药物的应用
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CN109957565A (zh) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 一种修饰的siRNA分子及其应用
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