SiRNA and antibody conjugate, product and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to siRNA and an antibody conjugate, a product and application thereof. A positive-sense strand of the siRNA is a sequence as shown in SEQ ID NO: 17, and an antisense strand of the siRNA is a sequence as shown in SEQ ID NO: 18; the invention also provides a siRNA antibody conjugate prepared by a fixed-point coupling method, the siRNA antibody conjugate comprises an antibody, a connexon and siRNA, and the siRNA is preferably modified siRNA; the modified nucleotide is selected from one or more of 2 '-position carbohydrate part modification, phosphate group modification or nucleotide analogue modification. The siRNA antibody conjugate can reduce expression of DMPK mRNA in human skeletal muscle cells HSKMC and human bladder transitional cell papilloma cells RT4, and has great significance in treatment of DM1 type ankylosing muscular dystrophy diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to siRNA and antibody conjugates thereof, products and applications. Background Art

[0002] RNA interference refers to a gene silencing phenomenon induced by double-stranded RNA in molecular biology. Its mechanism is to inhibit gene expression by hindering the transcription or translation of specific genes. When double-stranded RNA homologous to the coding region of endogenous messenger RNA (mRNA) is introduced into cells, the mRNA is degraded and gene expression is silenced. Small interfering RNA (siRNA) with a length of 20-25nt can trigger RNAi, which can specifically downregulate or shut down the expression of specific genes. It has the characteristics of high efficiency, easy synthesis and easy operation, so this technology has been widely used in the field of exploring gene function and gene therapy for infectious diseases and malignant tumors.

[0003] Antibody-oligonucleotide conjugate (AOC) is a new type of chimeric biomolecule synthesized by coupling an antibody with an oligonucleotide. The three main components of AOC are antibody, oligonucleotide payload, and a chemical linker connecting the two.

[0004] Myotonic dystrophy (DM) is an autosomal dominant hereditary disease caused by DMPK gene mutations. The cause is that DMPK gene mutations lead to overactivation of DMPK protein, causing muscle atrophy, weakness and myotonia, and damage to multiple organs outside the skeletal muscle system. DM includes type 1 and type 2. DM1 is caused by the expansion of the CTG sequence in the DM kinase (myotoninprotein, DMPK) gene, while DM2 is caused by the expansion of CCTG repeats in the cellular nucleic acid-binding protein (cellular nucleic acid-binding protein, CNBP) gene. The normal allele CTG repeat number is 5-37; the repeat number of mild patients is in the range of 50-100. As the number of repeats increases, the symptoms become more severe. DM1 type can be divided into: adult type, congenital type and childhood type according to the clinical phenotype, among which the adult type is the most common.

[0005] DM1 can occur at any age, but is more common after puberty. The average age of onset is about 25 years old, and the main symptoms are muscle stiffness, muscle atrophy and weakness. Muscle atrophy first appears in the distal limbs and gradually develops to the tongue muscles, masseter muscles, eyelid muscles, papillary muscles and neck muscles. Tendon reflexes weaken or disappear, and muscle stiffness is often limited to the upper limb muscles and tongue muscles. Its distribution is not as extensive and severe as congenital myotonia. Muscle weakness and atrophy are more obvious than muscle stiffness, and all skeletal muscles in the body can be affected. Most patients have electrocardiogram changes, forehead baldness, sweating, decreased basal metabolic rate, weight loss, impotence or irregular menstruation, infertility, endocrine changes, heart abnormalities, congenital defects and cataracts, etc., sometimes accompanied by intellectual disability or even dementia. Skull photos can reveal thickening of the skull. There may also be abnormal electroencephalograms and enlarged ventricles.

[0006] There is no effective symptomatic treatment for DM1. Existing membrane stabilizers such as phenytoin sodium and carbamazepine can promote sodium pump activity, reduce intracellular sodium ion concentration, and increase resting potential, thereby improving myotonia symptoms.

[0007] However, the current main DM1 therapies have problems such as poor single-drug efficacy, mainly relieving symptoms, large side effects, long treatment cycles, and poor patient compliance for daily medication. In addition, the pathogenesis of DM1 is at the transcriptional level, and small molecule inhibitors are difficult to develop into drugs. Therefore, there is an urgent need to provide a siRNA antibody conjugate for the treatment of DM. Summary of the invention

[0008] In order to solve the above problems, the present invention provides siRNA antibody conjugates, which combine the specific effects of antibody drugs and the precision of small nucleic acid drugs, so that small nucleic acid drugs can enter cells and target mRNA. At the same time, the stability of small nucleic acids is improved by using the stable structure of antibody drugs, ultimately promoting the safe, effective and selective treatment of diseases by small nucleic acid drugs.

[0009] In one aspect, the present invention provides an siRNA, characterized in that the sense strand of the siRNA may be the sequence shown in SEQ ID NO: 17, and the antisense strand may be the sequence shown in SEQ ID NO: 18; At least one of the siRNAs is a modified nucleotide; The modification is selected from one or more of 2' position sugar moiety modification, phosphate group modification or nucleotide analog modification.

[0010] Specifically, the modification of the sugar moiety at the 2' position includes but is not limited to: one or more of 2'-O-methyl modification, 2'-O-methoxyethyl modification, 2'-O-aminopropyl modification, 2'-deoxy modification, 2'-fluoro modification, 2'-O-dimethylaminoethyl modification, 2'-O-dimethylaminopropyl modification, TO-dimethylaminoethoxyethyl modification or 2'-ON-methylacetamido modification.

[0011] Preferably, the sugar moiety modification may be one or both of a 2'-O-methyl modification and a 2'-fluoro modification.

[0012] Specifically, the phosphate modification may be that at least one oxygen atom in the phosphodiester bond is replaced by a sulfur atom to form a thiophosphate group.

[0013] The phosphate modification may be a phosphorothioate group formed by replacing 2-8 oxygen atoms in the phosphodiester bond with sulfur atoms.

[0014] Specifically, the nucleotide analogs may be one or more of isonucleotide modifications, LNA modifications, ENA modifications, cET BNA modifications, UNA modifications, and GNA modifications.

[0015] Preferably, the nucleotide analogue is GNA modified.

[0016] More specifically, the modified sequence of the sense chain can be the sequence shown in SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:27; the antisense chain can be the sequence shown in SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.

[0017] In yet another aspect, the present invention provides the aforementioned siRNA-antibody conjugate.

[0018] Specifically, the antibodies in the siRNA-antibody conjugate include, but are not limited to: monoclonal antibodies or antibody binding fragments; The monoclonal antibodies include, but are not limited to, humanized antibodies or chimeric antibodies.

[0019] The antibody binding fragments include, but are not limited to, monovalent Fab', divalent Fab2, single chain variable fragment (scFv), diabody, minibody or single domain antibody (sdAb).

[0020] The single domain antibody (sdAb) includes but is not limited to: nanobodies, shark antibodies or camelid antibodies.

[0021] Preferably, the antibody is a TFR1 antibody or a structural mutant thereof.

[0022] Preferably, the heavy chain of the TFR1 antibody is as shown in SEQ ID NO:47, and the light chain is as shown in SEQ ID NO:48.

[0023] In further detail, the siRNA and the antibody are connected via a linker. In some cases, the linker is a covalent bond or a non-polymeric linker, connecting the antibody and the nucleic acid. In some cases, the nucleic acid linker is a functional group C1-C8 alkyl and / or a PEG functional group and a reactive linking structure. Further, the degree of polymerization n of the PEG functional group is 1-8. Further, the antibody linker includes the homobifunctional linker or heterobifunctional linker described above. Specifically, the linker is composed of linker1 and linker2, including but not limited to linker1-01, linker1-02, linker1-03, linker1-04, linker1-05, linker1-06, linker1-07, linker1-08, linker1-09, linker1-10, linker1-11, linker1-12 or linker1-13. The linker1 series linker alkynyl group is conjugated with the linker2 azido group through a Click reaction, and the other end is conjugated to the 5' end or 3' end of the nucleic acid. The linker2 is coupled to the antibody through MTGase enzyme catalysis.

[0024] The structural formula of the linker is shown below: linker1-01, linker1-02, linker1-03, linker1-04, linker1-05, linker1-06, linker1-07, linker1-08, linker1-09, linker1-10, linker1-11, linker1-12, linker1-13 or linker2.

[0025] Preferably, the linker1 linker is linker1-02, n=3.

[0026] Preferably, the 5' end of the sense strand of the siRNA is connected by C6-NH 2 Conjugated with the linker1 linker.

[0027] Specifically, the antibody is an antibody in which a reactive site is exposed after the conserved N-glycosylation site is hydrolyzed by an active enzyme.

[0028] More specifically, the structure of the antibody N-glycosylation site is a biantennary pentasaccharide molecule comprising 3 Man and 2 GlcNAc.

[0029] Specifically, the active enzyme includes but is not limited to: SiaT, PNGase, GalT, GALas, ENGase or EndoS2.

[0030] Preferably, the active enzyme is PNGase.

[0031] Preferably, the reactive site is glutamine at position 295 of the heavy chain.

[0032] In yet another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned siRNA or siRNA-antibody conjugate.

[0033] Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0034] The pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0035] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0036] In yet another aspect, the present invention provides use of the aforementioned siRNA or siRNA-antibody conjugate or pharmaceutical composition in the preparation of a drug for treating myotonic dystrophy.

[0037] Specifically, the myotonic dystrophy may be myotonic dystrophy type 1 and / or myotonic dystrophy type 2.

[0038] Preferably, the myotonic dystrophy is myotonic dystrophy type 1.

[0039] The technical effects achieved by the present invention are: The present invention designs corresponding siRNA for DMPK and interferes with its mRNA, which can effectively reduce the DMPK pre-mRNA with excessive CUG repeat sequences in the 3' non-coding region. At the same time, by coupling with TFR1 antibody, siRNA can be effectively delivered to muscle tissue, further reducing the systemic toxicity of siRNA drugs, thereby playing a role in treating DM1 type myotonic dystrophy. In addition, as a new type of "two-in-one" drug, AOC has the following advantages: (1) Through the targeting ability of specific antibodies, it can accurately identify target cells or tissues, thereby increasing bioavailability and reducing side effects; (2) Using the stable structure of antibody drugs, the stability of siRNA drugs is improved; (3) Due to the special mechanism of action of siRNA drugs, AOC can continue to act in the body and reduce the frequency of medication; (4) The tissue specificity of antibodies can solve the problem of extrahepatic delivery of current siRNA drugs, so that AOC can be used in the treatment of various diseases and has a wide range of application prospects; (5) Compared with random coupling, it has more significant efficacy; (6) AOC can be personalized for the patient's genotype and phenotype at the same time, so it can further improve the therapeutic effect and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing the effect of naked siRNA sequence in inhibiting DMPK mRNA expression in Hela cells.

[0041] Figure 2 This is a diagram showing the effect of the modified siRNA sequence in inhibiting DMPK mRNA expression in Hela cells.

[0042] Figure 3 is the IC of the modified siRNA sequence in Hela cells 50 .

[0043] Figure 4 This is the IEX-HPLC test result of the site-specific coupling AOC reaction solution.

[0044] Figure 5This is the LC-MS chart for molecular weight identification of site-directed conjugated AOC YJH-010-937 Ab-OAR1ss.

[0045] Figure 6 This is a diagram showing the effect of non-site-coupled AOC and site-coupled AOC in inhibiting DMPK mRNA expression in RT4 cells.

[0046] Figure 7 IC for site-directed conjugation of AOC YJH-010-937 Ab-OAR1ss 50 Test results.

[0047] Figure 8 Schematic diagram of the composition of YJH-010-937 Ab-OAR1ss.

[0048] Fig. 9 Schematic diagram of the composition of YJH-010-937 Ab-OAR1.

[0049] Fig.10 Schematic diagram of the composition of YJH-010-937 Ab-OAR2. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0051] Example 1 Knockdown effect experiment of naked siRNA sequence (1) Human cervical cancer cells (HeLa, Shanghai Biopharmaceuticals) in the logarithmic growth phase were digested with trypsin, and DMEM medium containing 10% (wt) FBS was added to terminate the digestion. The cells were collected by centrifugation, and DMEM medium containing 10% (wt) FBS was added. The cells were counted using a hemocytometer, and then 60,000 cells were added to each well of a 24-well plate for culture.

[0052] (2) Preparation of liposome transfection agent (LipoRNAiMAX, invitrogen) and siRNA mixture: Use the sequence in Table 1.

[0053] Table 1

[0054] Table 1

[0055] Note: m indicates that the nucleotide is modified with 2'-methoxy; s indicates that the two nucleotides are connected by a thiophosphate backbone; f indicates that the nucleotide is modified with 2'-fluoro.

[0056] 10 nM / well of the above siRNA and 1.5 μL of LipoRNAiMAX transfection agent were diluted in 25 μL of serum-free culture medium (Opti-MEM, Gibco), and then the above siRNA solution and LipoRNAiMAX solution were mixed and allowed to stand at room temperature for 5 minutes.

[0057] (3) Add 50 μL of the corresponding group of siRNA and LipoRNAiMAX mixed solution to each well. The blank group was not treated with sample (siRNA).

[0058] (4) After 48 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution. Use a nucleic acid extractor and arrange the well plates in order according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bioer, batch number c692302005) and use the BSC69 program to extract RNA.

[0059] (5) Prepare the qPCR system on ice and use the Novagen One-Step RT-qPCR Kit for detection. Add 1 μL of One Step SYBR Green Mix (Novagen), 10 μL of 2× One Step SYBR Green Mix (Novagen), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL of enzyme-free sterile water (RNase ddH2O, Novagen) and add to the wells. Mix well and place in the qPCR instrument for reaction.

[0060] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0061] PCR primers: hYJH-010-2PF (SEQ ID NO: 33): NF04_170903, hYJH-010-2PR (SEQ ID NO: 34): NF04_170904. See Table 2: Table 2

[0062] One-step qPCR Novagen kit: lot number 7E711D3.

[0063] Table 3 Inhibition rate of DMPK mRNA in Hela cells after sequence transfection

[0064] The results show that, as shown in Table 3 and Figure 1 As shown, compared with the blank control group and the NC group, after LipoRNAiMAX transfection, except for YJH-010-545, the other 11 sequences showed obvious inhibitory effects. Compared with YJH-010-3m and YJH-010-4m in the prior art, the two sequences YJH-010-709 and YJH-010-937 were better, so the two sequences were further modified and optimized to improve their stability and prolong their half-life.

[0065] Example 2 Knockdown effect experiment of YJH-010 modified sequence in Hela cells (1) Hela cells (Cybikang, Catalog No. iCell-h088) in the logarithmic growth phase were digested with trypsin, and DMEM medium containing 10% (wt) FBS was added to terminate the digestion. The cells were collected by centrifugation, and DMEM medium containing 10% (wt) FBS was added. The cells were counted using a hemocytometer, and then 60,000 cells were added to each well of a 24-well plate for culture.

[0066] (2) Preparation of LipoRNAiMAX and siRNA mixture: Use the sequence in Table 4 Table 4

[0067] Note: m means the nucleotide is modified by 2'-methoxy; s means the two nucleotides are connected by a thiophosphate backbone; f means the nucleotide is modified by 2'-fluoro; GNA means ethylene glycol nucleic acid modification.

[0068] 10 nM / well siRNA and 1.5 μL LipoRNAiMAX were diluted in 25 μL serum-free culture medium (Opti-MEM, Gibco), respectively. Then, the siRNA solution and LipoRNAiMAX solution were mixed and allowed to stand at room temperature for 5 minutes.

[0069] (3) Add 50 μL of the corresponding group of siRNA and LipoRNAiMAX mixed solution to each well. The blank group was not treated with sample (siRNA).

[0070] (4) After 48 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution, use a nucleic acid extractor, and arrange the well plate in order according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer: Bioer, product number: BSC69M1E), and use the BSC69 program to extract RNA.

[0071] (5) Prepare the qPCR system and use the Novagen One-Step RT-qPCR Kit for detection on ice. Add 1 μL of One Step SYBR Green Mix, 10 μL of 2× One Step SYBR Green Mix, 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL of enzyme-free sterile water (RNase ddH2O, Novagen) and add to the wells. Mix well and place in the qPCR instrument for reaction. PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0072] PCR primers: hYJH-010-2PF (SEQ ID NO: 33): NF04_170903, hYJH-010-2PR (SEQ ID NO: 34): NF04_170904. See Table 2.

[0073] One-step qPCR Novagen kit: lot number 7E711D37E610k2.

[0074] Table 5 Inhibition rate of DMPK mRNA in Hela cells after sequence transfection

[0075] The results show that, as shown in Table 5 and Figure 2 As shown, after LipoRNAiMAX transfection, compared with the blank control group and the NC group, all optimized or modified sequences had inhibitory effects. Compared with the prior art YJH-010-3m and YJH-010-4m, the optimized or modified sequences of YJH-010-937mS and YJH-010-937mE had the best inhibitory effects, with inhibition rates of 76.33% and 79.00%, respectively.

[0076] Example 3 IC of YJH-010 modified sequences YJH-010-937mS and YJH-010-937mE 50 Detection (1) Hela cells in the logarithmic growth phase were digested with trypsin, and DMEM medium containing 10% (wt) FBS was added to terminate the digestion. The cells were collected by centrifugation, and DMEM medium containing 10% (wt) FBS was added. The cells were counted using a hemocytometer, and then 60,000 cells were added to each well of a 24-well plate for culture.

[0077] (2) Preparation of LipoRNAiMAX and siRNA mixture: Use the sequence in Table 6.

[0078] Table 6

[0079] Note: m indicates that the nucleotide is modified with 2'-methoxy; s indicates that the two nucleotides are connected by a thiophosphate backbone; f indicates that the nucleotide is modified with 2'-fluoro.

[0080] The transfection concentration was set to 50nM, 5nM, 0.5nM, and 0.05nM according to the molecular weight of each sequence. Each sequence was transfected into the corresponding well using the transfection reagent LipoRNAiMAX, and 50nM, 5nM, 0.5nM, and 0.05nM NC sequences were transfected at the same time as controls. Each group was transfected with 3 replicates, and the cell plate was placed in a 37°C, 5% CO2 incubator for 48h after transfection.

[0081] (3) After 48 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine it with the binding solution, use a nucleic acid extractor, and according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer Bioer, product number: BSC69M1E), arrange the well plate in order and use the BSC69 program to extract RNA.

[0082] (5) Prepare the qPCR system on ice and use the Novagen One-Step RT-qPCR Kit for detection. Add 1 μL of One Step SYBR Green Mix (Novagen), 10 μL of 2× One Step SYBR Green Mix (Novagen), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL of enzyme-free sterile water (RNase ddH2O, Novagen) and add to the wells. Mix well and place in the qPCR instrument for reaction. PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0083] PCR primers: hYJH-010-2PF (SEQ ID NO: 33): NF04_170903, hYJH-010-2PR (SEQ ID NO: 34): NF04_170904. See Table 2.

[0084] One-step qPCR Novagen kit: lot number 7E711D3.

[0085] Table 7 IC after sequence transfection into Hela cells 50 (DMPK mRNA half inhibited)

[0086] As shown in Table 7 and Figure 3 As shown, the IC of YJH-010-937 and YJH-010-937 mE 50 All are nM level, IC of YJH-010-937mS 50 Less than 1nM, significantly better than the IC of the existing technology YJH-010-3m 50 The AOC coupling was performed with the YJH-010-937mS sequence.

[0087] Example 4 Investigation of stability of modified sequence of YJH-010 (1) Preparation of spiked solution Spiked solution-1: Take 5OD of each of the four siRNA sequences to be investigated, add an appropriate amount of enzyme-free ultrapure water to dissolve until the concentration is about 1 mg / mL measured by a micro-spectrophotometer. The measured concentrations of each sequence are shown in Table 8: Table 8

[0088] Spiked solution-2: Take appropriate amount of the spiked solution-1 and enzyme-free ultrapure water, dilute to make a solution with a concentration of about 0.32 mg / mL. The amount of spiked solution-1 and enzyme-free ultrapure water used in each sequence is shown in Table 9: Table 9

[0089] Spiked solution-3: Take appropriate amount of the spiked solution-1 and enzyme-free ultrapure water, dilute to make a solution with a concentration of about 75 μg / mL. The amount of spiked solution-1 and enzyme-free ultrapure water used in each sequence is shown in Table 10: Table 10

[0090] (2) Preparation of nuclease system solution 250U / μL RNase T1 solution: Take 2.5μL of RNase T1, add 7.5μL of enzyme-free ultrapure water, and mix well.

[0091] Nuclease solution: Take 120μL of 5M NaCl solution, 20μl of 1M pH7.4 Tris-HCl buffer, 20μl of 0.5M EDTA pH8.0 + 8μl of RNase A + 1μL of 250U / μl RNase T1 solution, add water to 2mL and mix well.

[0092] Nuclease working solution: Take 160 μL of the above nuclease solution, add 4.96 mL of enzyme-free sterile water, and mix well.

[0093] Blank (nuclease): Take 320 μL of the above nuclease working solution, 20 μL of RNA extraction reagent (TRIzol Reagent), and 80 μL of enzyme-free sterile water, mix well, and use as blank exonuclease.

[0094] Test solution (nuclease): Add nuclease working solution and TRIzol Reagent as described in Table 11, mix well, then add the above-mentioned spiked solution -3, mix well, and use this as the nuclease 0h test solution.

[0095] Table 11

[0096] 24h test solution (nuclease): Add nuclease working solution and spike solution-3 as described in Table 12, mix well, incubate at 37℃ for 24h, take out, add TRIzol Reagent, mix well, and use as nuclease 24h test solution.

[0097] Table 12

[0098] (3) Determination Take the blank, 0h test solution, and 24h test solution of the above nuclease respectively, detect them by high performance liquid chromatography (HPLC), and record the chromatogram. Calculate the percentage of 24h relative to 0h. As shown in Table 13: Table 13

[0099] The following conclusions can be drawn from the above experimental results: the unmodified sequence (YJH-010-937) is unstable in the presence of nucleases; in the nuclease system, the stability of the two modified sequences (YJH-010-937 mS and YJH-010-937 mE) is greatly improved, and there is basically no difference in stability with the positive sequence (YJH-010-m1).

[0100] Example 5 AOC Preparation (1) Preparation of YJH-010-937mS+linker1 Solid phase synthesis: The single strands are completely assembled on the solid phase using standard phosphoramidite chemistry and purified using HPLC. The last monomer at the 5' end of the siRNA sense strand is C6-NH 2 Modification, further through C6-NH 2 Linker 1 is connected to obtain YJH-010-937mS+linker 1, and the coupling reaction includes but is not limited to linker 1-01, linker 1-02, linker 1-03, linker 1-04, linker 1-05, linker 1-06, linker 1-07, linker 1-08, linker 1-09, linker 1-10, linker 1-11, linker 1-12 or linker 1-13. Linker 1 is preferably linker 1-02.

[0101] (2) Preparation of deglycosylated antibodies IgG antibodies have a conserved N-glycosylation site at Asn-297 in the Fc segment, and its core structure is a biantennary pentasaccharide molecule composed of three mannose (Man) and two N-acetylglucosamine (GlcNAc) molecules. After being hydrolyzed by PNGase, the conformation of the CH2 domain in the Fc region is partially unfolded, exposing the reactive glutamine-295 (Q-295) site that was originally shielded. The specific operation is as follows: At room temperature, 200mM phosphate buffer (pH 7.0) is used to prepare a 1mg / mL antibody solution. PNGase enzyme (750000U / mL) is added to the antibody solution at a ratio of 10:1 (antibody / enzyme, v / v) for desugaring treatment, and the solution is placed in a reactor for incubation for 20 hours. The reaction solution is buffer exchanged and concentrated with 200mM phosphate buffer to a concentration of 12.5mg / mL.

[0102] The purity of the antibody after desugaring was 96.47% as detected by SEC-HPLC, and the molecular weight of the heavy chain was 48718.32 as detected by LC-MS.

[0103] (3) Linker2 coupling Through the action of MTGase (transglutaminase), the desugared antibody was coupled with linker2. The specific operation was as follows: five times the equivalent of linker2 was added to the desugared antibody in step (2), and incubated at room temperature for 2 hours. After purification, the SEC-HPLC concentration was detected to be 98.35%. LC-MS analysis of the products coupled with linker showed that most of them were one heavy chain specifically coupled with one linker2.

[0104] (4) Preparation of AOC conjugate YJH-010-937 Ab-OAR1ss Antibody and siRNA coupling: The antibody with linker2 was coupled with siRNA YJH-010-937mS+linker1 through Click reaction, and the siRNA / antibody ratio was controlled to be 0.9. The reaction was placed in a reactor for 2 hours. After the reaction, the reaction products were detected by SEC-HPLC and IEX-HPLC respectively. As shown in the test results, IEX-HPLC is more suitable for the analysis of AOC, and the product separation is better ( Figure 4 ). Among them, the product with an antibody oligonucleotide ratio (OAR) of 1 accounted for about 56.84% of the crude product.

[0105] Separation and purification of reaction products: The reaction products were separated and purified using an ion exchange chromatography Q column. The eluates were combined according to the results of the simulated merger (Minipool). After the merger, the SEC-HPLC purity was 98.24%, and the IEX-HPLC purity was 100%, both of which met the purity requirements. Amplification and merger were performed, and the test results after merger were as follows: The SEC-HPLC purity of YJH-10-937 Ab-OAR1ss was 98.00%, and the IEX-HPLC purity was 100.00%. After deconvolution analysis of the molecular weight of YJH-010-937 Ab-OAR1ss by LC-MS, there was only a molecular ion peak of the heavy chain connected to an siRNA sense chain. Unlike non-site-specific coupling, this proves that the siRNA is connected to the desugared Q-295 site of the heavy chain through site-specific coupling. Molecular weight identification is as follows Figure 5 shown.

[0106] The schematic diagram of the structure of YJH-010-937 Ab-OAR1ss prepared in this example is as follows Figure 8 shown.

[0107] In this example, the antibody is TFR1 antibody, which is a full-length antibody, and the sequence is shown in Table 14, which includes a heavy chain (mAb-1H) and a light chain (mAb-1L), and the underlined regions represent the respective CDRs: Table 14 Antibody sequences

[0108] Example 6 Evaluation of knockdown of DMPK mRNA in RT4 cells by AOC conjugate YJH-010-937 Ab-OAR1ss (1) Human bladder cancer cell line RT4 cells (Shanghai Institute of the Chinese Academy of Sciences, Catalog No.: TChu226) frozen in liquid nitrogen were quickly thawed in a 37°C water bath. The thawed cells were added to 10 mL of preheated MEM complete medium, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, MEM complete medium was added again, the resuspended cells were transferred to a culture flask, and placed at 37°C, 5% CO 2 Cultivate in an incubator; (2) Collect the normally growing cells in the culture flask by trypsin digestion, calculate the cell concentration using a cell counting plate, and inoculate the cells into a 24-well cell culture plate according to the result and the appropriate number of plates, and place the plates at 37°C and 5% CO. 2Culture in an incubator. Transfection was performed when the cells in the 24-well cell culture plate grew to 30%-40%. The transfection concentration was adjusted to 10nM according to the molecular weight of the siRNA in each AOC. Each AOC was transfected into the corresponding well by free uptake or using the transfection reagent LipoRNAiMAX. At the same time, 10nM NC sequence was transfected as a control. The blank group was not treated with sample addition. Each group had 3 replicate wells. After transfection or direct incubation, the cell plate was placed at 37°C and 5% CO 2 The cells were cultured in the incubator for 96 h.

[0109] The composition diagram of Lip-YJH-010-937 Ab-OAR1, YJH-010-937 Ab-OAR1, Lip-YJH-010-937 Ab-OAR2, YJH-010-937 Ab-OAR2, Lip-YJH-010-937 Ab-OAR1ss, and YJH-010-937 Ab-OAR1ss is shown in Figure 8-Figure 10 As shown, Lip means transfection with LipoRNAiMAX. YJH-010-937 Ab-OAR1 and YJH-010-937 Ab-OAR2 are non-site-specific conjugated AOC conjugates with OAR values ​​of 1 and 2, respectively; YJH-010-937 Ab-OAR1ss is a site-specific conjugated AOC conjugate with an OAR value of 1.

[0110] (3) After 96 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution, use a nucleic acid extractor, and according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bioer, product number: BSC69M1E), arrange the well plate in order and use the BSC69 program to extract RNA.

[0111] (4) Prepare the qPCR system on ice and use the Novagen One-Step RT-qPCR Kit for detection. Add 1 μL of One Step SYBR Green Mix (Novagen), 10 μL of 2× One Step SYBR Green Mix (Novagen), 0.4 μL of hYJH-010-3PF, and 0.4 μL of hYJH-010-3PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL of enzyme-free sterile water (RNase ddH2O, Novagen) and add to the wells. Mix well and place in the qPCR instrument for reaction.

[0112] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0113] PCR primers: hYJH-010-3PF (SEQ ID NO: 49): NF04_170905, hYJH-010-3PR (SEQ ID NO: 50): NF04_170906, see Table 15: Table 15

[0114] One-step qPCR Novagen kit: lot number 7E711D3.

[0115] Table 16 Inhibition rate of DMPK mRNA in RT4 cells after transfection or self-uptake in each group

[0116] The results show that, as shown in Table 16 and Figure 6 As shown, compared with the blank control group and the NC group, the non-site-coupled AOC and the site-coupled AOC had significant inhibitory effects after LipoRNAiMAX transfection, and the inhibitory effect was slightly weakened when freely taken up without the addition of transfection reagent; among them, the site-coupled AOCYJH-010-937 Ab-OAR1ss performed best, with an inhibition rate of 52.01±4.64%, which was better than the non-site-coupled one, indicating that the site-coupled method improved the knockdown efficiency while reducing the heterogeneity of the conjugate.

[0117] Example 7 YJH-010-937 Ab-OAR1ss IC 50 Detection (1) Thaw the RT4 cells frozen in liquid nitrogen in a 37°C water bath quickly, add the thawed cells to 10 mL of preheated MEM complete medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, re-add MEM complete medium, transfer the resuspended cells to a culture flask, and place at 37°C, 5% CO 2 The cells growing normally in the culture flask were collected by trypsin digestion, and the cell concentration was calculated by cell counting plate. According to the results, the cells were inoculated into 24-well cell culture plates according to the appropriate number of plating, and placed at 37°C, 5% CO 2 Culture in an incubator.

[0118] (2) When the cells in the 24-well cell culture plate grow to 30%-40%, transfection is performed. The transfection concentration is set to 80nM, 40nM, 10nM, 2nM, 0.04nM, and 0.01nM according to the molecular weight of each sequence, and each sequence is transfected into the corresponding well in the form of free uptake. Each group is transfected with 3 replicates. After transfection, the cell plate is placed at 37°C and 5% CO 2 The cells were cultured in the incubator for 96 hours.

[0119] (3) After 96 hours of culture, discard the culture medium, wash twice with enzyme-free PBS, add lysis solution (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake and mix, let stand at room temperature for 2-3 minutes, centrifuge, transfer the layered supernatant to the well plate and combine with the binding solution, use a nucleic acid extractor, and according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bioer, product number: BSC69M1E), arrange the well plate in order and use the BSC69 program to extract RNA.

[0120] (5) Prepare the qPCR system on ice and use the Novagen One-Step RT-qPCR Kit for detection. Add 1 μL of One Step SYBR Green Mix (Novagen), 10 μL of 2× One Step SYBR Green Mix (Novagen), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL of enzyme-free sterile water (RNase ddH2O, Novagen) and add to the wells. Mix well and place in the qPCR instrument for reaction. PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0121] PCR primers: hYJH-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906, see Table 15.

[0122] One-step qPCR Novagen kit: lot number 7E711D3.

[0123] Table 17 IC of YJH-010-937 Ab-OAR1ss after free uptake by RT4 cells 50 Test results

[0124] As shown in Table 17 and Figure 7 The results showed that the IC of YJH-010-937 Ab-OAR1ss 50 It is 0.26nM.

[0125] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Those skilled in the art can design many other modifications and implementation methods, which will fall within the scope and spirit of the principles disclosed in this application. More specifically, within the scope of the present disclosure, the drawings and the claims, various modifications and improvements can be made to the components and / or layout of the subject combination layout. In addition to the modifications and improvements made to the components and / or layout, other uses will also be obvious to those skilled in the art.

Claims

1. A siRNA, characterized in that The sense strand of the siRNA is the sequence shown in SEQ ID NO: 17, and the antisense strand is the sequence shown in SEQ ID NO: 18; At least one of the siRNAs is a modified nucleotide; The modification is selected from one or more of 2' position sugar moiety modification, phosphate group modification or nucleotide analog modification.

2. The siRNA according to claim 1, characterized in that The 2'-position sugar moiety modification comprises one or more of 2'-O-methyl modification, 2'-O-methoxyethyl modification, 2'-O-aminopropyl modification, 2'-deoxy, 2'-fluoro modification, 2'-O-dimethylaminoethyl modification, 2'-O-dimethylaminopropyl modification, 2'-O-dimethylaminoethoxyethyl modification or 2'-ON-methylacetamido modification.

3. The siRNA according to claim 1, characterized in that The phosphate group is modified such that at least one oxygen atom in the phosphodiester bond is replaced by a sulfur atom to form a thiophosphate group; The nucleotide analog modification is one or more of isonucleotide modification, LNA modification, ENA modification, cET BNA modification, UNA modification and GNA modification.

4. The siRNA according to any one of claims 1 to 3, characterized in that The modified sequence of the sense strand of the siRNA is the sequence shown in SEQ ID NO:41, and the antisense strand is the sequence shown in SEQ ID NO:42; or the modified sequence of the sense strand of the siRNA is the sequence shown in SEQ ID NO:43, and the antisense strand is the sequence shown in SEQ ID NO:

44.

5. A siRNA-antibody conjugate comprising the siRNA according to any one of claims 1 to 4.

6. The siRNA-antibody conjugate according to claim 5, characterized in that: The antibody in the siRNA-antibody conjugate is a monoclonal antibody or an antibody binding fragment; The antibody is a TFR1 antibody or a structural mutant thereof.

7. The siRNA-antibody conjugate according to claim 6, characterized in that: The heavy chain of the TFR1 antibody is shown in SEQ ID NO:47, and the light chain is shown in SEQ ID NO:

48.

8. The siRNA-antibody conjugate according to claim 7, characterized in that: The siRNA and the antibody are connected via a linker, and the 5' end of the positive strand of the siRNA is conjugated to the linker.

9. The siRNA-antibody conjugate according to claim 8, characterized in that: The linker is composed of linker1 and linker2, wherein linker1 is linker1-01, linker1-02, linker1-03, linker1-04, linker1-05, linker1-06, linker1-07, linker1-08, linker1-09, linker1-10, linker1-11, linker1-12 or linker1-13; the structural formula of the linker is as follows: linker1-01、 link1-02、 link1-03、 link1-04、 link1-05、 link1-06、 linker1-07、 linker1-08、 link1-09、 links1-10、 links1-11、 links1-12、 linker1-13 or linker2。 10. The siRNA-antibody conjugate according to any one of claims 6 to 9, characterized in that: The antibody is an antibody in which a reactive site is exposed after the conserved N-glycosylation site is hydrolyzed by an active enzyme.

11. The siRNA-antibody conjugate according to claim 10, characterized in that: The active enzymes include SiaT, PNGase, GalT, GALas, ENGase or EndoS2.

12. The siRNA-antibody conjugate according to claim 11, characterized in that: The reactive site is glutamine at position 295 of the heavy chain.

13. A pharmaceutical composition, characterized in that The invention comprises the siRNA according to any one of claims 1 to 4 or the siRNA-antibody conjugate according to any one of claims 5 to 12.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

15. Use of the siRNA according to any one of claims 1 to 4, the siRNA-antibody conjugate according to any one of claims 5 to 12, or the pharmaceutical composition according to any one of claims 13 to 14 in the preparation of a drug for treating myotonic dystrophy.

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