A siRNA and antibody conjugate, product and application thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to an siRNA, its antibody conjugate, product and application. The sense strand of the siRNA of the present invention is the sequence shown in SEQ ID NO: 17, and the antisense strand is the sequence shown in SEQ ID NO: 18; the present invention also provides an siRNA antibody conjugate prepared by a site-specific conjugation method, including an antibody, a linker and siRNA, and the siRNA is preferably a modified siRNA; the modified nucleotide is selected from one or more of 2'-position sugar moiety modification, phosphate group modification or nucleotide analogue modification. The siRNA antibody conjugate of the present invention can reduce the expression of DMPK mRNA in human skeletal muscle cells HSKMC and human bladder transitional cell papilloma cells RT4, and has great significance for the treatment of DM1 type myotonic dystrophy disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an siRNA, an antibody conjugate thereof, a product and an application. Background Art

[0002] RNA interference refers to a phenomenon of gene silencing 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, resulting in gene expression silencing. Small interfering RNA (siRNA) with a length of 20-25 nt can trigger RNAi, specifically down-regulate or turn off the expression of specific genes, and has the characteristics of high efficiency, easy synthesis and easy operation. Therefore, this technology has been widely used in the fields of exploring gene functions and gene therapy for infectious diseases and malignant tumors.

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

[0004] Myotonic dystrophy (DM) is an autosomal dominant hereditary disease caused by mutations in the DMPK gene. The cause is that mutations in the DMPK gene lead to overactivation of the DMPK protein, resulting in muscle atrophy, weakness, myotonia, and damage to multiple organs other than the skeletal muscle system. DM includes type 1 and type 2. DM type 1 is due to the amplification of the CTG sequence of the DM kinase (myotonin protein, DMPK) gene, while DM type 2 is caused by the amplification of the CCTG repeat in the cellular nucleic acid-binding protein (CNBP) gene. The normal allele CTG repeat number is 5-37; the repeat number in mild patients is in the range of 50-100, and the symptoms become more severe as the repeat number increases. DM type 1 can be divided into adult type, congenital type, and childhood type according to clinical phenotypes, 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 myotonia, muscle atrophy and weakness. Muscle atrophy first appears in the distal extremities and gradually develops to the tongue muscles, chewing muscles, eyelid muscles, papillary muscles and neck muscles, etc. The tendon reflex is weakened or disappeared. Myotonia is often limited to the upper limb muscles and tongue muscles, and its distribution is not as extensive and severe as that of congenital myotonia. Muscle weakness and atrophy are more obvious than myotonia, and all skeletal muscles of the body can be involved. Most patients have electrocardiogram changes, frontal alopecia, hyperhidrosis, decreased basal metabolic rate, weight loss, impotence or menstrual disorders, infertility, endocrine changes, heart abnormalities, congenital defects and cataracts, etc. Sometimes it is accompanied by mental retardation or even dementia. Skull radiographs can show thickening of the skull. There may also be abnormal electroencephalogram and enlargement of the ventricles, etc.

[0006] There is no effective symptomatic treatment method for DM1 at present. Existing membrane stabilizers such as phenytoin sodium and carbamazepine can promote the activity of sodium pumps, reduce the intracellular sodium ion concentration, increase the resting potential, and thus improve the symptoms of myotonia.

[0007] However, the current main DM1 therapies have problems such as poor efficacy of single drugs, mainly relieving symptoms, large side effects, long treatment cycles, and poor compliance of patients taking medicine every day. In addition, the pathogenesis of DM1 lies at the transcriptional level, and it is difficult for small molecule inhibitors to be developed into drugs. Therefore, there is an urgent need to provide an siRNA antibody conjugate for the treatment of DM. Summary of the Invention

[0008] In order to solve the above problems, the present invention provides an siRNA antibody conjugate, which combines the specific action of antibody drugs and the precision of small nucleic acid drugs, enables the small nucleic acid drug to enter the cell and target mRNA, and at the same time uses the stable structure of antibody drugs to improve the stability of small nucleic acids, and finally promotes the safe, effective and selective treatment of diseases by small nucleic acid drugs.

[0009] On the one hand, the present invention provides an siRNA, characterized in that the sense strand of the siRNA can be the sequence shown in SEQ ID NO: 17, and the antisense strand is the sequence shown in SEQ ID NO: 18;

[0010] At least one of the siRNAs is a modified nucleotide;

[0011] The modification is selected from one or more of 2'-position sugar moiety modification, phosphate group modification or nucleotide analog modification.

[0012] Specifically, the sugar moiety modification 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, 2'-O-dimethylaminoethoxyethyl modification, or 2'-O-N-methylacetamido modification.

[0013] Preferably, the sugar moiety modification may be one or both of 2'-O-methyl modification or 2'-fluoro modification.

[0014] Specifically, the phosphate group modification may be that at least one oxygen atom in the phosphodiester bond is replaced by a sulfur atom to form a phosphorothioate group.

[0015] The phosphate group modification may be a phosphorothioate group formed by replacing 2-8 oxygen atoms in the phosphodiester bond with sulfur atoms.

[0016] Specifically, the nucleotide analog may be one or more of heteronucleotide modification, LNA modification, ENA modification, cET BNA modification, UNA modification, and GNA modification.

[0017] Preferably, the nucleotide analog is GNA modification.

[0018] More specifically, the sequence of the sense strand after modification may be the sequences shown in SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27; the antisense strand may be the sequences shown in SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28.

[0019] In another aspect, the present invention provides an siRNA antibody conjugate of the aforementioned siRNA.

[0020] Specifically, the antibody in the siRNA antibody conjugate includes but is not limited to: monoclonal antibody or antibody binding fragment;

[0021] The monoclonal antibody includes but is not limited to: humanized antibody or chimeric antibody.

[0022] The antibody binding fragment includes but is not limited to: monovalent Fab', bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, or single-domain antibody (sdAb).

[0023] The single-domain antibody (sdAb) includes, but is not limited to: nanobody, shark antibody, or camelid antibody.

[0024] Preferably, the antibody is a TFR1 antibody or a structural mutant thereof.

[0025] 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.

[0026] More specifically, the siRNA and the antibody are connected by a linker. In some cases, the linker is a covalent bond or a non-polymeric linker that connects the antibody and the nucleic acid. In some cases, the nucleic acid linker is a functional group C1-C8 alkyl and / or 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.

[0027] 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 alkyne group of the linker1 series and the azide group of linker2 are conjugated by 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 by MTGase enzyme catalysis.

[0028] The structural formula of the linker is as follows:

[0029] linker1-01,

[0030] linker1-02,

[0031] linker1-03,

[0032] linker1-04,

[0033] linker1-05,

[0034] linker1-06,

[0035] linker1-07,

[0036] linker1-08,

[0037] linker1-09,

[0038] linker1-10,

[0039] linker1-11,

[0040] linker1-12,

[0041] linker1-13 or

[0042] linker2.

[0043] Preferably, the linker1 linker is linker1-02 and n = 3.

[0044] Preferably, the 5'-end of the sense strand of the siRNA is conjugated to the linker1 linker via C6-NH2.

[0045] Specifically, the antibody is an antibody that exposes a reactive site after being hydrolyzed by an active enzyme at a conserved N-glycosylation site.

[0046] More specifically, the structure of the N-glycosylation site of the antibody is a bi-antennary pentasaccharide molecule containing 3 Man and 2 GlcNAc.

[0047] Specifically, the active enzyme includes but is not limited to: SiaT, PNGase, GalT, GALas, ENGase or EndoS2.

[0048] Preferably, the active enzyme is PNGase.

[0049] Preferably, the reactive site is glutamine at position 295 of the heavy chain.

[0050] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned siRNA or siRNA-antibody conjugate.

[0051] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0052] The pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifying agents, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0053] Preferably, the pharmaceutically acceptable excipients are selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0054] In another aspect, the present invention provides the use of the aforementioned siRNA or siRNA antibody conjugate or pharmaceutical composition in the preparation of a drug for treating myotonic dystrophy.

[0055] Specifically, the myotonic dystrophy may be myotonic dystrophy type 1 and / or myotonic dystrophy type 2.

[0056] Preferably, the myotonic dystrophy is myotonic dystrophy type 1.

[0057] The technical effects achieved by the present invention:

[0058] 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 conjugating with the TFR1 antibody, the siRNA can be effectively delivered to muscle tissue, further reducing the systemic toxicity of the siRNA drug, and thus 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) It can accurately recognize target cells or tissues through the targeting ability of specific antibodies, thereby increasing bioavailability and reducing side effects; (2) Utilize the stable structure of antibody drugs to improve the stability of siRNA drugs; (3) Due to the special mechanism of action of siRNA drugs, AOC can act continuously in the body and reduce the frequency of drug administration; (4) The tissue specificity of antibodies can solve the problem of extrahepatic delivery of current siRNA drugs, enabling AOC to be applied to the treatment of various diseases and having broad application prospects; (5) It has more significant pharmacodynamic effects compared to random conjugation; (6) AOC can be personalized designed according to the genotype and phenotype of patients, so as to further improve the treatment effect and safety. Brief Description of the Drawings

[0059] Figure 1 It is the effect diagram of inhibiting DMPK mRNA expression by siRNA naked sequence in Hela cells.

[0060] Figure 2 The effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in Hela cells.

[0061] Figure 3 The IC of the modified siRNA sequence in Hela cells 50 .

[0062] Figure 4 The IEX-HPLC detection result of the site-specific conjugation AOC reaction solution.

[0063] Figure 5 The LC-MS diagram of the molecular weight identification of the site-specific conjugation AOC YJH-010-937 Ab-OAR1ss.

[0064] Figure 6 The effect diagram of non-site-specific conjugation AOC and site-specific conjugation AOC inhibiting DMPK mRNA expression in RT4 cells.

[0065] Figure 7 The IC of the site-specific conjugation AOC YJH-010-937 Ab-OAR1ss 50 detection result.

[0066] Figure 8 The composition schematic diagram of YJH-010-937 Ab-OAR1ss.

[0067] Figure 9 The composition schematic diagram of YJH-010-937 Ab-OAR1.

[0068] Figure 10 The composition schematic diagram of YJH-010-937 Ab-OAR2. Specific implementation manners

[0069] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0070] Example 1 Knockdown effect experiment of siRNA naked sequence

[0071] (1) Digest human cervical cancer cells (Hela, Shanghai Saibakang) in the logarithmic growth phase with trypsin. After terminating the digestion with DMEM medium supplemented with 10% (wt) FBS, centrifuge to collect the cells. Add DMEM medium supplemented with 10% (wt) FBS, count the cells using a hemocytometer, and then add 60,000 cells to each well of a 24-well plate for culture.

[0072] (2) Preparation of the liposome transfection reagent (LipoRNAiMAX, invitrogen) and siRNA mixture: Use the sequences in Table 1.

[0073] Table 1

[0074]

[0075] Continued Table 1

[0076]

[0077] Note: m indicates that the nucleotide is modified with 2'-methoxy; s indicates that the two adjacent nucleotides are linked by a phosphorothioate backbone; f indicates that the nucleotide is modified with 2'-fluoro.

[0078] Dilute the above siRNA at 10 nM / well and 1.5 μL of the LipoRNAiMAX transfection reagent separately in 25 μL of serum-free culture medium (Opti-MEM, Gibco), and then mix the siRNA solution with the LipoRNAiMAX solution and let it stand at room temperature for 5 minutes.

[0079] (3) Add 50 μL of the siRNA and LipoRNAiMAX mixture solution corresponding to each group to each well. The blank group is not treated with the addition of samples (siRNA).

[0080] (4) After culturing for 48 hours, discard the culture medium, wash twice with enzyme-free PBS, add lysis buffer (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake well and let it stand at room temperature for 2 - 3 minutes, centrifuge, and transfer the supernatant after stratification to a well plate to bind with the binding solution. Use a nucleic acid extractor, and according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer Bori, batch number c692302005), place the well plate in order and use the BSC69 program to extract RNA.

[0081] (5) Prepare the qPCR system on ice and detect it using the Novoprotein One Step RT-qPCR Kit. Add 1 μL of the One Step Premix Reagent (One Step SYBR Green Mix, Novoprotein), 10 μL of the 2× One Step Premix Reagent (One Step SYBR Green Mix, Novoprotein), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR to each well. Dilute 30 ng or 20 ng of RNA in 8.2 μL of enzyme-free and sterile water (RNase ddH2O, Novoprotein) and add it to the well. Mix well and place it in the qPCR instrument for reaction.

[0082] PCR reaction conditions: Pre-denaturation at 50 °C for 15 minutes, 95 °C for 1 minute, annealing at 95 °C for 15 seconds, extension at 60 °C for 1 minute, 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] Table 2

[0085]

[0086] Novoprotein One Step qPCR Kit: Lot number 7E711D3.

[0087] Table 3 Inhibition rate of DMPK mRNA in Hela cells after sequence transfection

[0088]

[0089] The results show that, as shown in Table 3 and Figure 1 as indicated, compared with the blank control group and the NC group, after transfection with LipoRNAiMAX, except for YJH-010-545, the other 11 sequences all showed obvious inhibitory effects. Compared with the existing technologies YJH-010-3m and YJH-010-4m, the two sequences YJH-010-709 and YJH-010-937 are superior. Therefore, further modification and optimization of these two sequences are carried out to improve their stability and extend their half-life.

[0090] Example 2 Knockdown effect experiment of YJH-010 modified sequences in Hela cells

[0091] (1) Digest Hela cells (Saibakang, product number iCell-h088) in the logarithmic growth phase with trypsin. After adding DMEM medium containing 10% (wt) FBS to terminate digestion, centrifuge to collect the cells. Add DMEM medium containing 10% (wt) FBS, count the cells with a hemocytometer, and then add 60,000 cells to each well of a 24-well plate for culture.

[0092] (2) Preparation of the LipoRNAiMAX and siRNA mixture: Select the sequences in Table 4 for

[0093] Table 4

[0094]

[0095] Note: m indicates that the nucleotide is modified with 2'-methoxy; s indicates that the two nucleotides before and after are connected by a phosphorothioate backbone; f indicates that the nucleotide is modified with 2'-fluoro; GNA is glycol nucleic acid modification.

[0096] Dilute 10 nM / well of siRNA and 1.5 μL of LipoRNAiMAX in 25 μL of serum-free culture medium (Opti-MEM, Gibco), respectively. Then mix the above siRNA solution and LipoRNAiMAX solution and let it stand at room temperature for 5 minutes.

[0097] (3) Add 50 μL of the siRNA and LipoRNAiMAX mixture corresponding to each group to each well. The blank group is not treated with sample addition (addition of siRNA).

[0098] (4) After culturing for 48 hours, discard the culture medium, wash twice with enzyme-free PBS, add lysis buffer (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, shake well and let it stand at room temperature for 2 - 3 minutes, centrifuge, transfer the supernatant after layering to a well plate to bind with the binding solution, use a nucleic acid extractor, and place the well plate in order according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer Bioer, product number: BSC69M1E), and then use the BSC69 program to extract RNA.

[0099] (5) Prepare the qPCR system and detect it using the Novizan one-step RT-qPCR kit. Conduct the operation on ice. Add 1 μL of the one-step premixed reagent (One Step SYBR Green Mix), 10 μL of the 2× one-step premixed reagent (One Step SYBR Green Mix, Novizan), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR into each well. Dilute 30 ng of RNA or 20 ng of RNA in 8.2 μL of enzyme-free and sterile water (RNase ddH2O, Novizan) and add it into the well. Mix well and place it in the qPCR instrument for reaction.

[0100] PCR reaction conditions: Pre-denaturation at 50 °C for 15 minutes, 95 °C for 1 minute, annealing at 95 °C for 15 seconds, extension at 60 °C for 1 minute, and conduct 39 cycles.

[0101] PCR primers: hYJH-010-2PF (SEQ ID NO:33): NF04_170903, hYJH-010-2PR (SEQ ID NO:34): NF04_170904. See Table 2.

[0102] One-step qPCR Novizan kit: Lot number 7E711D37E610k2.

[0103] Table 5 Inhibition rate of DMPK mRNA in Hela cells after sequence transfection

[0104]

[0105] The results show that, as shown in Table 5 and Figure 2 as indicated, after transfection with LipoRNAiMAX, compared with the blank control group and the NC group, all optimized or modified sequences have inhibitory effects. Compared with the existing technologies YJH-010-3m and YJH-010-4m, the optimized or modified sequences of YJH-010-937mS and YJH-010-937mE have the best inhibitory effects, and the inhibition rates are 76.33% and 79.00% respectively.

[0106] Example 3 IC of YJH-010 modified sequences YJH-010-937mS and YJH-010-937mE 50 Detection

[0107] (1) Digest Hela cells in the logarithmic growth phase with trypsin. After adding DMEM medium containing 10% (wt) FBS to terminate digestion, centrifuge to collect the cells. Add DMEM medium containing 10% (wt) FBS, count the cells using a hemocytometer, and then add 60,000 cells into each well of a 24-well plate for culture.

[0108] (2) Preparation of the mixture of LipoRNAiMAX and siRNA: Use the sequences in Table 6 for preparation.

[0109] Table 6

[0110]

[0111] Note: m indicates that the nucleotide is modified with 2'-methoxy; s indicates that the two nucleotides before and after are connected by a phosphorothioate backbone; f indicates that the nucleotide is modified with 2'-fluoro.

[0112] Set the transfection concentrations to 50 nM, 5 nM, 0.5 nM, and 0.05 nM according to the molecular weights of the respective sequences. Use the transfection reagent LipoRNAiMAX to transfect each sequence into the corresponding wells, and at the same time transfect the 50 nM, 5 nM, 0.5 nM, and 0.05 nM NC sequences as controls. Transfect 3 replicate wells for each group. After transfection, place the cell plate in an incubator at 37°C and 5% CO2 and continue to culture for 48 h.

[0113] (3) After culturing for 48 hours, discard the culture medium, wash twice with enzyme-free PBS, then add lysis buffer (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, mix well by shaking and let stand at room temperature for 2 - 3 minutes, centrifuge, transfer the supernatant after layering to a well plate to bind with the binding solution, use a nucleic acid extractor, and place the well plate in order according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer: Bori, catalog number: BSC69M1E), and then use the BSC69 program to extract RNA.

[0114] (5) Prepare the qPCR system on ice, and use the Novizan one-step RT-qPCR kit for detection. Add 1 μL of the one-step premix reagent (One Step SYBR Green Mix, Novizan), 10 μL of the 2× one-step premix reagent (One Step SYBR Green Mix, Novizan), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR to each well. Dilute 30 ng of RNA or 20 ng of RNA in 8.2 μL of enzyme-free and sterile water (RNase ddH2O, Novizan) and add it to the well, mix well and put it into the qPCR instrument for reaction.

[0115] PCR reaction conditions: Pre-denaturation at 50°C for 15 minutes, 95°C for 1 minute, annealing at 95°C for 15 seconds, extension at 60°C for 1 minute, and perform 39 cycles.

[0116] PCR primers: hYJH-010-2PF (SEQ ID NO:33): NF04_170903, hYJH-010-2PR (SEQ ID NO:34): NF04_170904. See Table 2.

[0117] One-step qPCR Novoprotein kit: lot number 7E711D3.

[0118] Table 7 IC after sequence transfection of Hela cells 50 (Half-maximal inhibitory concentration of DMPK mRNA)

[0119]

[0120] As shown in Table 7 and Figure 3 shown, the ICs of YJH-010-937 and YJH-010-937 mE 50 are both at the nM level, and the IC of YJH-010-937mS 50 is below 1 nM, significantly better than the IC of the prior art YJH-010-3m 50 which is 319.40 nM. Therefore, the YJH-010-937mS sequence is preferably used for AOC conjugation.

[0121] Example 4 Investigation of the stability of YJH-010 modified sequences

[0122] (1) Preparation of spiked solutions

[0123] Spiked solution -1: Take 5 OD of each of the 4 siRNA sequences to be investigated above, add an appropriate amount of enzyme-free ultrapure water to dissolve, and adjust the concentration to about 1 mg / mL as measured by a micro-spectrophotometer. The measured concentration of each sequence is shown in Table 8:

[0124] Table 8

[0125]

[0126] Spiked solution -2: Take an appropriate amount of the above spiked solution -1 and enzyme-free ultrapure water, and dilute to a solution with a concentration of about 0.32 mg / mL. The amounts of spiked solution -1 and enzyme-free ultrapure water taken for each sequence are shown in Table 9:

[0127] Table 9

[0128]

[0129] Spiked solution -3: Take an appropriate amount of the above spiked solution -1 and enzyme-free ultrapure water, and dilute to a solution with a concentration of about 75 μg / mL. The amounts of spiked solution -1 and enzyme-free ultrapure water taken for each sequence are shown in Table 10:

[0130] Table 10

[0131]

[0132] (2) Preparation of Nuclease System Solution

[0133] 250 U / μL RNase T1 Solution: Take 2.5 μL of RNase T1, add 7.5 μL of enzyme-free ultrapure water, and mix well.

[0134] Nuclease Solution: Take 120 μL of 5 M NaCl solution, 20 μL of 1 M Tris-HCl buffer (pH 7.4), 20 μL of 0.5 M EDTA (pH 8.0), 8 μL of RNase A, and 1 μL of 250 U / μL RNase T1 solution, add water to 2 mL, and mix well.

[0135] Nuclease Working Solution: Take 160 μL of the above nuclease solution, add 4.96 mL of enzyme-free sterile water, and mix well.

[0136] 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 it as the blank exo- and endonuclease.

[0137] Test Solution (Nuclease): Add the nuclease working solution and TRIzol Reagent respectively according to Table 11, mix well, then add the above spiked solution -3, mix well, and use it as the nuclease 0 h test solution.

[0138] Table 11

[0139]

[0140] 24 h Test Solution (Nuclease): Add the nuclease working solution and spiked solution -3 respectively according to Table 12, mix well, incubate at 37 °C for 24 h, take out, add TRIzol Reagent, mix well, and use it as the nuclease 24 h test solution.

[0141] Table 12

[0142]

[0143] (3) Determination

[0144] Respectively take the blank, 0 h test solution, and 24 h test solution of the above nuclease, detect them by high performance liquid chromatography (HPLC), and record the chromatogram. Calculate the percentage of 24 h relative to 0 h. As shown in Table 13:

[0145] Table 13

[0146]

[0147] The following conclusions can be drawn from the above experimental results: The unmodified sequence (YJH-010-937) is unstable to nuclease; in the nuclease system, the stability of the two modified sequences (YJH-010-937 mS and YJH-010-937 mE) is significantly improved, and there is basically no difference in stability from the positive sequence (YJH-010-m1).

[0148] Example 5 Preparation of AOC

[0149] (1) Preparation of YJH-010-937mS+linker1

[0150] Solid-phase synthesis: The single strand was completely assembled on the solid phase using standard phosphoramidite chemistry and purified by HPLC. The last monomer at the 5' end of the siRNA sense strand was modified with C6-NH2, and linker1 was further linked through C6-NH2 to obtain YJH-010-937mS+linker1. The coupling reaction includes but is 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. Linker1 is preferably linker1-02.

[0151] (2) Preparation of deglycosylated antibody

[0152] IgG antibodies have a conserved N-glycosylation site at Asn-297 in the Fc region, and its core structure is a biantennary pentasaccharide molecule composed of three mannose (Man) and two N-acetylglucosamine (GlcNAc) molecules. After hydrolysis with PNGase, the conformation of the CH2 domain in the Fc region is locally unfolded, exposing the originally masked reactive glutamine-295 (Q-295) site. The specific operation is as follows: At room temperature, a 1 mg / mL antibody solution was prepared using 200 mM phosphate buffer (pH 7.0). PNGase enzyme (750000 U / mL) was added to the antibody solution at a ratio of 10:1 (antibody / enzyme, v / v) for deglycosylation treatment, and incubated in a reactor for 20 hours. The reaction solution was buffer-exchanged and concentrated with 200 mM phosphate buffer to a concentration of 12.5 mg / mL.

[0153] The purity of the deglycosylated antibody detected by SEC-HPLC was 96.47%, and the molecular weight of the heavy chain detected by LC-MS was 48718.32.

[0154] (3) Coupling of linker2 linker

[0155] Through the action of MTGase (transglutaminase), the deglycosylated antibody was conjugated with linker2. The specific operation is as follows: Five-fold equivalent of linker2 was added to the deglycosylated antibody in step (2), and incubated at room temperature for 2 h. After purification, the SEC-HPLC concentration was detected to be 98.35%. LC-MS analysis of the product conjugated with linker found that most of them were one linker2 specifically conjugated to one heavy chain.

[0156] (4)Preparation of AOC conjugate YJH-010-937 Ab-OAR1ss

[0157] Antibody-siRNA conjugation: The antibody with linker2 was conjugated with siRNA YJH-010-937mS+linker1 by Click reaction, controlling the siRNA / antibody ratio to be 0.9, and placed in a reactor for reaction for 2 h. After the reaction, the reaction products were detected by SEC-HPLC and IEX-HPLC respectively. As the detection results showed, IEX-HPLC was more suitable for the analysis of AOC, and the product had good resolution ( Figure 4 ). Among them, the proportion of the product with an antibody-oligonucleotide ratio (OAR) of 1 in the crude product was about 56.84%.

[0158] Separation and purification of the reaction products: The reaction products were separated and purified using a Q column for ion exchange chromatography. The eluates were combined according to the results of simulated pooling (Minipool). After combination, the SEC-HPLC purity was 98.24%, and the IEX-HPLC purity was 100%, both meeting the purity requirements. Then, amplification and combination were carried out, and the detection results after combination 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 one siRNA sense strand linked to the heavy chain. Different from non-site-specific conjugation, it can be proved that siRNA was linked to the deglycosylated Q-295 site of the heavy chain by site-specific conjugation. The molecular weight identification is as Figure 5 shown.

[0159] The schematic structural diagram of YJH-010-937 Ab-OAR1ss prepared in this example is as Figure 8 shown.

[0160] In this example, the antibody is a TFR1 antibody, which is a full-length antibody. The sequence is shown in Table 14. It contains a heavy chain (mAb-1H) and a light chain (mAb-1L). The underlined regions represent their respective CDRs:

[0161] Table 14 Antibody Sequences

[0162]

[0163] Example 6 Evaluation of the knockdown of DMPK mRNA in RT4 cells by the AOC conjugate YJH-010-937 Ab-OAR1ss

[0164] (1) The human bladder cancer cell line RT4 cells (Shanghai Institute of the Chinese Academy of Sciences, catalog number: TChu226) frozen in liquid nitrogen were quickly thawed in a 37°C water bath. The thawed cells were added to 10 mL of pre-warmed MEM complete medium, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and MEM complete medium was added again. The resuspended cells were transferred to a culture flask and cultured in a 37°C, 5% CO2 incubator;

[0165] (2) The cells growing normally in the culture flask were collected by trypsin digestion. The cell concentration was calculated using a cell counting plate, and the cells were seeded into a 24-well cell culture plate according to the appropriate seeding number based on the results and cultured in a 37°C, 5% CO2 incubator. When the cells in the 24-well cell culture plate grew to 30%-40%, transfection was performed. According to the molecular weight of the siRNA in each AOC, the transfection concentration was adjusted to 10 nM, and each AOC was transfected into the corresponding wells by free uptake or using the transfection reagent LipoRNAiMAX. At the same time, 10 nM NC sequence was transfected as a control, and no sample was added to the blank group. Each group had 3 replicates. After transfection or direct incubation, the cell plate was placed in a 37°C, 5% CO2 incubator and cultured for another 96 h.

[0166] Schematic diagrams of the compositions 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 are as Figures 8 - 10 shown, where Lip indicates transfection with LipoRNAiMAX. YJH-010-937 Ab-OAR1 and YJH-010-937 Ab-OAR2 are AOC conjugates with non-site-specific coupling and OAR values of 1 and 2 respectively; YJH-010-937 Ab-OAR1ss is a site-specific conjugate with an OAR value of 1.

[0167] (3) After culturing for 96 hours, discard the culture medium. Wash twice with enzyme-free PBS and then add lysis buffer (BioFlux) to lyse the cells. Add chloroform (MREDA) for extraction. After shaking and mixing evenly, let it stand at room temperature for 2 - 3 minutes, then centrifuge. Transfer the supernatant after stratification to a well plate and bind it with the binding solution. Use a nucleic acid extractor, and according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer: Bori, product number: BSC69M1E), place the well plate in order and then use the BSC69 program to extract RNA.

[0168] (4) Prepare the qPCR system on ice. Use the Novoprotein One-Step RT-qPCR Kit for detection. Add 1 μL of the one-step premix reagent (One Step SYBR Green Mix, Novoprotein), 10 μL of the 2× one-step premix reagent (One Step SYBR Green Mix, Novoprotein), 0.4 μL of hYJH-010-3PF, and 0.4 μL of hYJH-010-3PR to each well. Dilute 30 ng of RNA or 20 ng of RNA in 8.2 μL of enzyme-free and sterile water (RNase ddH2O, Novoprotein) and add it to the well. Mix well and put it into the qPCR instrument for reaction.

[0169] PCR reaction conditions: Pre-denaturation at 50 °C for 15 minutes, 95 °C for 1 minute, annealing at 95 °C for 15 seconds, extension at 60 °C for 1 minute, and perform 39 cycles.

[0170] PCR primers: hYJH-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906, see Table 15:

[0171] Table 15

[0172]

[0173] Novoprotein One-Step qPCR Kit: Lot number 7E711D3.

[0174] Table 16 Inhibition rates of DMPK mRNA in RT4 cells after transfection or autophagocytosis in each group

[0175]

[0176] The results show that as shown in Table 16 and Figure 6As shown, compared with the blank control group and the NC group, non-site-specific conjugated AOC and site-specific conjugated AOC transfected with LipoRNAiMAX had significant inhibitory effects, and the inhibitory effect was slightly weakened when free uptake without transfection reagent was used. Among them, site-specific conjugated AOC YJH-010-937 Ab-OAR1ss showed the best performance, with an inhibition rate of 52.01±4.64%, which was better than non-site-specific conjugation, indicating that the site-specific conjugation method improved the knockdown efficiency while reducing the heterogeneity of the conjugate.

[0177] Example 7 YJH-010-937 Ab-OAR1ss IC 50 Detection

[0178] (1) Place the RT4 cells frozen in liquid nitrogen in a 37°C water bath for rapid thawing. Add the thawed cells to 10 mL of pre-warmed 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 them in a 37°C, 5% CO2 incubator for culture. Collect the cells growing normally 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 appropriate seeding number based on the results, and place them in a 37°C, 5% CO2 incubator for culture.

[0179] (2) When the cells in the 24-well cell culture plate grow to 30%-40%, perform transfection. Set the transfection concentrations to 80 nM, 40 nM, 10 nM, 2 nM, 0.04 nM, and 0.01 nM according to the molecular weights of each sequence, and transfect each sequence into the corresponding wells in the form of free uptake. Transfect 3 replicate wells for each group. After transfection, place the cell plate in a 37°C, 5% CO2 incubator and continue to culture for 96 hours.

[0180] (3) After culturing for 96 hours, discard the culture medium, wash twice with enzyme-free PBS, add lysis buffer (BioFlux) to lyse the cells, add chloroform (MREDA) for extraction, mix well by shaking and let stand at room temperature for 2-3 minutes, centrifuge, transfer the supernatant after stratification to a well plate to bind with the binding solution, use a nucleic acid extractor, and place the well plate in order according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer Bioer, product number: BSC69M1E), and then use the BSC69 program to extract RNA.

[0181] (5) Prepare the qPCR system on ice and use the Novoprotein One Step RT-qPCR Kit for detection. Add 1 μL of the One Step Premix Reagent (One Step SYBR Green Mix, Novoprotein), 10 μL of the 2× One Step Premix Reagent (One Step SYBR Green Mix, Novoprotein), 0.4 μL of hYJH-010-2PF, and 0.4 μL of hYJH-010-2PR into each well. Dilute 30 ng of RNA or 20 ng of RNA in 8.2 μL of enzyme-free and sterile water (RNase ddH2O, Novoprotein) and add it into the well. Mix well and place it in the qPCR instrument for reaction.

[0182] PCR reaction conditions: pre-denaturation at 50 °C for 15 minutes, 95 °C for 1 minute, annealing at 95 °C for 15 seconds, extension at 60 °C for 1 minute, and perform 39 cycles.

[0183] PCR primers: hYJH-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906, see Table 15.

[0184] Novoprotein One Step qPCR Kit: Lot number 7E711D3.

[0185] Table 17 IC of YJH-010-937 Ab-OAR1ss after free uptake by RT4 cells 50 Test results

[0186]

[0187] As shown in Table 17 and Figure 7 The results show that the IC of YJH-010-937 Ab-OAR1ss 50 is 0.26 nM.

[0188] The series of detailed descriptions listed above are only specific descriptions of the feasible implementation modes of the present invention, and they are not intended to limit the protection scope of the present invention. Those skilled in the art can design many other modifications and implementation modes, and these modifications and implementation modes will fall within the scope of the principles and spirit disclosed in this application. More specifically, within the scope of the present invention disclosure, drawings and claims, various variations and improvements can be made to the components and / or layout of the subject combination layout. In addition to the variations 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 sequence of the sense strand of the siRNA is the sequence shown in SEQ ID NO:41, and the sequence of the antisense strand is the sequence shown in SEQ ID NO:42; or the sequence of the sense strand of the siRNA is the sequence shown in SEQ ID NO:43, and the sequence of the antisense strand is the sequence shown in SEQ ID NO:

44.

2. A siRNA-antibody conjugate comprising the siRNA according to claim 1, characterized in that: The antibody is TFR1 antibody; 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; the TFR1 antibody is an antibody in which the reactive site is exposed after the conserved N-glycosylation site is hydrolyzed by an active enzyme.

3. The siRNA-antibody conjugate according to claim 2, 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.

4. The siRNA-antibody conjugate according to claim 3, 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、 linker1-09、 links1-10、 links1-11、 links1-12、 linker1-13 or linker2。 5. The siRNA-antibody conjugate according to claim 4, characterized in that: The active enzymes include SiaT, PNGase, GalT, GALas, ENGase or EndoS2.

6. The siRNA-antibody conjugate according to claim 5, characterized in that: The reactive site is glutamine at position 295 of the heavy chain.

7. A pharmaceutical composition, characterized in that It comprises the siRNA according to claim 1 or the siRNA-antibody conjugate according to any one of claims 2-6.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. Use of the siRNA according to claim 1, the siRNA-antibody conjugate according to any one of claims 2 to 6, or the pharmaceutical composition according to any one of claims 7 to 8 in the preparation of a drug for treating myotonic dystrophy.

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