Preparation method and application of siRNA antibody conjugate

Abstract

The invention discloses a preparation method of an siRNA antibody conjugate, which comprises the following steps: synthesizing a DMPK siRNA double-chain body and modifying, conjugating the modified sequence with a linker to obtain a linker-siRNA conjugate, then conjugating an antibody to obtain an siRNA antibody conjugate of DAR1 or DAR2, then separating and purifying, and evaluating the purity of the siRNA antibody conjugate. The siRNA antibody conjugate prepared by the method is applied to a medicine for treating DM1 type ankylosing muscular dystrophy, and the expression of a target gene can be effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a preparation method and use of an siRNA antibody conjugate. 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 blocking 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 degrades, resulting in gene expression silencing. Small interfering RNAs with a length of 20-25 nt can trigger RNAi, specifically downregulate or turn off the expression of specific genes, and have 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 type of novel chimeric biomolecule derived from antibody-drug conjugate (ADC), also known as siRNA antibody conjugate, which is synthesized by conjugating an antibody with an oligonucleotide. Similar to ADC, the three main components of AOC are an antibody, an oligonucleotide payload, and a chemical linker connecting the two. AOC effectively combines an antibody and siRNA to achieve targeted therapy. Compared with traditional siRNA therapy, AOC has better pharmacokinetic properties and more specific biodistribution. 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 vivo, reducing 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) AOC can be personalized designed according to the genotype and phenotype of patients, so it can further improve the therapeutic effect and safety.

[0004] Myotonic dystrophy (DM) is a common autosomal dominant hereditary myopathy with a prevalence of approximately 10 / 100,000, and there are about 500,000 patients globally. DM mostly presents as progressive muscle weakness, myotonia, and damage to multiple organs outside 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 myotonin protein kinase (DMPK) gene, while DM type 2 is caused by the amplification of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene. The normal allele has 5 - 37 CTG repeats; in mild patients, the repeat number 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] Currently, the main DM1 therapies have problems such as poor efficacy of single drugs, mainly alleviating 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 a need to provide new therapeutic drugs for improving drug efficacy and their preparation methods. Summary of the Invention

[0006] To achieve the above object, the present invention adopts the following technical solutions.

[0007] One object of the present invention provides a method for preparing an siRNA antibody conjugate, comprising the following steps:

[0008] (1) Using a DMPK siRNA duplex, the single strands are fully assembled and modified on a solid phase using standard phosphoramidite chemistry to obtain a sense strand and an antisense strand and perform modifications;

[0009] (2) Conjugate a C 6 -NH 2 linking site at the 5'-end of the sense strand of the siRNA, and connect 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester to obtain a linker-siRNA conjugate;

[0010] (3) Connect an antibody to one linker-siRNA conjugate or two linker-siRNA conjugates to obtain an siRNA antibody conjugate with DAR1 or DAR2;

[0011] (4) Purification: Separate the fraction containing the siRNA antibody conjugate with DAR1 or DAR2, concentrate it, and perform buffer exchange with phosphate buffer, and evaluate the purity of the siRNA antibody conjugate by size exclusion chromatography (SEC).

[0012] Preferably, the synthesis steps of the siRNA antibody conjugate further include:

[0013] (a1) Buffer exchange the antibody with 50 mM phosphate buffer at pH 7.0;

[0014] (b1) Add an equivalent amount of TCEP in the same phosphate buffer to (a1) and incubate at 22 °C for 2 hours to obtain an antibody-TCEP mixture;

[0015] (c1) Combine the obtained antibody-TCEP mixture and the solution of the linker-siRNA conjugate in 10 mM acetate buffer at pH 6.0, and incubate at 22 °C for 1 hour.

[0016] Preferably, the synthesis steps of the siRNA antibody conjugate further include:

[0017] (a2) Mix the linker-siRNA conjugate and NEM (N-ethylmaleimide) to obtain a linker-siRNA-NEM mixture and perform buffer exchange on the antibody with 50 mM phosphate buffer at pH 7.0;

[0018] (b2) Add an equivalent amount of TCEP in the same phosphate buffer to (a2) and incubate at 22 °C for 2 hours to obtain an antibody-TCEP mixture;

[0019] (c2) Combine the antibody-TCEP mixture and the solution of the linker-siRNA-NEM mixture in 10 mM acetate buffer at pH 6.0, and incubate at 22 °C for 1 hour.

[0020] Preferably, the amount of the linker-siRNA conjugate or the linker-siRNA-NEM mixture is 1 to 3 times the equivalent amount of the antibody, and the amount of the TCEP is 1 to 3 times the equivalent amount of the antibody.

[0021] Preferably, the ratio of the linker-siRNA conjugate to NEM is 1:1.

[0022] Preferably, the concentration reached after the buffer exchange is 2 to 9 mg / ml.

[0023] Preferably, the amount of the TCEP is the same as the amount of the linker-siRNA conjugate.

[0024] Preferably, the amount of the TCEP is the same as the amount of the linker-siRNA-NEM mixture.

[0025] Preferably, the sense strand is the sequence shown in SEQ ID NO: 31, 15, 35, 37, 39, 17, 41, 43, 45, and the antisense strand is the sequence shown in SEQ ID NO: 32, 16, 36, 38, 40, 18, 42, 44, 46.

[0026] The linker-siRNA conjugate is YJH-010-937mER, and its structural formula is as follows:

[0027]

[0028] The siRNA antibody conjugate of DAR1 or DAR2 is YJH-010-937Ab (DAR1) or YJH-010-937Ab (DAR2), wherein,

[0029] The structural formula of the YJH-010-937Ab (DAR1) is as follows:

[0030]

[0031] The structural formula of the YJH-010-937Ab (DAR2) is as follows:

[0032]

[0033] The second object of this application is the use of the siRNA antibody conjugate as a drug for treating myotonic dystrophy type 1 (DM1). Beneficial effects

[0034] The present invention designs corresponding siRNA for DMPK to interfere 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 the TFR1 antibody through the method of the targeted siRNA antibody conjugate, the siRNA can be effectively delivered to muscle tissues, further reducing the systemic toxicity of the siRNA drug, and thus playing a role in treating myotonic dystrophy type 1 (DM1), and further improving the treatment effect and safety. Brief description of the drawings

[0035] The following further details the specific embodiments of the present invention with reference to the drawings.

[0036] Figure 1 It is the effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in Hela cells.

[0037] Figure 2 It is the effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in human skeletal muscle cells HSKMC.

[0038] Figure 3 Effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in mouse myoblast C2C12 cells.

[0039] Figure 4 Effect diagram of the in vitro stability of the modified siRNA sequence and the naked sequence.

[0040] Figure 5 Effect diagram of the siRNA antibody conjugate inhibiting DMPK mRNA expression in human skeletal muscle cells HSKMC.

[0041] Figure 6 Result diagram of the in vivo mRNA knockdown experiment of the siRNA antibody conjugate. Detailed implementation mode

[0042] Example 1: Preparation of siRNA antibody conjugate

[0043] 1. Preparation of YJH-010-937mER

[0044] The linker is a linker or a non-polymeric linker. In some cases, the linker is a C1-C6 alkyl group. Further, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. Further, the linker includes the homobifunctional linker or heterobifunctional linker described above. More specifically, the linker includes N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB, CAS: 79886-55-8), N-succinimidyl 11-maleimidoundecanoate (KMUS, CAS: 87981-04-2), N-succinimidyl 3-maleimidobenzoate (MBS, CAS: 58626-38-3), N-γ-maleimidobutyryloxy succinimide ester (GMBS, CAS: 80307-12-6), N-α-maleimidoacetyloxy succinimide ester (AMAS, CAS: 55750-61-3), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, CAS: 64987-85-5), sodium N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfonate (Sulfo-SMCC, CAS: 92921-24-9), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-(6-aminocaproate) (LC-SMCC, CAS: 125559-00-4), etc. The linker is conjugated to the 5'-end or 3'-end of the nucleic acid, and the other end of the linker is conjugated to the antibody.

[0045] Table 1: Abbreviations and structural formulas of each linker

[0046]

[0047] For the siRNA-antibody conjugate used in this experiment, DMPK siRNA duplexes were used.

[0048] Steps for DMPK siRNA duplexes:

[0049] (1) Design of siRNA sequence: When designing siRNA, first select the target site of the target gene, and then design a double-stranded RNA sequence according to the basic principles of ATCG. The obtained siRNA sequence consists of 21 - 27 nucleotides, including a 21-nt perfectly matched region and two 2-nt overhangs at the 5' or 3' ends.

[0050] (2) Synthesis of single strands: The designed siRNA sequence was synthesized into two complementary RNA single strands respectively. Common RNA synthesis methods include chemical synthesis method and enzymatic cleavage method, etc.

[0051] (3) Purification of polymerization products: The two complementary RNA single strands were mixed together to form double-stranded RNA, and methods such as gel electrophoresis, dialysis or HPLC were used to purify and refine the products.

[0052] (4) siRNA transfection: The purified siRNA was transfected into target cells to achieve gene-targeted silencing through RNA interference technology.

[0053] The DMPK siRNA duplexes were synthesized by solid-phase synthesis method. The single strands were fully assembled on the solid phase using the standard phosphoramidite chemistry method (also known as the phosphoramidite method) and purified by HPLC. The synthesis of DMPK siRNA duplexes is prior art and can also be completed by entrusting a nucleic acid synthesis company. The synthesized sequences are shown in Table 2:

[0054] Table 2

[0055]

[0056] The modified sequences are shown in Table 3:

[0057] Table 3

[0058] 2. Synthesis of linker-siRNA conjugate: The sense strand of siRNA contains a C6-NH 2 linking site at the 5' end, and the linking site was further linked to N-(4-(maleimidomethyl)cyclohexane-1-carboxamido)succinimide to obtain YJH-010-937mER (i.e., linker-siRNA conjugate).

[0059] YJH-010-937mER is as follows:

[0060] 3. The specific synthesis steps of YJH-010-937Ab (DAR1, DAR2) are as follows:

[0061] Synthesis of siRNA antibody conjugate: Connect an antibody to one YJH-010-937mER to obtain YJH-010-937Ab (DAR1); connect two YJH-010-937mERs to one antibody to obtain YJH-010-937Ab (DAR2). YJH-010-937Ab (DAR1) and YJH-010-937Ab (DAR2) are called siRNA antibody conjugates.

[0062] Step 1: Reduce the antibody with TCEP

[0063] To synthesize DAR1, first, at room temperature, mix YJH-010-937mER with N-ethylmaleimide (NEM) at a ratio of 1:1 to obtain a mixture of YJH-010-937mER and NEM; perform buffer exchange on the antibody with 50 mM phosphate buffer (pH 7.0) and adjust its concentration to 2 - 5 mg / ml, preferably 2.83 mg / ml. Add 1 - 3 equivalents of TCEP (tris(2-carboxyethyl)phosphine hydrochloride), preferably 1.5 equivalents, in the same phosphate buffer to the above solution, and incubate at 22 °C for 2 hours to obtain an antibody-TCEP mixture. At room temperature, combine the antibody-TCEP mixture with a solution of 1 - 3 equivalents (preferably 1.5 equivalents) of the mixture of YJH-010-937mER and NEM in 10 mM acetate buffer at pH 6.0, and incubate at 22 °C for 1 hour.

[0064] To synthesize DAR2, perform buffer exchange on the antibody with 50 mM phosphate buffer (pH 7.0) and adjust its concentration to 2 - 9 mg / ml, preferably 7 mg / ml. Add 1 - 3 equivalents of TCEP, preferably 1.2 equivalents, in the same phosphate buffer to the above solution, and incubate at 22 °C for 2 hours to obtain an antibody-TCEP mixture. At room temperature, combine the obtained antibody-TCEP mixture with a solution of 1 - 3 equivalents (preferably 1.2 equivalents) of YJH-010-937mER in 10 mM acetate buffer at pH 6.0, and incubate at 22 °C for 1 hour.

[0065] Since two free sulfhydryl groups are generated from a pair of disulfide bonds after the reduction of the antibody's internal disulfide bonds by TCEP, and both can conjugate with N-maleimide (NEM)-modified siRNA, generally, the conjugation method using TCEP reduction can obtain an AOC with DAR≥2; while when the NEM-modified siRNA is premixed with NEM in a certain ratio and then reacts with the free sulfhydryl groups on the antibody, the free NEM will compete with the NEM-modified siRNA, and each will react to bind to one sulfhydryl group, thus obtaining an AOC with DAR<2.

[0066] Step 2: Purification

[0067] Analysis of the prepared siRNA-antibody conjugate by ion exchange chromatography (IEX) showed the siRNA-antibody conjugate, as well as the unreacted antibody and siRNA. The fraction containing the siRNA-antibody conjugate with DAR1 or DAR2 was separated from the siRNA-antibody conjugate, concentrated, and buffer-exchanged with phosphate buffer at pH 7.5. The purity of the siRNA-antibody conjugate was evaluated by size exclusion chromatography (SEC).

[0068] Table 4: Purity of siRNA-antibody conjugate

[0069]

[0070] The antibody includes a humanized antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab’, a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a shark antibody or a camelid antibody or its binding fragment. The antibody is also a transferrin receptor antibody or its binding fragment.

[0071] In this example, the antibody of YJH-010-937Ab is a TFR1 antibody, which is a full-length antibody. The sequence is shown in Table 5. It contains a heavy chain (mAb-1H) and a light chain (mAb-1L), and the underlined regions represent their respective CDRs.

[0072] Table 5

[0073]

[0074] The structural formula of the finally obtained siRNA-antibody conjugate is as follows.

[0075] YJH-010-937Ab (DAR1):

[0076]

[0077] YJH-010-937Ab (DAR2):

[0078]

[0079] Example 2: Experiment on the knockdown effect of the YJH-010 modification sequence in Hela cells

[0080] 1. Digest Hela cells in the logarithmic growth phase with trypsin. After adding DMEM medium containing 10% FBS to terminate digestion, centrifuge to collect the cells. Add DMEM medium containing 10% FBS, count the cells with a hemocytometer, and then add 60,000 cells to each well of a 24-well plate for culture.

[0081] 2. Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture: Select the sequences in Table 3, where

[0082] m represents that the nucleotide is modified with 2'-methoxy; s represents that the two nucleotides before and after are connected by a phosphorothioate backbone; f represents that the nucleotide is modified with 2'-fluoro; GNA is glycol nucleic acid.

[0083] Dilute 10 nM / well of siRNA and 1.5 μL of LipoRNAiMAX (invitrogen) separately in 25 μL of serum-free culture medium (Opti-MEM, gibco). Then mix the above siRNA solution and LipoRNAiMAX (invitrogen) solution and let it stand at room temperature for 5 minutes.

[0084] 3. Add 50 μL of the siRNA and LipoRNAiMAX (invitrogen) mixed solution corresponding to each group to each well.

[0085] 4. After culturing for 48 hours, discard the culture medium. Wash the cells twice with enzyme-free PBS, then 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, then centrifuge. 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 Bioer, catalog number: BSC69M1E), place the well plate in order and use the BSC69 program to extract RNA.

[0086] 5. Prepare the qPCR system on ice. Add 1 μL of One Step SYBR Green Mix (Novoprotein), 10 μL of 2*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 RNase ddH2O (Novoprotein) and add it to the well. Mix well and place it in the qPCR instrument for reaction.

[0087] 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.

[0088] PCR primers: hYJH-010-2PF (SEQ ID NO:47): NF04_170903, hYJH-010-2PR (SEQ ID NO:48): NF04_170904.

[0089]

[0090] Novoprotein kit: Lot number 7E610k2.

[0091] The results showed that, as Figure 1 shown, after transfection with LipoRNAiMAX, compared with the NC group, all optimized or modified sequences had an inhibitory effect. Compared with YJH-010-3m and YJH-010-4m, the optimized or modified sequence of YJH-010-937mE had the best inhibitory effect, with an inhibition rate reaching 79%.

[0092] Example 3: Knockdown effect experiment of YJH-010 modified sequence in HSKMC cells

[0093] 1. Digest human skeletal muscle cells (HSKMC, Shanghai Qingqi Biotech) in the logarithmic growth phase with trypsin. Add DMEM medium containing 10% FBS to terminate digestion, then centrifuge to collect the cells. Add DMEM medium containing 10% FBS, count the cells with a hemocytometer, and then add 80,000 cells to each well of a 24-well plate for culture.

[0094] 2. Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture: Select the sequences in Table 6 for

[0095] Table 6

[0096]

[0097] Dilute 10 nM / well of siRNA and 1.5 μL of LipoRNAiMAX (Invitrogen) separately in 25 μL of serum-free culture medium (Opti-MEM, Gibco), and then mix the above siRNA solution with the LipoRNAiMAX (Invitrogen) solution and let it stand at room temperature for 5 minutes.

[0098] 3. Add 50 μL of the siRNA and LipoRNAiMAX (Invitrogen) mixed solution corresponding to each group to each well.

[0099] 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 stratification to the well plate to bind with the binding solution, use a nucleic acid extractor, 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.

[0100] 5. Prepare the qPCR system on ice. Add 1 μL of One Step SYBR Green Mix (Vazyme), 10 μL of 2*One Step SYBR Green Mix (Vazyme), 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 RNase ddH2O (Vazyme) and add it to the well, mix well and put it into the qPCR instrument for reaction.

[0101] 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.

[0102] PCR primers: hYJH-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906.

[0103]

[0104] Vazyme kit: Batch number 7E610k2.

[0105] The results show that, as Figure 2As shown, after transfection with LipoRNAiMAX, compared with the NC group, the sequences YJH-010-937 and YJH-010-937mE had extremely significant inhibitory effects, with inhibition rates of 50.67% and 54%; the sequences YJH-010-709 and YJH-010-4m had inhibitory effects of 25% and 13.67% respectively. Therefore, the sequence YJH-010-937mE was selected for the next experiment.

[0106] Example 4: Knockdown effect experiment of YJH-010 modified sequence in C2C12 cells

[0107] 1. Trypsinize mouse myoblasts (C2C12, Shanghai Saibakang) in the logarithmic growth phase, add DMEM medium with 10% FBS to terminate digestion, then centrifuge to collect the cells, add DMEM medium with 10% FBS, count the cells using a hemocytometer, and then add 80,000 cells to each well of a 24-well plate for culture.

[0108] 2. Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture: Select the sequences in Table 7 for

[0109] Table 7

[0110]

[0111] Dilute 10 nM / well of siRNA and 1.5 μL of LipoRNAiMAX (invitrogen) separately in 25 μL of serum-free culture medium (Opti-MEM, purchased from gibco), then mix the above siRNA solution with the LipoRNAiMAX (invitrogen) solution and let it stand at room temperature for 5 minutes.

[0112] 3. Add 50 μL of the siRNA and LipoRNAiMAX (invitrogen) mixed solution corresponding to each group to each well.

[0113] 4. 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, 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 according to the kit instructions (MagaBio plus total RNA purification kit II, manufacturer Bori, product number: BSC69M1E), place the well plate in order and use the BSC69 program to extract RNA.

[0114] 5. Prepare the qPCR system on ice. Add 1 μL of One Step SYBR Green Mix (Novoprotein), 10 μL of 2*One Step SYBR Green Mix (Novoprotein), 0.4 μL of mYJH-010-1PF, and 0.4 μL of mYJH-010-1PR to each well. Dilute 30 ng of RNA or 20 ng of RNA in 8.2 μL of RNase ddH2O (Novoprotein) and add it to the well. Mix well and place it in 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: mYJH-010-1PF (SEQ ID NO:51): NF06_190704, mYJH-010-1PR (SEQ ID NO:52): NF06_190705.

[0117]

[0118] Novoprotein kit: Lot number 7E610k2.

[0119] The results showed that, as Figure 3 shown, after transfection with LipoRNAiMAX, compared with the NC group, all sequences had a significant inhibitory effect on the DMPK mRNA level in C2C12 cells. Compared with YJH-010-2m, YJH-010-3m, and YJH-010-4m, YJH-010-937mE had the best inhibitory effect. The inhibition rates of YJH-010-937mE, YJH-010-709mE, YJH-010-2m, YJH-010-3m, and YJH-010-4m were 51.00%, 41.33%, 35.00%, 40.00%, and 36.00% respectively.

[0120] Example 5: In vitro stability experiment of YJH-010 sequence.

[0121] 1. Preparation of exonuclease solution: 120 μL of 5M NaCl (MREDA), 20 μL of 1M Tris-HCl (pH 7.4) (solarbio), 20 μL of 0.5M disodium EDTA (Xiangerkang), 1 μL of RNase T1 (250 U / μL) (Thermoscientific), and add nuclease-free sterile water (Meilunbio) to 2 mL.

[0122] 2. Use the sequences in Table 8 as the objects:

[0123] Table 8

[0124]

[0125] Dilute them to 0.02 mg / mL with enzyme-free sterile water (Meilunbio) respectively, and prepare them according to 40 μL siRNA solution + 4 μL exonuclease solution + 156 μL enzyme-free sterile water. Place the sample solutions in a thermostatic metal bath (Youning Instruments) and incubate them at 37 °C for 6 h, 2 h, and 0 h respectively.

[0126] 3. Prepare the control BLK siRNA solution. Take 4 μL of 0.02 mg / mL siRNA solution and add 16 μL of enzyme-free sterile water to prepare a 4 μg / mL siRNA solution.

[0127] 4. Weigh 2 g of agarose (solarbio) and add it to 200 mL of 1×TAE buffer (solarbio). Heat to dissolve the agarose, then add 10 μL of nucleic acid dye (solarbio), mix well, and pour it out while it is hot and let it cool to make a gel.

[0128] 5. After the sequence incubation is completed, remove the samples from the 37 °C environment.

[0129] 6. Prepare the loading solution by mixing 10 μL of the sample solution with 10 μL of RNA loading Buffer (solarbio). Load 18 μL into each well, and perform gel electrophoresis on a gel electrophoresis apparatus (BIORAD) at 120 V for 25 min. After electrophoresis, take a photo using a gel imager (SAGECREATION).

[0130] The results are as Figure 4 shown. The stabilities of the modified YJH-010-937mS, YJH-010-937mE, and YJH-010-937mE+ sequences in vitro for 6 h are not very different. However, the results of the knockdown effect experiments in Hela, HSKMC, and C2C12 cells show that the inhibitory effect of YJH-010-937mE is the best. Therefore, considering comprehensively, the YJH-010-937mE sequence is selected.

[0131] Example 6: Knockdown effect experiment of YJH-010-937Ab in HSKMC cells with or without transfection reagent

[0132] 1. Digest human skeletal muscle cells (HSKMC, Shanghai Qingqi Bio) in the logarithmic growth phase with trypsin. After adding DMEM medium containing 10% FBS to terminate the digestion, centrifuge to collect the cells. Add DMEM medium containing 10% FBS, count the cells using a hemocytometer, and then add 100,000 cells to each well of a 24-well plate for culture.

[0133] 2. Preparation (transfection) of the mixture of LipoRNAiMAX (invitrogen) and siRNA and the mixture of siRNA conjugate:

[0134] Table 9

[0135]

[0136] Dilute 10 nM / well of YJH-010-937mE, YJH-010-937mER, YJH-010-937Ab (DAR2), YJH-010-937Ab (DAR1) and 1.5 μL of LipoRNAiMAX (invitrogen) separately in 25 μL of serum-free culture medium (Opti-MEM, gibco). Then mix the above YJH-010-937mE, YJH-010-937mER, YJH-010-937Ab (DAR2) and YJH-010-937Ab (DAR1) with the LipoRNAiMAX (invitrogen) solution respectively and let stand at room temperature for 5 minutes.

[0137] Preparation (free uptake) of the siRNA mixture and the siRNA conjugate mixture without LipoRNAiMAX: Dilute 10 nM / well of YJH-010-937mE, YJH-010-937mER, YJH-010-937Ab (DAR2) and YJH-010-937Ab (DAR1) separately in 50 μL of serum-free culture medium (Opti-MEM, gibco) and let stand at room temperature for 5 minutes.

[0138] 3. Add 50 μL of the above mixed solutions of YJH-010-937mE, YJH-010-937mER, YJH-010-937Ab (DAR2) and YJH-010-937Ab (DAR1) transfected with LipoRNAiMAX (invitrogen) and not transfected with LipoRNAiMAX (invitrogen) to each well.

[0139] 4. 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, shake well and let stand at room temperature for 2 - 3 minutes, centrifuge, transfer the supernatant after stratification to the well plate to bind with the binding solution, and use a nucleic acid extractor. According to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer Bioer, product number: BSC69M1E), place the well plate in order and use the BSC69 program to extract RNA.

[0140] 5. Prepare the qPCR system on ice. Add 1 μL of One Step SYBR Green Mix (Novoprotein), 10 μL of 2*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 RNase ddH2O (Novoprotein) and add it to the well. Mix well and place it in the qPCR instrument for reaction.

[0141] 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.

[0142] PCR primers: hYJH-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906.

[0143]

[0144] Novoprotein kit: Lot number 7E610k2.

[0145] The results show that, as Figure 5 shown, after transfection with LipoRNAiMAX, compared with the NC group, the YJH-010-937mE and YJH-010-937mER sequences have significant inhibitory effects after transfection with LipoRNAiMAX, and the inhibition rates are 45% and 56% respectively. However, the inhibitory effects of these two sequences are weakened when the transfection reagent is not added. In addition, the YJH-010-937Ab sequence has significant inhibitory effects whether transfected with LipoRNAiMAX or not, and the inhibitory effects are consistent, indicating that the conjugated antibody can effectively promote the transfection of the siRNA sequence. It shows that the siRNA antibody conjugate prepared by this method has good inhibitory effects.

[0146] Example 7: In vivo mRNA knockdown experiment of siRNA antibody conjugate

[0147] 1. Evaluate the siRNA antibody conjugate using hTFR1 humanized mice. Intravenous injection of 3 mg / kg (calculated based on the siRNA dose) of the siRNA antibody conjugate to mice, n = 3. Euthanize the mice on the 7th day after administration and isolate liver, heart, brain, quadriceps, and gastrocnemius tissues.

[0148] 2. Extract the RNA from each tissue and perform RT-qPCR detection. 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.

[0149] PCR primers: mYJH-010-1PF (SEQ ID NO:51): NF06_190704, mYJH-010-1PR (SEQ ID NO:52): NF06_190705.

[0150]

[0151] The results showed that, as Figure 6 shown, after intravenous administration of the siRNA antibody conjugate, compared with the blank control group, the DMPK mRNA levels in the brain tissue, gastrocnemius muscle, and quadriceps muscle of the administered group of mice decreased by 21.33%, 23.33%, and 51.67% respectively, while there was no decrease in the liver and heart. It was shown that the delivery of siRNA by the TFR1 antibody was muscle-specific.

[0152] The above examples can illustrate that conjugating an antibody on the YJH-010-937mE sequence can achieve an effect similar to that of the transfected sequence without transfection, and can reduce the expression of DMPK mRNA in human skeletal muscle cells HSKMC; the untransfected YJH-010-937Ab sequence also has an equivalent effect to the transfected YJH-010-937mE sequence, while the YJH-010-937mE modified sequence without antibody conjugation does not have an inhibitory effect without the assistance of a transfection reagent for delivery. That is to say, the transfected YJH-010-937mE sequence cannot be delivered into the body to exert an inhibitory effect, while the YJH-010-937Ab sequence with an antibody can achieve the transfection effect and be delivered into the body. Therefore, the siRNA antibody conjugate prepared by this method can effectively inhibit the expression of the DMPK gene.

[0153] 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 disclosure, drawings, and claims of this application, 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 method for preparing a siRNA-antibody conjugate, characterized in that: The steps include: (1) Using DMPK siRNA duplexes, the single strands were fully assembled and modified on a solid phase using standard phosphoramidite chemistry to obtain and modify the sense and antisense strands; (2) conjugating a C6-NH2 linking site to the 5' end of the sense strand of the siRNA, and connecting it to 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester to obtain a linker-siRNA conjugate; (3) connecting an antibody to one linker-siRNA conjugate or two linker-siRNA conjugates to obtain a siRNA-antibody conjugate of DAR1 or DAR2; (4) Purification: The fractions containing DAR1 or DAR2 siRNA-antibody conjugates were separated, concentrated, and buffer exchanged with phosphate buffer. The purity of the siRNA-antibody conjugates was evaluated by size exclusion chromatography (SEC).

2. The method according to claim 1, characterized in that The synthesis step of the siRNA antibody conjugate also includes: (a1) Buffer exchange of the antibody with 50 mM phosphate buffer, pH 7.0; (b1) adding an equivalent amount of TCEP in the same phosphate buffer to (a1), and incubating at 22°C for 2 hours to obtain an antibody-TCEP mixture; (c1) The antibody-TCEP mixture and the linker-siRNA conjugate were combined in 10 mM acetate buffer at pH 6.0 and incubated at 22°C for 1 hour.

3. The method according to claim 1, characterized in that The synthesis step of the siRNA antibody conjugate also includes: (a2) mixing the linker-siRNA conjugate and NEM (N-ethylmaleimide) to obtain a linker-siRNA-NEM mixture and exchanging the antibody buffer with 50 mM phosphate buffer at pH 7.0; (b2) adding an equivalent amount of TCEP in the same phosphate buffer to (a2) and incubating at 22°C for 2 hours to obtain an antibody-TCEP mixture; (c2) The antibody-TCEP mixture and the linker-siRNA-NEM mixture in 10 mM acetate buffer at pH 6.0 were combined and incubated at 22°C for 1 hour.

4. The method according to claim 2 or 3, characterized in that: The amount of the linker-siRNA conjugate or linker-siRNA-NEM mixture is 1 to 3 times the equivalent of the antibody, and the amount of TCEP is 1 to 3 times the equivalent of the antibody.

5. The method according to claim 3, characterized in that: The ratio of the linker-siRNA conjugate to NEM was 1:

1.

6. The method according to claim 2 or 3, characterized in that: The concentration reached after the buffer exchange is 2-9 mg / ml.

7. The method according to claim 2, characterized in that The amount of TCEP is the same as that of the linker-siRNA conjugate.

8. The method according to claim 3, characterized in that The amount of TCEP was the same as that of the linker-siRNA-NEM mixture.

9. The method according to claim 1, characterized in that: The sense strand is the sequence shown in SEQ ID NO:31, 15, 35, 37, 39, 17, 41, 43, 45, and the antisense strand is the sequence shown in SEQ ID NO:32, 16, 36, 38, 40, 18, 42, 44, 46.

10. Use of the siRNA-antibody conjugate prepared according to the method of claim 9 as a drug for treating DM1 myotonic dystrophy.

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