SiRNA antibody conjugate and application thereof

Abstract

The invention discloses a siRNA antibody conjugate and application thereof, and the siRNA antibody conjugate comprises an antibody, a connexon and siRNA, the 3'end of an antisense strand of the siRNA has 1-3 protruding ends, the 5 'end of a sense strand of the siRNA has 1-3 protruding ends, at least one siRNA in the sense strand or the antisense strand is modified siRNA, and the modified siRNA is selected from one or more of phosphate groups or nucleotide analogues with sugar part modification at the 2' position or with at least one phosphate group containing a modification group. The siRNA antibody conjugate provided by the invention can reduce the expression of DMPK mRNA in human skeletal muscle cells HSKMC, and has great significance in treating DM1 type ankylosing muscular dystrophy diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an siRNA antibody conjugate and its use. 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 is degraded, 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 novel chimeric biomolecule derived from antibody-drug conjugate (ADC) and 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.

[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 is mostly manifested 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 caused by 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, with the adult type being 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. Tendon reflexes are weakened or absent. 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 there is mental retardation or even dementia. Skull radiographs can show thickening of the skull. There may also be abnormal electroencephalograms and enlargement of the ventricles, etc. Patients often die of sudden cardiac death. The progression of DM1 is slow, with a disease course of 20 - 25 years, and most patients die at the age of 45 - 50.

[0006] There is currently no effective symptomatic treatment for DM1. 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] Mexiletine has been proven to significantly reduce non - dystrophic myotonia. Therefore, for patients with myotonic dystrophy with functionally limiting myotonia, mexiletine has become a first - line drug. However, mexiletine can exacerbate arrhythmia in patients with ventricular arrhythmia in a few cases, so the drug is contraindicated in patients with second - or third - degree atrioventricular block; experts recommend consulting a cardiologist before starting mexiletine treatment, especially for patients with abnormal electrocardiograms.

[0008] ① Myotonia can be treated with membrane - stabilizing drugs, which can promote the activity of sodium pumps, reduce the sodium ion concentration inside the membrane, increase the resting potential, and improve the state of myotonia, such as quinine sulfate, procainamide, and methoin; phenytoin may be the first choice for myotonic dystrophy because other drugs have adverse effects on cardiac conduction;

[0009] ② There is currently no treatment for muscle weakness. Muscle atrophy can be treated with nandrolone phenylpropionate to enhance protein anabolic metabolism;

[0010] ③ Other non - drug treatment methods mainly include strengthening muscle exercise, braces, mobility aids, etc., aiming to improve the patient's lifestyle and quality of life.

[0011] The current 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 become drugs. Summary of the Invention

[0012] To achieve the above - mentioned objectives, the present invention adopts the following technical solutions:

[0013] One object of the present invention is to provide an siRNA, comprising a sense strand and an antisense strand, wherein the antisense strand is reverse complementary to a segment on a target gene, the length of the sense strand or the antisense strand of the siRNA is 21-27 bp, the sense strand of the siRNA is the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or a sequence having more than 90% homology with the sense strand; the antisense strand is the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or a sequence having more than 90% homology with the antisense strand, and the siRNA is used to inhibit the DMPK gene.

[0014] The 3'-end of the antisense strand has 1-3 overhangs, and the 5'-end of the sense strand has 1-3 overhangs.

[0015] Preferably, at least one of the siRNAs is a modified nucleotide, and the modified nucleotide is selected from one or more of a modification of the sugar moiety at the 2'-position, a phosphodiester group in which at least one phosphate group is a phosphate group containing a modified group, or a nucleotide analogue.

[0016] Preferably, the modification of the sugar moiety includes one or more of 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), T-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) modification.

[0017] Preferably, the phosphate group containing a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom.

[0018] Preferably, the nucleotide analogue is one of a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a cET bridged nucleic acid (BNA), an unlocked nucleic acid (UNA), and a glycol nucleic acid (GNA).

[0019] Preferably, the sense strand of the modified sequence is the sequence shown in SEQ ID NO: 35, 37, 39, 41, 43, 45; the antisense strand is the sequence shown in SEQ ID NO: 36, 38, 40, 42, 44, 46.

[0020] The second object of the present invention is to provide an siRNA antibody conjugate, which comprises a linker, an antibody and the siRNA according to any one of claims 1-7. The 5'-end of the sense strand of the siRNA is conjugated to the linker, and the linker is conjugated to the antibody.

[0021] Preferably, the number of the siRNA and the linker is one or two.

[0022] Preferably, the linker comprises SMCC, Sulfo-SMCC, LC-SMCC, SMPB, KMUS, MBS, GMBS, AMAS.

[0023] Preferably, the antibody comprises a heavy chain and a light chain. The heavy chain is as shown by mAb-1H of SEQ ID NO:53, and the light chain is as shown by mAb-1L of SEQ ID NO:54.

[0024] The third object of the present invention is to provide a pharmaceutical composition, which comprises: the siRNA antibody conjugate according to claim 11; and a pharmaceutically acceptable excipient. The pharmaceutical composition is formulated as a nanoparticle preparation or a liquid preparation, and is formulated for parenteral, oral, intranasal, buccal, rectal or transdermal administration. Parenteral administration includes intravenous, subcutaneous and intramuscular administration.

[0025] Preferably, the siRNA or the siRNA antibody conjugate is used for the treatment of myotonic dystrophy type 1 (DM1). Beneficial effects

[0026] 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'-untranslated region. At the same time, by conjugating the TFR1 antibody, the siRNA can be effectively delivered to muscle tissues, further reducing the systemic toxicity of the siRNA drug, and thus playing a role in the treatment of myotonic dystrophy type 1 (DM1). In addition, as a new type of "two-in-one" drug, AOC has the following advantages: 1) It can accurately identify 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 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) AOC can be personalized designed according to the genotype and phenotype of patients, so as to further improve the therapeutic effect and safety. Description of the drawings

[0027] The following further describes in detail the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0028] Figure 1 It is the effect diagram of siRNA naked sequence inhibiting DMPK mRNA expression in Hela cells.

[0029] Figure 2 It is the effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in Hela cells.

[0030] Figure 3 It is the effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in human skeletal muscle cells HSKMC.

[0031] Figure 4 It is the effect diagram of the modified siRNA sequence inhibiting DMPK mRNA expression in mouse myoblast C2C12.

[0032] Figure 5 It is the effect diagram of the in vitro stability of the modified siRNA sequence and the naked sequence.

[0033] Figure 6 It is the effect diagram of the siRNA antibody conjugate inhibiting DMPK mRNA expression in human skeletal muscle cells HSKMC.

[0034] Figure 7 It is the result diagram of the in vivo mRNA knockdown experiment of the siRNA antibody conjugate. Specific Embodiments

[0035] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] As used in the present invention, the term "connection", when referring to the connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (for example, a hydrogen bond or an ionic bond).

[0037] As used in the present invention, an "oligonucleotide" is a nucleotide sequence containing 10 - 50 nucleotides or nucleotide base pairs. In some embodiments of the present invention, the oligonucleotide has a nucleobase sequence that is at least partially complementary to the coding sequence in the target gene expressed in the cell. The nucleotides can be optionally modified. In some embodiments of the present invention, after the oligonucleotide is delivered to the cell expressing the gene, the oligonucleotide can inhibit or block the expression of the gene in vitro or in vivo.

[0038] As used herein, the term "inhibition", when referring to the expression of a given gene, means that the gene expression is reduced when the cell, cell population or tissue is treated with the siRNA, pharmaceutical composition and siRNA conjugate of the present invention, as compared to a cell, cell population or tissue that has not been treated.

[0039] The term "inhibition" as used herein is interchangeable with "reduction", "silencing", "downregulation", "suppression" and other similar terms, and includes any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0040] Each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide. In the context of the present invention, unless otherwise specified, "conjugation" means the connection of two or more chemical moieties each having a specific function to each other in a covalent linkage; accordingly, a "conjugate" means a compound formed by the covalent linkage between the respective chemical moieties. Further, an "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties having specific functions to an siRNA.

[0041] In the foregoing and the following, unless otherwise specified, generally, "G", "C", "A", "T" and "U" each represent a nucleotide containing guanine, cytosine, adenine, thymine and uracil as bases. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, a nucleotide surrogate replacement moiety, as further detailed below.

[0042] In the context of the present invention, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meanings well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions do not pair in a complementary form.

[0043] As used above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between two nucleotide sequences involved; "essentially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "fully reverse complementary" means that there is no base mismatch between two nucleotide sequences. As used above and below, that a nucleotide sequence has "nucleotide differences" from another nucleotide sequence means that, compared with the latter, the base type of the nucleotide at the same position has changed. For example, when a nucleobase in the latter is A, if the corresponding nucleobase at the same position in the former is U, C, G or T, it is determined that there are nucleotide differences between the two nucleotide sequences at this position. In some embodiments, when a nucleotide at the original position is replaced with a non-base nucleotide or its equivalent, it can also be considered that nucleotide differences are generated at this position.

[0044] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. For example, for the experimental methods without specific conditions in the following examples, they are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified. For example, the sequences can be commissioned to existing nucleic acid synthesis companies for synthesis.

[0045] Example 1: Experiment on the knockdown effect of naked siRNA sequences

[0046] 1. Digest human cervical cancer cells (Hela, Shanghai Saibakang) 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.

[0047] 2. Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture: Select the sequences in Table 1 for:

[0048]

[0049] Dilute 10 nM / well of the above siRNA and 1.5 μL of the LipoRNAiMAX (invitrogen) transfection reagent respectively in 25 μL of serum-free culture medium (Opti-MEM, gibco), and then mix the above siRNA solution and the LipoRNAiMAX (invitrogen) solution, and let it stand at room temperature for 5 minutes.

[0050] 3. Add 50 μL of the mixed solution of siRNA and LipoRNAiMAX (invitrogen) corresponding to each group into each well.

[0051] 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, mix well by shaking and let stand at room temperature for 2 - 3 minutes, centrifuge, and 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, batch number c692302005), and then use the BSC69 program to extract total RNA.

[0052] 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-010-2PF, 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 RNase ddH2O (Vazyme) and add it into the well, mix well and put it into the qPCR instrument for reaction.

[0053] 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.

[0054] PCR primers: hYJH-010-2PF (SEQ ID NO:47): NF04_170903, hYJH-010-2PR (SEQ ID NO:48): NF04_170904.

[0055]

[0056] Vazyme kit: Batch number 7E610k2.

[0057] Table 2: Naked sequence inhibition rate table (take the average of three times under the same batch)

[0058]

[0059] The results show that as shown in Table 2 and Figure 1As shown, after transfection with LipoRNAiMAX, except for YJH-010-545, the other 11 sequences all showed obvious inhibitory effects. Compared with YJH-010-3m and YJH-010-4m, the two sequences YJH-010-709 and YJH-010-937 were better, so these two sequences were modified.

[0060] Example 2: Experiment on the knockdown effect of YJH-010 modified sequences in Hela cells

[0061] 1. Digest Hela cells in the logarithmic growth phase with trypsin, terminate digestion with DMEM medium containing 10% FBS, centrifuge to collect 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.

[0062] 2. Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture: Select the sequences in Table 3 for:

[0063] Table 3

[0064]

[0065] 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.

[0066] 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.

[0067] 3. Add 50 μL of the siRNA and LipoRNAiMAX (invitrogen) mixed solution corresponding to each group to each well.

[0068] 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 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, product number: BSC69M1E), and then use the BSC69 program to extract RNA.

[0069] 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 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.

[0070] 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.

[0071] PCR primers: hYJH-010-2PF (SEQ ID NO:47): NF04_170903, hYJH-010-2PR (SEQ ID NO:48): NF04_170904.

[0072]

[0073] Novoprotein kit: Lot number 7E610k2.

[0074] Table 4: Inhibition rate table of the screened naked sequences and their modified sequences (taking the average of three times under the same batch)

[0075]

[0076] The results show that, as shown in Table 4 and Figure 2 as shown, after transfection with LipoRNAiMAX, compared with the NC group, all optimized or modified sequences have inhibitory effects. Compared with YJH-010-3m and YJH-010-4m, the optimized or modified sequences of YJH-010-937mE have the best inhibitory effect.

[0077] It should be noted that there are differences between the naked sequences in this part and the results of Example 1, mainly because the two experiments were not carried out in the same batch. Even under the same conditions in biological experiments, there will be significant differences in different batches, which is a normal phenomenon and does not affect the experimental plan and subsequent screening.

[0078] Example 3: Knockdown effect experiment of YJH-010 modified sequence in HSKMC cells

[0079] Digest human skeletal muscle cells (HSKMC, Shanghai Qingqi Biotechnology) 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 using a hemocytometer, and then add 80,000 cells to each well of a 24-well plate for culture.

[0080] Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture: The following sequences were selected:

[0081] Table 5

[0082]

[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 with the 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, mix well by shaking, 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, place the well plate in order according to the kit instructions (MagaBio plus Total RNA Purification Kit II, manufacturer: Bori, product number: BSC69M1E), and then use the BSC69 program to extract RNA.

[0086] 5. Prepare the qPCR system on ice. Add 1 μL of One Step SYBR Green Mix (Novizan), 10 μL of 2*One Step SYBR Green Mix (Novizan), 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 (Novizan) and add it to the well, mix well, and put it into a 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-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906.

[0089]

[0090] Novoprotein kit: batch number 7E610k2.

[0091] The results showed that, as Figure 3 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.

[0092] Example 4: Experiment on the knockdown effect of YJH-010 modified sequence in C2C12 cells.

[0093] 1. Digest mouse myoblasts (C2C12, Shanghai Saibakang) in 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 mixture of LipoRNAiMAX (invitrogen) and siRNA: The following sequences were selected:

[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, purchased from gibco), then mix the above siRNA solution and LipoRNAiMAX (invitrogen) solution, and let it stand at room temperature for 5 minutes.

[0098] 3. Add 50 μL of the mixed solution of siRNA and LipoRNAiMAX (invitrogen) 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, 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: Bori, catalog number: BSC69M1E), then use the BSC69 program to extract RNA.

[0100] 5. Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novoprotein), 10 μL 2*One Step SYBR Green Mix (Novoprotein), 0.4 μL mYJH-010-1PF, and 0.4 μL mYJH-010-1PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL RNase ddH2O (Novoprotein) and add it to the well, mix well and put it into a 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: mYJH-010-1PF (SEQ ID NO:51): NF06_190704, mYJH-010-1PR (SEQ ID NO:52): NF06_190705.

[0103]

[0104] Novoprotein kit: batch number 7E610k2.

[0105] The results showed that, as Figure 4 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.

[0106] Example 5: In vitro stability experiment of YJH-010 sequence.

[0107] 1. Preparation of the exonuclease digestion solution: 120 μL of 5 M NaCl (MREDA), 20 μL of 1 M Tris-HCl (pH 7.4) (solarbio), 20 μL of 0.5 M disodium EDTA (Xiangerkang), 1 μL of RNase T1 (250 U / μL) (Thermoscientific), and add enzyme-free and sterile water (Meilunbio) to make up to 2 mL.

[0108] 2. Using the following sequences as the targets:

[0109] Table 7

[0110] Dilute them to 0.02 mg / mL with enzyme-free and sterile water (Meilunbio) respectively, and prepare according to 40 μL of siRNA solution + 4 μL of exonuclease digestion solution + 156 μL of enzyme-free and sterile water. Place the sample solutions in a thermostatic metal bath (Youning Instruments) and incubate at 37 °C for 6 h, 2 h, and 0 h respectively.

[0111] 3. Prepare the control BLK siRNA solution. Take 4 μL of 0.02 mg / mL siRNA solution and add 16 μL of enzyme-free and sterile water to prepare a 4 μg / mL siRNA solution.

[0112] 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.

[0113] 5. After the sequences are incubated, remove the samples from the 37 °C environment.

[0114] 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 using a gel electrophoresis apparatus (BIORAD) at 120 V for 25 min. After electrophoresis, take a photo using a gel imaging apparatus (SAGECREATION).

[0115] The results are as Figure 5 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 experimental results of the knockdown effects 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.

[0116] Example 6: Experiment on the knockdown effect of the YJH-010 modification sequence in HSKMC cells with or without transfection reagent

[0117] 1. Preparation of YJH-010-937mER and YJH-010-937Ab:

[0118] 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 microbody, 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.

[0119] The linker is a bond 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, as shown in Table 8, 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 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide ester (Sulfo-SMCC, CAS: 92921-24-9), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-(6-aminohexanoate) (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.

[0120] Table 8: Abbreviations and structures of each linker

[0121]

[0122] For the siRNA antibody conjugate used in this experiment, a DMPK siRNA duplex was used.

[0123] Solid-phase synthesis: The single strand was fully assembled on the solid phase using standard phosphoramidite chemistry and purified by HPLC. The sense strand of the siRNA contains a C6-NH at the 5' end 2 linking site, and the linking site was further conjugated with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate to obtain YJH-010-937mER. Then an antibody was conjugated to YJH-010-937mER to obtain YJH-010-937Ab (DAR1); two YJH-010-937mER were conjugated to the antibody to obtain YJH-010-937Ab (DAR2). YJH-010-937mER is a linker and siRNA conjugate, and YJH-010-937Ab (DAR1) and YJH-010-937Ab (DAR2) are siRNA-antibody conjugates.

[0124] YJH-010-937mER is as follows:

[0125]

[0126] YJH-010-937Ab (DAR1) is as follows:

[0127]

[0128] YJH-010-937Ab (DAR2) is as follows:

[0129]

[0130] The specific synthesis steps of YJH-010-937Ab (DAR1, DAR2) are as follows:

[0131] Step 1: Reduce the antibody with TCEP

[0132] To synthesize DAR1, first, at room temperature, YJH-010-937mER was mixed with N-ethylmaleimide (NEM) at a ratio of 1:1 to obtain a mixture of YJH-010-937mER and NEM; the buffer of the antibody was exchanged with 50 mM phosphate buffer (pH 7.0) and its concentration was adjusted to 2.83 mg / ml. 1.5 equivalents of TCEP of the antibody in the same phosphate buffer was added to the above solution and incubated at 22 °C for 2 hours to obtain a mixture of the antibody and TCEP. At room temperature, the mixture of the antibody and TCEP, and a solution of 1.5 equivalents of the mixture of YJH-010-937mER and NEM of the antibody in 10 mM acetate buffer at pH 6.0 were combined and incubated at 22 °C for 1 hour.

[0133] Synthesize DAR2, perform buffer exchange on the antibody with 50 mM phosphate buffer (pH 7.0) and adjust its concentration to 7 mg / ml. Add 1.2 equivalents of TCEP of the antibody 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 1.2 equivalents of YJH-010-937mER of the antibody in 10 mM acetate buffer at pH 6.0 and incubate at 22 °C for 1 hour.

[0134] Since two free sulfhydryl groups are generated from a pair of disulfide bonds after TCEP reduces the disulfide bonds in the antibody, and both can be coupled with N-maleimide (NEM)-modified siRNA, generally, coupling by the reduction method of TCEP 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 of them reacts and binds to one sulfhydryl group, thus obtaining an AOC with DAR<2.

[0135] Step 2: Purification

[0136] Analysis of the reaction mixture by ion exchange chromatography (IEX) showed the siRNA-antibody conjugate as well as unreacted antibody and siRNA. The crude reaction mixture was separated to obtain fractions containing the siRNA-antibody conjugate with DAR1 or DAR2, 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), as shown in Table 9.

[0137] Table 9: Purity of siRNA-antibody conjugate

[0138]

[0139] In this example, the antibody of YJH-010-937Ab is the TFR1 antibody, which is a full-length antibody. The sequence is shown below. It contains a heavy chain (mAb-1H) and a light chain (mAb-1L), and the underlined regions represent their respective CDRs.

[0140] Table 10.

[0141]

[0142] 2. Trypsinize human skeletal muscle cells (HSKMC, Shanghai Qingqi Biotechnology) in the logarithmic growth phase. After adding DMEM medium containing 10% FBS to terminate 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.

[0143] 3. Preparation of the LipoRNAiMAX (invitrogen) and siRNA mixture and the siRNA conjugate mixture (transfection):

[0144] Table 11

[0145] 。

[0146] 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.

[0147] 4. Preparation of the siRNA mixture and the siRNA conjugate mixture without LipoRNAiMAX (free uptake): 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.

[0148] 5. Add 50 μL of the above YJH-010-937mE, YJH-010-937mER, YJH-010-937Ab (DAR2) and YJH-010-937Ab (DAR1) mixed solutions that have been transfected with LipoRNAiMAX (invitrogen) and those without LipoRNAiMAX (invitrogen) to each well.

[0149] 6. 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, 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: Bori, product number: BSC69M1E), and then use the BSC69 program to extract RNA.

[0150] 7. Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novoprotein), 10 μL 2*One Step SYBR Green Mix (Novoprotein), 0.4 μL hYJH-010-3PF, and 0.4 μL hYJH-010-3PR to each well. Dilute 30 ng RNA or 20 ng RNA in 8.2 μL RNase ddH2O (Novoprotein) and add it to the well, mix well and put it into a qPCR instrument for reaction.

[0151] 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.

[0152] PCR primers: hYJH-010-3PF (SEQ ID NO:49): NF04_170905, hYJH-010-3PR (SEQ ID NO:50): NF04_170906.

[0153]

[0154] Novoprotein kit: batch number 7E610k2.

[0155] The results showed that, as Figure 6 shown, after transfection with LipoRNAiMAX, compared with the NC group, the YJH-010-937mE and YJH-010-937mER sequences had significant inhibitory effects after transfection with LipoRNAiMAX, and the inhibition rates were 45% and 56% respectively. However, the inhibitory effects of these two sequences weakened when the transfection reagent was not added. In addition, the YJH-010-937Ab sequence had significant inhibitory effects with or without transfection with LipoRNAiMAX, and the inhibitory effects remained consistent, indicating that the conjugated antibody can effectively promote the transfection of the siRNA sequence.

[0156] Example 7: In vivo mRNA knockdown experiment of YJH-010-937Ab (DAR2)

[0157] Assess the siRNA antibody conjugate using hTFR1 humanized mice. Administer the siRNA antibody conjugate at a dose of 3 mg / kg (calculated based on the siRNA dose) to the mice via intravenous injection, n = 3. Euthanize the mice on the 7th day after administration and isolate the liver, heart, brain, quadriceps, and gastrocnemius tissues.

[0158] 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, for 39 cycles.

[0159] PCR primers: mYJH-010-1PF (SEQ ID NO:51): NF06_190704, mYJH-010-1PR (SEQ ID NO:52): NF06_190705.

[0160]

[0161] The results showed that, as Figure 7 shown, after intravenous administration of the siRNA antibody conjugate, compared with the blank control group, the DMPK mRNA levels in the brain tissue, gastrocnemius, and quadriceps 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 shows that the delivery of siRNA by the TFR1 antibody is muscle-specific.

[0162] The above examples can illustrate that conjugating an antibody to 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 effect equivalent to that of 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 untransfected 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 studied in the present invention can deliver siRNA into the interior of TFR1-highly expressed cells and exert its inhibitory effect on mRNA.

[0163] The detailed descriptions listed above are only specific descriptions of the feasible implementation manners 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 manners, and these modifications and implementation manners will fall within the principle scope 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 siRNA, characterized in that The sense strand of the siRNA is the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23; the antisense strand is the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24.

2. The siRNA according to claim 1, characterized in that The 3' end of the antisense strand has 1-3 overhangs, and the 5' end of the sense strand has 1-3 overhangs.

3. The siRNA according to claim 2, characterized in that At least one of the siRNAs is a modified nucleotide, and the modified nucleotide is selected from one or more of a phosphate group or a nucleotide analogue containing a modified group and a sugar part modified at the 2' position.

4. The siRNA according to claim 3, characterized in that The modifications of the sugar moiety include one or more of 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), T-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modifications.

5. The siRNA according to claim 4, characterized in that The phosphate group containing a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom.

6. The siRNA according to claim 5, characterized in that The nucleotide analogue is one of isonucleotide, LNA, ENA, cET BNA, UNA and GNA.

7. The siRNA according to claim 6, characterized in that The sense strand of the modified sequence is the sequence shown in SEQ ID NO: 35, 37, 39, 41, 43, 45; the antisense strand is the sequence shown in SEQ ID NO: 36, 38, 40, 42, 44, 46.

8. A siRNA-antibody conjugate, characterized in that: The siRNA-antibody conjugate comprises a linker, an antibody and the siRNA according to any one of claims 1 or 7, wherein the 5' end of the sense strand of the siRNA is conjugated to the linker, and the linker is conjugated to the antibody.

9. The siRNA-antibody conjugate according to claim 8, characterized in that: The number of the siRNA and the linker is one or two.

10. The siRNA-antibody conjugate according to claim 9, characterized in that: The linkers include SMCC, Sulfo-SMCC, LC-SMCC, SMPB, KMUS, MBS, GMBS, and AMAS.

11. The siRNA-antibody conjugate according to claim 10, characterized in that: The antibody comprises a heavy chain and a light chain, the heavy chain being shown as mAb-1H in SEQ ID NO:53, and the light chain being shown as mAb-1L in SEQ ID NO:

54.

12. A pharmaceutical composition comprising: The siRNA-antibody conjugate of claim 11; and a pharmaceutically acceptable excipient or other components.

13. The siRNA according to any one of claims 1 to 7 or the siRNA-antibody conjugate according to any one of claims 8 to 11, characterized in that: The siRNA or the siRNA-antibody conjugate is used as a drug for treating DM1 myotonic dystrophy.

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