Single-stranded nucleic acid or double-stranded nucleic acid and use thereof in preparing medicine for treating Duchenne muscular dystrophy

Abstract

The present invention discloses a single-stranded nucleic acid or a double-stranded nucleic acid and its application in the preparation of a drug for treating Duchenne muscular dystrophy, belonging to the technical field of nucleic acid drugs. The single-stranded nucleic acid is a modified antisense nucleic acid chain; the double-stranded nucleic acid is composed of ① a modified antisense nucleic acid chain and ② a complementary chain or a modified complementary chain, the nucleotide sequence of the antisense nucleic acid chain is shown in SEQ ID NO.1; the nucleotide sequence of the complementary chain is shown in SEQ ID NO.2. A carrier, such as α-tocopherol, may also be connected to the complementary chain. The application of the single-stranded nucleic acid or the double-stranded nucleic acid in the preparation of a drug for treating Duchenne muscular dystrophy. The present invention also discloses a pharmaceutical composition for treating Duchenne muscular dystrophy. The single-stranded nucleic acid or the double-stranded nucleic acid of the present invention can achieve exon skipping, increase the expression of dystrophin, and promote the recovery of muscle strength in patients with Duchenne muscular dystrophy.

Description

Technical Field

[0001] The invention relates to a single-stranded nucleic acid or a double-stranded nucleic acid and application thereof in preparing a medicine for treating Duchenne muscular dystrophy, belonging to the technical field of nucleic acid medicines. Background Art

[0002] Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal rare genetic disease, usually caused by gene mutations, and its pathogenesis is complex and diverse. DMD is more common in children, accounting for 1 / 3500 to 1 / 4000 in male newborns, while women are often carriers and rarely develop the disease. The disease-causing gene, the dystrophin gene, is the largest gene in humans, located in the Xp21.2 region, encoding dystrophin. The protein produced after mutation cannot maintain the integrity of skeletal muscle and myocardial cell membranes. It usually develops latently between the ages of three to five, with the gastrocnemius being the most obvious, and most accompanied by myocardial damage. Most patients die of respiratory infections and heart failure between the ages of twenty and thirty. At present, there is no complete cure for DMD, and new treatment strategies are urgently needed in the clinic.

[0003] Nucleic acid drugs have high therapeutic efficiency, strong specificity, low drug toxicity and a wide range of applications. They have become a hot spot in precision biomedicine and tumor and cancer treatment, and are becoming a leader in the third wave of new drug research and development.

[0004] Antisense oligonucleotide-mediated exon skipping therapy can bind to complementary sequences on mRNA precursors and produce effective target exon skipping by regulating mRNA precursor splicing, thereby restoring the transcription reading frame and producing a shortened but partially functional dystrophin protein. It has been proven that it can restore the dystrophin gene expression function of DMD patients in current clinical treatment. Chinese invention patent CN 102203253 A discloses a multi-exon skipping composition for DMD, provides antisense molecules that can bind to selected target sites in the human dystrophin gene to induce exon skipping, and methods of use thereof. The disclosed morpholino oligomer contains 20 to 35 morpholino subunits, has a large base sequence, and has a high production cost. Summary of the invention

[0005] In view of the above prior art, the present invention provides a double single-stranded nucleic acid or double-stranded nucleic acid, and its use in the preparation of a drug for treating Duchenne muscular dystrophy. The single-stranded nucleic acid or double-stranded nucleic acid of the present invention can significantly jump exon 23, does not require a morpholino oligomer structure, has great application potential, and has broad application prospects.

[0006] The present invention is achieved through the following technical solutions:

[0007] A single-stranded nucleic acid or a double-stranded nucleic acid, wherein the single-stranded nucleic acid is a modified antisense nucleic acid strand; the double-stranded nucleic acid is composed of ① a modified antisense nucleic acid strand (also called a sense strand, template strand, parent strand, main strand) and ② a complementary strand (also called a side strand) or a modified complementary strand, wherein the nucleotide sequence of the antisense nucleic acid strand is shown in SEQ ID NO.1; the nucleotide sequence of the complementary strand is shown in SEQ ID NO.2; the antisense nucleic acid strand and the complementary strand constitute a heterologous double-stranded nucleic acid complex;

[0008] The modified antisense nucleic acid strand is:

[0009] 5'-t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a-3', where capital letters represent RNA; lowercase letters represent DNA; (+) represents locked nucleic acid modification (the 2'-O on the ribose is linked to the 4' carbon), and the number of locked nucleic acid modifications accounts for 20% to 80% of the total number of nucleotides in this chain; m represents 5' methylation modification of cytosine (5'-O-methylation) (i.e. mC represents 5-methylcytosine, which is a common RNA modification); ^ represents phosphorothioate diester bond modification.

[0010] Furthermore, the modification method is selected from any one or more of locked nucleic acid modification, methylation modification (including conventional 2' methylation modification and 5' methylation modification of cytosine), sulfation modification, DNA nucleic acid chain chimeric RNA modification, etc. The purpose of the modification is to enhance the therapeutic effect of single-stranded nucleic acid or double-stranded nucleic acid or make it more stable or play other positive roles.

[0011] Furthermore, the modified complementary chain is: 5'-U^U^C^GAAAAUU^U^C^A-3', or: 5'- U^U^C^gaaaatt^U^C^A-3'.

[0012] Furthermore, a carrier is connected to the complementary chain, and the carrier is selected from a viral vector, a nanomaterial, a liposome, an exosome, etc., preferably α-tocopherol.

[0013] Furthermore, the α-tocopherol is linked to the 5' end of the complementary chain.

[0014] The single-stranded nucleic acid or double-stranded nucleic acid is used in preparing a drug for treating Duchenne muscular dystrophy, and in preparing a drug for generating exon skipping in the processing of mRNA pre-processed by the dystrophin gene.

[0015] A pharmaceutical composition for treating Duchenne muscular dystrophy, wherein the active ingredient is the above-mentioned single-stranded nucleic acid or double-stranded nucleic acid or its derivatives, and may also include commonly used pharmaceutical excipients such as solvents, fillers, etc.

[0016] The single-stranded nucleic acid or double-stranded nucleic acid of the present invention can achieve exon skipping, increase the expression of dystrophin, and promote the recovery of muscle strength in patients with Duchenne muscular dystrophy. In clinical applications, the subjects are mammals including humans. The in vivo animal experiment or clinical administration route is intravenous administration, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intracerebroventricular injection and other parenteral routes or gastrointestinal administration (oral, gastrointestinal nutrition tube, anal plug, etc.), the dosage is 0.01-100 mg / kg / day, and the administration can be repeated.

[0017] The double-stranded nucleic acid of the present invention has been experimentally studied to show that when the main chain is specifically modified (including main chain spacer locked nucleic acid modification) and the complementary chain is not methylated, the double-stranded nucleic acid can better exert its inhibitory effect on the target gene (inhibit the following exon), and when the base sequence or modification method is changed, the double-stranded nucleic acid does not play the preset role (cannot play the role of exon skipping). Moreover, it is generally believed in the prior art that the complementary chain is methylated and modified to have a better effect, but the present invention unexpectedly found that this sequence is better in the case of non-methylation. In addition, the present invention also found through experimental studies that when the complementary chain is DNA, the carrier α-tocopherol has the best effect when connected to the 5' end of the complementary chain (connected at other positions, such as the 3' end of the sense chain, the 3' end of the complementary chain, the 5' end of the sense chain, and when the complementary chain is methylated, the effect is not good).

[0018] The present invention solves the following technical problems and achieves the following technical effects:

[0019] (1) The technical difficulties of nucleic acid medicine have been solved. Through research, the present invention has found that after the single-stranded nucleic acid or double-stranded nucleic acid of the present invention is injected into the muscle of an animal (mice), the exon skipping effect and the expression of anti-dystrophin can be significantly increased, and it can be used as a potential therapeutic drug for the treatment of Duchenne muscular dystrophy. (2) The problem of high cost of double-stranded nucleic acid has been solved. The present invention uses heterologous double-stranded nucleic acid, and the second chain does not need to be methylated, which greatly reduces the cost of nucleic acid synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Schematic diagram of electrophoresis detection results of exon 23 exon skipping Figure 1 .

[0021] Figure 2 :Exon 23 exon skipping electrophoresis detection results Figure 2 .

[0022] Figure 3 : Schematic diagram of the results of determination of exon skipping efficiency (Example 2).

[0023] Figure 4 : Schematic diagram of the results of determination of exon skipping efficiency (Example 3).

[0024] Figure 5 : Schematic diagram of the results of determination of exon skipping efficiency (Example 4).

[0025] Figure 6 :Determination results of exon skipping efficiency Figure 1 (Example 5).

[0026] Figure 7 :Determination results of exon skipping efficiency Figure 2 (Example 5).

[0027] Figure 8 : Schematic diagram of Western Blot detection results of dystrophin (Example 6).

[0028] Fig. 9 : Schematic diagram of dystrophin expression levels (Example 6).

[0029] Fig.10 : Schematic diagram of Western Blot detection results of dystrophin (Example 7).

[0030] Fig.11 : Schematic diagram of dystrophin expression levels (Example 7).

[0031] Fig.12 : Schematic diagram of the results of determination of exon skipping efficiency (Example 8).

[0032] Fig.13 : Schematic diagram of the results of determination of exon skipping efficiency (Example 9).

[0033] Fig.14 : Schematic diagram of the results of determination of exon skipping efficiency (Example 10). DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0035] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0036] The single-stranded nucleic acid and double-stranded nucleic acid of the present invention can be completely or partially prepared and synthesized by chemical synthesis methods (currently mainly using automated synthesizers) or by enzymatic reaction processes (including but not limited to polymerase, ligase or restriction reactions).

[0037] The locked nucleic acid LNA mentioned in the present invention is a kind of oligonucleotide derivative, in which the 2'-O and 4'-C positions of β-D-ribofuranose are connected by hydration to form a rigid structure.

[0038] The present invention adopts the conventional phosphoramidite triester synthesis method to prepare nucleic acids (antisense nucleic acid chain, complementary chain). This method is the most widely used oligonucleotide synthesis method and is also the synthesis method adopted by mainstream synthesizers at home and abroad. This method has the characteristics of high efficiency, rapid coupling and relatively stable starting reactants. First, RNA monomers and DNA monomers are synthesized by conventional methods. RNA methylation, DNA thiolation, locked nucleic acid (bridging nucleic acid) and the like used in the present invention are all conventional methods well known to those skilled in the art. RNA monomers, DNA monomers or locked nucleic acids are fixed on a solid phase carrier to complete the synthesis of RNA or DNA chains, and the synthesis is extended from the 3' end to the 5' end of the primer to be synthesized, and adjacent nucleotides are connected by 3'→5' phosphodiester bonds. According to the predetermined base sequence, the corresponding phosphoramidite synthesis monomers and other necessary chemical reagents are added to the solid phase carrier with surface modification in advance in sequence through the liquid path system to complete the synthesis of the specified oligonucleotide sequence. After the synthesis is completed, the product is cut off and collected from the solid phase carrier by ammonia or other alkaline conditions, and the oligonucleotide of the target base sequence is obtained. The specific operation can be as follows:

[0039] Synthesis parameters: Synthesis scale: 200 umol; Coupling time: 12 min; Ammonolysis conditions: 55°C, concentrated ammonia solution (25% ammonia solution).

[0040] Reagent information: as shown in Table 1.

[0041] Table 1

[0042] Reagents formula ACN ACN Deblock DCA (3% in DCM) Activator BTT(0.3 M in ACN) CAP A Ac2O / THF=10 / 90 (v / v) CAP B Py / NMI / THF=10 / 16 / 74 Thio-reagents DDTT (0.05 M ,ACN / Py=6 / 4)

[0043] Monomer information: dA, dG, dT, dC, LNA-A, LNA-T, LNA-(5Me)C, LNA-G, MOE-A, MOE-T, MOE-C(5Me) are all from Wuhu Huaren Technology Co., Ltd.

[0044] Instrumental synthesis reaction steps:

[0045] The first step is deprotection. Trichloroacetic acid is used to remove the DMT on the nucleoside connected to CPG to expose the 5' hydroxyl group for the next coupling step. In this step, it should be noted that TCA is a strong acid and may have a depurinating effect, so the contact time between TCA and oligonucleotide should not exceed the specified time of 3 minutes.

[0046] The second step is activation. Before coupling, the monomer is mixed with tetrazole and enters the synthesis column. At this time, tetrazole provides a proton to the N atom of the diisopropylamine group on the 3' phosphate. The protonated diisopropylamine is a good free group, which forms a phosphoramidite tetrazole, an active intermediate, with tetrazole. The excess tetrazole in this step ensures that the monomer is fully activated.

[0047] The third step is coupling. When the phosphoramidite tetrazole collides with the nucleotide connected to CPG, it undergoes a nucleophilic reaction with its 5' hydroxyl group, coupling occurs and the tetrazole is removed, and the synthesized oligonucleotide chain is extended by one. The excess of monomers in this step relative to the 5'-hydroxyl group on the nucleotide connected to CPG ensures high coupling efficiency.

[0048] The fourth step is capping. In order to prevent the unreacted 5' hydroxyl group connected to CPG from being extended in the subsequent cycles, it is necessary to block it after the coupling reaction is fully carried out. Acetylation is often used to block this hydroxyl group. Before use, acetic anhydride and N-methylimidazole are mixed to form a highly active acetylation reagent, which forms an ester bond with a small amount of 5' hydroxyl group that does not participate in the coupling reaction. Since there are few hydroxyl groups to be blocked and the acetylation reagent is highly active and fully excessive, the reaction speed of this step is very fast, and a few seconds are enough. Too long capping time may cause acetylation reaction at unexpected positions, and increase the risk of acetic anhydride and trace water forming acid to attack the newly formed phosphite bond.

[0049] Step 5 - Thiosulfation. After the coupling reaction, the newly added nucleotide is connected to the oligonucleotide chain on the CPG through a phosphite bond (phosphorus is trivalent). This phosphite bond is unstable and easily hydrolyzed by acid and alkali, so the trivalent phosphorus here needs to be sulfurized to pentavalent phosphorus. Use DDTT (0.05 M, ACN / Py=6 / 4). The reaction speed of this step is also very fast.

[0050] The above steps 1 to 5 are repeated until the oligonucleotide chain is extended to the desired length.

[0051] Cleavage and deprotection: Add concentrated ammonia (25% ammonia solution) to the support (PS), heat to 55°C and react overnight for aminolysis. Then filter the support and precipitate with ethanol before preparation.

[0052] Analytical instrument: Waters Acquity UPLC QDA.

[0053] ssRNA method: Buffer A: HFIP / DIEA / EDTA in water. Buffer B: HFIP / DIEA / EDTA in MeOH (80%) / water (20%). Gradient: B 25-45% in 6 min, hold 85% B for 2 min.

[0054] The product was prepared by ionization, desalting and freeze-drying.

[0055] Attachment of α-tocopherol: α-tocopherol is covalently attached to the 5' or 3' end of the complementary chain using conventional methods.

[0056] Preparation of double-chain compounds: After synthesizing the main chain and the side chain using conventional methods, the main chain and the side chain were mixed in the same amount of substances, the mixture was heated at 95°C for 5 minutes, and allowed to stand at a constant temperature of 37°C for 1 hour to allow the chains to renature, and stored in an environment of 4°C.

[0057] Unless otherwise specified, the mice used in the experiments mentioned in the present invention were all 7-week-old C57 / BL mice (male mice), weighing about 20 g. Unless otherwise specified, the number of mice used in each group was 3.

[0058] Example 1 Screening of single-stranded sequences

[0059] 1. Construction of Minigene Model

[0060] (1) The full gene sequence of DMD was found through the NCBI and USCS databases. Genomic DNA was extracted from HEK293 cells and amplified by PCR to obtain exons 21, 23, 24, 25 of the DMD gene and some of their flanking introns. Since the exon 23 on this minigene was subsequently detected, it was named Mini-DMD23.

[0061] (2) Use SLIC (sequence and ligase independent clonging) technology for cloning, and then use PCR to add sequences that overlap with the two ends of the vector (usually 25 bp in length) to both ends of the inserted fragment, and then use T4 DNA polymerase to construct a recombinant vector.

[0062] (3) Polyethylenimine (PEI) transfection.

[0063] (II) Screening out target nucleic acid sequences with better effects on Exon 23 skipping

[0064] (1) Lipofectamin 2000 transfection.

[0065] (2) hExon21-25HEK293 in good condition was used for in vitro screening experiments. The medium was replaced after 4 hours. After 24 hours, qPCR and PCR electrophoresis were used to detect the expression levels of exon 23 mRNA and protein, respectively, to screen for effective target nucleic acid sequences.

[0066] Results: Eight effective target nucleic acid sequences that can achieve skipping therapy for Exon 23 were screened out, as shown in Table 2. The electrophoresis test results of the reverse transcribed DNA of the extracted mRNA are shown in Figure 1 , Figure 2 As shown ( Figure 1 , Figure 2 The bands are not from the same batch). As can be seen from the figure, only DYS-4 is weakly developed, while the others are not developed.

[0067] Table 2 8 effective target nucleic acid sequences

[0068] serial number DNA sequence Number in the sequence listing DYS-1 5'-cttacctgaaatt-3' SEQ ID NO.3 DYS-2 5'-tacctgaaatttt-3' SEQ ID NO.4 DYS-3 5'-cctgaaattttcg-3' SEQ ID NO.5 DYS-4 5'-tgaaattttcgaa-3' SEQ ID NO.1 DYS-5 5'-aaattttcgaagt-3' SEQ ID NO.6 DYS-6 5'-attttcgaagttt-3' SEQ ID NO.7 DYS-7 5'-tttcgaagtttat-3' SEQ ID NO.8 DYS-8 5'-ttacctgaaattt-3' SEQ ID NO.9

[0069] Example 2 Effects of different numbers of locked nucleic acids on exon skipping effects

[0070] In previous studies, it has been proven that modified DNA or RNA is more effective and stable than naked DNA or RNA. Therefore, the present invention modifies the DNA molecule shown in DYS-4 and compares the effects of different modified nucleic acid molecules on exon skipping.

[0071] First, some deoxyribobases were replaced with ribobases to perform locked nucleic acid modification, and the number of locked nucleic acids was 4, 6, and 8, respectively. At the same time, sulfation modification and methylation modification (including conventional methylation modification, i.e., 2'-O-methylation, and methylation modification of cytosine, i.e., 5-methylcytosine) were performed, and a total of 6 nucleic acid molecules were obtained after modification (as shown in Table 3), and these 6 nucleic acid molecules were artificially synthesized.

[0072] Table 3 Six modified nucleic acid molecules (direction 5'-3')

[0073] DYS-4-LNA4 t^G(+)^a^a^A(+)^t^t^T(+)^t^c^G(+)^a^a DYS-4-LNA6 t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a DYS-4-LNA8 t^G(+)^A(+)^a^A(+)^T(+)^t^T(+)^T(+)^c^G(+)^A(+)^a DYS-4-LNA4 / 2' U(M)^G(+)^A(M)^A(M)^A(+)^U(M)^U(M)^T(+)^U(M)^C(M)^G(+)^A(M)^A(M) DYS-4-LNA6 / 2' U(M)^G(+)^A(M)^A(+)^A(M)^T(+)^U(M)^T(+)^U(M)^mC(+)^G(M)^A(+)^A(M) DYS-4-LNA8 / 2' U(M)^G(+)^A(+)^A(M)^A(+)^T(+)^U(M)^T(+)^T(+)^C(M)^G(+)^A(+)^A(M)

[0074] Among them, capital letters represent RNA; lowercase letters represent DNA; (+) represents locked nucleic acid modification (2'-O and 4' carbon on ribose are linked together); (M) represents conventional methylation modification (2'-O-methylation); m represents 5' methylation modification of cytosine (5'-O-methylation); ^ represents phosphorothioate diester bond modification.

[0075] The above six nucleic acid molecules were tested in vivo in mice, and the administration method was intramuscular injection (tibialis anterior muscle). Seven-week-old C57 / BL mice were randomly divided into seven groups: control group, experimental groups 1 to 6. Among them, experimental group 1 was injected with DYS-4-LNA4, experimental group 2 was injected with DYS-4-LNA6, experimental group 3 was injected with DYS-4-LNA8, experimental group 4 was injected with DYS-4-LNA4 / 2', experimental group 5 was injected with DYS-4-LNA6 / 2', and experimental group 6 was injected with DYS-4-LNA8 / 2'.

[0076] The experimental group received intramuscular injection (tibialis anterior muscle) of the single-stranded nucleic acid molecules prepared above, diluted with PBS buffer to a concentration of 1000 μM and then injected. The injection dose was 2 nmol / head (referring to the dose of nucleic acid molecules) (calculated as 100 nmol / kg). The control group was injected with an equal amount of PBS buffer only.

[0077] 14 days after the injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative testing was performed, and the exon skipping efficiency was calculated.

[0078] The results of the determination of exon skipping efficiency are as follows Figure 3 As shown in the figure (the exon skipping efficiency of the control group was 0, so it is not shown in the figure). The results showed that the exon skipping effect of experimental group 2 was the best, significantly better than other experimental groups, and the data were statistically significant. It can be seen that DYS-4-LNA6 (the number of locked nucleic acid modifications was 6 and the modifications were spaced) had the best effect.

[0079] Example 3 Effects of different nucleic acid chain lengths on exon skipping effects

[0080] Although the target for designing the sequence was screened in Example 1, the length of the target does not represent the best length. It is generally believed in the art that the best effect can be achieved within 7 to 35 mer. The inventors of the present invention believe that the best effect can be achieved when the length is 13, 17, 20, and 25 mer based on previous research experience. Therefore, the length of DYS-4 was modified, and various modifications (including locked nucleic acid modification, methylation modification, sulfation modification, etc.) were performed at the same time to obtain 4 nucleic acid molecules (as shown in Table 4, where DYS-4-13 mer is DYS-4-LNA6 in Example 2), and these 4 nucleic acid molecules were artificially synthesized.

[0081] Table 4 Length and modified four nucleic acid molecules (direction 5'-3')

[0082] DYS-4-13mer t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a Dys-4-17 mer t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a^G(+)^t^T(+)^t Dys-4-20 mer t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a^G(+)^t^T(+)^t^A(+)^t^T(+) Dys-4-25mer t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a^G(+)^t^T(+)^t^A(+)^t^T(+)^c^A(+)^t^A(+)^t

[0083] The above four nucleic acid molecules were used for in vivo experiments in mice, and the administration method was intramuscular injection (tibialis anterior muscle). Seven-week-old C57 / BL mice were randomly divided into five groups: control group, experimental groups 1 to 4. Among them, experimental group 1 was injected with DYS-4-13 mer, experimental group 2 was injected with Dys-4-17 mer, experimental group 3 was injected with Dys-4-20 mer, and experimental group 4 was injected with Dys-4-25 mer.

[0084] The experimental group received intramuscular injection (tibialis anterior muscle) of the single-stranded nucleic acid molecules prepared above, diluted with PBS buffer to a concentration of 1000 μM and then injected. The injection dose was 2 nmol / head (referring to the dose of nucleic acid molecules) (calculated as 100 nmol / kg). The control group was injected with an equal amount of PBS buffer only.

[0085] 14 days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative detection was performed, and exon skipping efficiency was calculated.

[0086] The results of the determination of exon skipping efficiency are as follows Figure 4 As shown in the figure (the exon skipping efficiency of the control group is 0, so it is not shown in the figure). The results show that the exon skipping effect of experimental group 1 is the best, which is significantly better than that of other experimental groups, and the data are statistically significant. It can be seen that DYS-4-13 mer (nucleic acid chain length is 13 mer) has the best effect.

[0087] Example 4 Comparison of exon skipping effects of single-stranded nucleic acid and double-stranded nucleic acid (complementary strand without methylation modification)

[0088] It can be seen from the screening of Examples 2 and 3 that DYS-4-LNA6 has the best effect. Therefore, DYS-4-LNA6 was used as a single-stranded nucleic acid for mouse experiments. At the same time, the complementary chain of Dys-4 (5'-UUCGAAAAUUUCA-3') was modified (sulfation modification), and the modified complementary chain was: 5'-U^U^C^GAAAAUU^U^C^A-3', which was artificially synthesized. DYS-4-LNA6 was used as the main chain and combined with the modified complementary chain to prepare a double-stranded nucleic acid compound.

[0089] The above single-stranded nucleic acid and double-stranded nucleic acid compounds were tested in vivo in mice, and the administration method was intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice were randomly divided into 7 groups: control group, single-stranded experimental groups 1 to 3, and double-stranded nucleic acid compound experimental groups 1 to 3.

[0090] The double-stranded nucleic acid compound experimental groups were all injected intramuscularly (tibialis anterior muscle) with the double-stranded nucleic acid compound prepared above, diluted with PBS buffer to a concentration of 1000μM and then injected. The doses (referring to the dose of the compound) of the double-stranded nucleic acid compound experimental groups 1, 2, and 3 were 2 nmol / mouse, 4 nmol / mouse, and 8 nmol / mouse, respectively (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg).

[0091] The single-stranded nucleic acid was injected intramuscularly (into the tibialis anterior muscle) in all single-stranded experimental groups, diluted with PBS buffer to a concentration of 1000 μM before injection. The doses (referring to the dose of single-stranded nucleic acid) of single-stranded experimental groups 1, 2, and 3 were 2 nmol / mouse, 4 nmol / mouse, and 8 nmol / mouse, respectively (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg).

[0092] The control group was injected with an equal volume of PBS buffer only.

[0093] 14 days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative detection was performed, and exon skipping efficiency was calculated.

[0094] The results of the determination of exon skipping efficiency are as follows Figure 5 As shown in the figure (the exon skipping efficiency of the control group is 0, so it is not shown in the figure). The results show that the exon skipping effect of the double-stranded nucleic acid compound experimental group 3 is the best, which is significantly better than other experimental groups, and the data is statistically significant. It can be seen that the effect of double-stranded nucleic acid compounds (complementary chains without methylation modification) is significantly stronger than that of single-stranded nucleic acids.

[0095] Example 5 Comparison of exon skipping effects of single-stranded nucleic acid and double-stranded nucleic acid (complementary strand contains methylation modification)

[0096] In the prior art, it is generally believed that the complementary chain has a better effect after methylation modification. Therefore, based on Example 4, the present invention further performs methylation modification on the complementary chain. The modified complementary chain (referred to as complementary chain M) is: 5'-U(M)^U(M)^C(M)^GAAAAUU^U(M)^C(M)^A(M)-3'. DYS-4-LNA6 is used as the main chain and combined with the complementary chain M to prepare a double-stranded nucleic acid compound (referred to as double-stranded nucleic acid compound M).

[0097] The double-stranded nucleic acid compound M, as well as the single-stranded nucleic acid and double-stranded nucleic acid compounds of Example 4, were used for in vivo experiments in mice, and the administration method was intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice were randomly divided into 8 groups: control group, single-stranded group, double-stranded nucleic acid compound groups 1 to 3 (i.e., double-stranded groups 1 to 3), and double-stranded nucleic acid compound M groups 1 to 3 (i.e., double-stranded groups M1 to 3).

[0098] Double-stranded nucleic acid compound groups were injected intramuscularly (tibialis anterior muscle) with double-stranded nucleic acid compounds, diluted with PBS buffer to a concentration of 1000 μM before injection. The doses (referring to the dose of the compound) of double-stranded nucleic acid compound groups 1, 2, and 3 were 2 nmol / mouse, 4 nmol / mouse, and 8 nmol / mouse, respectively (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg).

[0099] The double-stranded nucleic acid compound M group was injected intramuscularly (tibialis anterior muscle) with the double-stranded nucleic acid compound M prepared above, diluted with PBS buffer to a concentration of 1000 μM and then injected. The doses (referring to the dose of the compound) of double-stranded nucleic acid compound M groups 1, 2, and 3 were 2 nmol / mouse, 4 nmol / mouse, and 8 nmol / mouse, respectively (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg).

[0100] The single-stranded nucleic acid was injected intramuscularly (into the tibialis anterior muscle) and diluted with PBS buffer to a concentration of 1000 μM before injection. The dose of the single-stranded nucleic acid in the single-stranded group (referring to the dose of the single-stranded nucleic acid) was 4 nmol / mouse (calculated as 200 nmol / kg).

[0101] The control group was injected with an equal volume of PBS buffer only.

[0102] 14 days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative detection was performed, and exon skipping efficiency was calculated.

[0103] The results of the determination of exon skipping efficiency are as follows Figure 6 , Figure 7 As shown (the exon skipping efficiency of the control group was 0, so it is not shown in the figure) (where, Figure 6 The double-stranded group M in the figure refers to the double-stranded group M2, and the double-stranded group refers to the double-stranded group 2). Figure 6 The results showed that under the same dosage conditions, the double-stranded nucleic acid compound (the complementary chain was not methylated) was significantly stronger than the double-stranded nucleic acid compound M (the complementary chain was methylated), and the results were statistically significant. Figure 7The results show that under different dosage conditions, the double-stranded nucleic acid compound (complementary chain without methylation modification) is significantly stronger than the double-stranded nucleic acid compound M (complementary chain with methylation modification), and there is a dose-effect dependence, and the results are statistically significant. The research results of this example show that the effect of methylation modification of the complementary chain is not good, which is contrary to the conventional cognition in the prior art. It can be seen that the double-stranded nucleic acid compound used to treat Duchenne muscular dystrophy has particularity.

[0104] Example 6 Effect of low-concentration nucleic acid intramuscular injection on the expression of dystrophin in animals

[0105] The single-stranded nucleic acid and double-stranded nucleic acid compounds prepared in Example 4 were used for mouse experiments. 7-week-old Duchenne muscular dystrophy model mice (20 g, 12 mice) were randomly divided into 3 groups: model animal group, single-stranded group, and double-stranded group. At the same time, normal 7-week-old C57 / BL mice (20 g, 4 mice) were used as the control group, and the internal reference protein tubulin was used as the internal reference protein, which is well known to those skilled in the art.

[0106] The model mice were purchased from Jackson Laboratory, raised in an SPF-grade laboratory, fed with a high-fat (6%) diet, and housed in a breeding room with a 12-h light-dark cycle and a stable temperature of (20±2)°C and humidity of (55±5)%.

[0107] The double-stranded group received an intramuscular injection (im) of the double-stranded nucleic acid compound diluted with PBS buffer to a concentration of 1000 μM, with a dose of 2 nmol / mouse (100 nmol / kg).

[0108] The single-stranded group was injected intramuscularly (im into the tibialis anterior muscle) with single-stranded nucleic acid diluted with PBS buffer to a concentration of 1000 μM. The dose was 2 nmol / mouse (100 nmol / kg).

[0109] The control group was injected with an equal volume of PBS buffer only.

[0110] The model animal group was injected with an equal amount of PBS buffer only.

[0111] Three days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion, and Western Blot was used to detect the expression level of anti-dystrophin.

[0112] Western Blot test results Figure 8 As shown, the expression level of dystrophin Fig. 9 As shown ( Figure 8 , 9The "normal mice (B10)" in the experiment refers to the control group. The results showed that under the same dosage conditions, both single-stranded nucleic acid and double-stranded nucleic acid compounds could significantly increase the expression of dystrophin in the model mice, and the effect of double-stranded nucleic acid compounds was significantly stronger than that of single-stranded nucleic acid, and the results were statistically significant.

[0113] Example 7 Effects of high-concentration nucleic acid intramuscular injection on dystrophin expression in animals

[0114] Same as Example 6, except that the injection dose of the single-stranded group and the double-stranded nucleic acid compound was 8 nmol / animal (400 nmol / kg). The control group and the model animal group were injected with only an equal amount of PBS buffer.

[0115] Three days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion, and Western Blot was used to detect the expression level of anti-dystrophin.

[0116] Western Blot test results Fig.10 As shown, the expression level of dystrophin Fig.11 As shown ( Fig.10 , 11 The "normal mice (B10)" in the experiment refers to the control group. The results showed that under the same dosage conditions, both single-stranded nucleic acid and double-stranded nucleic acid compounds could significantly increase the expression of dystrophin in the model mice, and the effect of double-stranded nucleic acid compounds was significantly stronger than that of single-stranded nucleic acid. At the same time, high doses also had better effects than low doses (compared with Example 6). The results were statistically significant.

[0117] Example 8 Exon skipping effect of double-stranded nucleic acid compound linked to vector 1

[0118] Vitamin E is a fat-soluble compound, which includes tocopherol and tocotrienol, among which α-tocopherol is the most widely distributed, most abundant and most active form of vitamin E in nature. In addition to the most well-known antioxidant function, the biological function of vitamin E also involves the regulation of enzyme activity, gene expression regulation and neurobiological function, etc. Vitamin E absorbed into the plasma through the digestive tract enters the liver cells through receptor mediation, and the vitamin E entering the liver cells enters the extrahepatic tissue under the transportation of vitamin E binding protein. Therefore, α-tocopherol has the possibility of becoming a carrier of gene medicine (to realize the organ-targeted delivery of gene medicine). This embodiment verifies this possibility.

[0119] DYS-4-LNA6 was used as the main chain of single-stranded nucleic acid and double-stranded nucleic acid compounds. At the same time, the complementary chain of Dys-4 (5'-UUCGAAAAUUUCA-3') was modified (sulfation modification, methylation modification, etc.), and α-tocopherol was connected to the 5' end or 3' end of the complementary chain to obtain 4 complementary chains (as shown in Table 5, Toc represents α-tocopherol), and these 4 complementary chains were artificially synthesized. Using DYS-4-LNA6 as the main chain, combined with these 4 complementary chains, 4 double-stranded nucleic acid compounds were prepared, which were respectively called: double-stranded nucleic acid compound 5'M, double-stranded nucleic acid compound 3'M, double-stranded nucleic acid compound 5', and double-stranded nucleic acid compound 3'.

[0120] Table 5 Four complementary chains

[0121] Complementary strand methylation (5'M-Toc) 5'-Toc-U(M)^U(M)^C(M)^gaaaatt^U(M)^C(M)^A(M)-3' Complementary strand methylation (3'M-Toc) 5'-U(M)^U(M)^C(M)^gaaaatt^U(M)^C(M)^A(M)-Toc-3' Complementary strand (5'-Toc) 5'-Toc-U^U^C^gaaaatt^U^C^A-3' Complementary strand (3'-Toc) 5'-U^U^C^gaaaatt^U^C^A-Toc-3'

[0122] The above single-stranded nucleic acid and double-stranded nucleic acid compounds were used for in vivo experiments in mice, and the administration method was intramuscular injection (tibialis anterior muscle). Seven-week-old C57 / BL mice were randomly divided into 6 groups: control group, single-stranded group, double-stranded group (5'M), double-stranded group (3'M), double-stranded group (5'), and double-stranded group (3'). Among them, the double-stranded group (5'M) was injected with double-stranded nucleic acid compound 5'M, the double-stranded group (3'M) was injected with double-stranded nucleic acid compound 3'M, the double-stranded group (5') was injected with double-stranded nucleic acid compound 5', and the double-stranded group (3') was injected with double-stranded nucleic acid compound 3'.

[0123] Each double-stranded group was injected intramuscularly (tibialis anterior muscle) with the double-stranded nucleic acid compound prepared above, diluted with PBS buffer to a concentration of 1000 μM, and then injected. The dose (referring to the dose of the compound) was 4 nmol / mouse (calculated as 200 nmol / kg).

[0124] The single-stranded group was injected intramuscularly (tibialis anterior muscle) with the above single-stranded nucleic acid, diluted with PBS buffer to a concentration of 1000 μM before injection. The dose of the single-stranded group (referring to the dose of single-stranded nucleic acid) was 4 nmol / mouse (calculated as 200 nmol / kg).

[0125] The control group was injected with an equal volume of PBS buffer only.

[0126] 14 days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative detection was performed, and exon skipping efficiency was calculated.

[0127] The results of the determination of exon skipping efficiency are as follows Fig.12As shown in the figure (the exon skipping efficiency of the control group was 0, so it is not shown in the figure). The results showed that under the same dosage conditions, the double-stranded nucleic acid compound 5' (the complementary chain is the DNA chain, and there is no methylation modification, and α-tocopherol is connected to the 5' end of the complementary chain) has the best effect, which is significantly better than other experimental groups. The results are statistically significant.

[0128] Example 9 Exon skipping effect of double-stranded nucleic acid ligation vector 2

[0129] To further compare the difference in the effect of the carrier connection position on nucleic acid exon skipping, DYS-4-LNA6 was used as the main chain of single-stranded nucleic acid and double-stranded nucleic acid compounds. α-Tocopherol was covalently linked to the 5' or 3' end of the main chain (as shown in Table 6), combined with the modified complementary chain (5'- U^U^C^gaaaatt^U^C^A -3') of Example 8, to prepare two double-stranded nucleic acid compounds, respectively referred to as: double-stranded nucleic acid compound 5' main and double-stranded nucleic acid compound 3' main.

[0130] Table 6 α-Tocopherol linked to the 5' or 3' end of the main chain

[0131] Main chain (5') 5'-Toc-t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a-3' Main chain (3') 5'-t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a-Toc-3' Complementary chain 5'-U^U^C^gaaaatt^U^C^A-3'

[0132] The above-mentioned single-stranded nucleic acid, double-stranded nucleic acid compound 5' main, double-stranded nucleic acid compound 3' main, and double-stranded nucleic acid compound 5' (prepared in Example 8) were used for in vivo experiments in mice, and the administration method was intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice were randomly divided into 5 groups: control group, single-stranded group, double-stranded group (5' main), double-stranded group (3' main), and double-stranded group (5'). Among them, the double-stranded group (5' main) was injected with double-stranded nucleic acid compound 5' main, the double-stranded group (3' main) was injected with double-stranded nucleic acid compound 3' main, and the double-stranded group (5') was injected with double-stranded nucleic acid compound 5'.

[0133] Each double-stranded group was injected intramuscularly (tibialis anterior muscle) with the above-mentioned double-stranded nucleic acid compound diluted with PBS buffer to a concentration of 1000 μM. The dose (referring to the dose of the compound) was 4 nmol / mouse (calculated as 200 nmol / kg).

[0134] The single-stranded group was injected intramuscularly (tibialis anterior muscle) with the above single-stranded nucleic acid, diluted with PBS buffer to a concentration of 1000 μM before injection. The dose of the single-stranded group (referring to the dose of single-stranded nucleic acid) was 4 nmol / mouse (calculated as 200 nmol / kg).

[0135] The control group was injected with an equal volume of PBS buffer only.

[0136] 14 days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative detection was performed, and exon skipping efficiency was calculated.

[0137] The results of the determination of exon skipping efficiency are as follows Fig.13 As shown in the figure (the exon skipping efficiency of the control group is 0, so it is not shown in the figure). The results show that under the same dosage conditions, the double-stranded nucleic acid compound 5' (the complementary chain is a DNA chain, and there is no methylation modification, and α-tocopherol is connected to the 5' end of the complementary chain) has the best effect, which is significantly better than other experimental groups (when α-tocopherol is connected to the 5' or 3' end of the main chain, the exon skipping effect is not as good as the double-stranded nucleic acid compound 5', nor as good as the single-stranded nucleic acid). The results are statistically significant.

[0138] Example 10 Dose-effect relationship of exon skipping effect of double-stranded nucleic acid ligation vector

[0139] To further compare the difference in the effect of vector position on nucleic acid exon skipping, single-stranded nucleic acid (DYS-4-LNA6) and double-stranded nucleic acid compound 5' prepared in Example 8 were used as drugs for in vivo mouse experiments, and the administration method was intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice were randomly divided into 4 groups: control group, double-stranded groups 1 to 3.

[0140] Double-stranded groups 1 to 3 were injected intramuscularly (tibialis anterior muscle) with the above double-stranded nucleic acid compound 5', which was diluted with PBS buffer to a concentration of 1000μM before injection. The doses (referring to the dose of the compound) of double-stranded groups 1 to 3 were 2 nmol / mouse, 4 nmol / mouse, and 8 nmol / mouse, respectively (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg).

[0141] The control group was injected with an equal volume of PBS buffer only.

[0142] 14 days after intramuscular injection, blood was collected from behind the eyeball of the anesthetized mouse, and serum was extracted for blood biochemical testing; then the heart, muscle, liver and other tissues were extracted by dissection and PBS perfusion. RNA was extracted, PCR quantitative detection was performed, and exon skipping efficiency was calculated.

[0143] The results of the determination of exon skipping efficiency are as follows Fig.14 As shown in the figure (the exon skipping efficiency of the control group was 0, so it is not shown in the figure). The results showed that as the dose increased, the 5' exon inhibition effect of the double-stranded nucleic acid compound also increased. The results were statistically significant.

[0144] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A double-stranded nucleic acid, characterized in that: The double-stranded nucleic acid is composed of ① a modified antisense nucleic acid strand and ② a modified complementary strand, wherein the nucleotide sequence of the antisense nucleic acid strand is shown in SEQ ID NO.1; the nucleotide sequence of the complementary strand is shown in SEQ ID NO.2; The modified antisense nucleic acid strand is: 5'-t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a-3', wherein capital letters represent RNA; lowercase letters represent DNA; (+) represents locked nucleic acid modification; m represents 5' methylation modification of cytosine; ^ represents phosphorothioate diester bond modification; The modified complementary chain is: 5'-U^U^C^GAAAAUU^U^C^A-3', or: 5'-U^U^C^gaaaatt^U^C^A-3'; When the modified complementary chain is 5'-U^U^C^gaaaatt^U^C^A-3', α-tocopherol is connected to the complementary chain, and the α-tocopherol is connected to the 5' end of the complementary chain.

2. Use of the double-stranded nucleic acid according to claim 1 in the preparation of a drug for treating Duchenne muscular dystrophy.

3. A pharmaceutical composition for treating Duchenne muscular dystrophy, characterized in that: The active ingredient is the double-stranded nucleic acid according to claim 1.

4. The pharmaceutical composition for treating Duchenne muscular dystrophy according to claim 3, characterized in that: Pharmaceutical solvents or fillers are also included.

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