Single-stranded nucleic acid or double-stranded nucleic acid and use thereof in preparation of drug for treating duchenne muscular dystrophy

Abstract

Provided are a single-stranded nucleic acid or a double-stranded nucleic acid and use thereof 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 strand; and the double-stranded nucleic acid consists of (1) a modified antisense nucleic acid strand and (2) a complementary strand or a modified complementary strand, wherein the nucleotide sequence of the antisense nucleic acid strand is set forth in SEQ ID NO. 1; and the nucleotide sequence of the complementary strand is set forth in SEQ ID NO. 2. A carrier, such as α-tocopherol, may also be linked to the complementary strand. Use of the single-stranded nucleic acid or the double-stranded nucleic acid in the preparation of a drug for treating Duchenne muscular dystrophy. Also provided is a pharmaceutical composition for treating Duchenne muscular dystrophy. The single-stranded nucleic acid or the double-stranded nucleic acid can achieve exon skipping, increase the expression of anti-muscular atrophy proteins, and promote the recovery of muscle strength in patients with Duchenne muscular dystrophy.

Description

Single or double stranded nucleic acid and application thereof in preparation of medicine for treating Duchenne muscular dystrophy TECHNICAL FIELD

[0001] The present application relates to a single or double stranded nucleic acid and application thereof in preparation of medicine for treating Duchenne muscular dystrophy, and belongs to the technical field of nucleic acid medicine. BACKGROUND

[0002] Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal genetic rare disease, which is usually caused by gene mutation and has complex pathogenesis. DMD is mostly seen in children, accounting for 1 / 3500-1 / 4000 in male newborns, while females are usually carriers and rarely get sick. The pathogenic gene of the disease, dystrophin gene, is the largest gene in human, located in Xp21.2 region, encoding dystrophin, and the protein produced after mutation cannot maintain the integrity of the membrane of skeletal muscle and myocardial cells. Usually, the onset is occult at three to five years old, and the most obvious is the gastrocnemius muscle, most of which are accompanied by myocardial damage, and most patients die of respiratory tract infection and heart failure at the age of 20 to 30. At present, there is no complete cure for DMD, and new treatment strategies are urgently needed in clinical practice.

[0003] Nucleic acid drugs have high treatment efficiency, strong specificity, small drug toxicity, and wide application field, and have become a hot spot in precise biomedical and tumor and cancer treatment, and are becoming the best 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 to restore the transcriptional reading frame and produce a shortened but partially functional dystrophin protein, which has been proven to restore dystrophin gene expression function in current clinical treatment of DMD patients. Chinese invention patent CN 102203253 A discloses a multi-exon skipping composition for DMD, which provides antisense molecules capable of binding to selected target sites in human dystrophin gene to induce exon skipping, and methods of use thereof, and the disclosed morpholino oligomer contains 20-35 morpholino subunits, has a large number of base sequences, and has high production cost. SUMMARY

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

[0006] The present application is realized by the following technical solutions:

[0007] A single-stranded nucleic acid or a double-stranded nucleic acid, 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 (also known as a sense chain, a template chain, a mother chain, and a main chain) or a modified antisense nucleic acid chain and ② a complementary chain (also known as a side chain) or a modified complementary chain, wherein 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; the antisense nucleic acid chain and the complementary chain constitute a heterologous double-stranded nucleic acid complex.

[0008] Further, the modified antisense nucleic acid chain is:

[0009] 5'-t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a-3', wherein the capital letter represents RNA; the lowercase letter represents DNA; (+) represents a locked nucleic acid modification (2'-0 and 4' carbon of ribose are connected together); the number of the locked nucleic acid modification accounts for 20% to 80% of the total number of nucleotides in the chain; m represents 5'methylation modification (5'-0-methylation) of cytosine (i.e., mC represents 5-methylcytosine, which is a common RNA modification); and ^ represents a phosphorothioate bond modification.

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

[0011] Further, 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] Further, the complementary chain is connected with a carrier, and the carrier is selected from a virus carrier, a nanomaterial, a liposome, an exosome, and the like, and preferably alpha-tocopherol.

[0013] Further, the alpha-tocopherol is connected to the 5' end of the complementary chain.

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

[0015] A pharmaceutical composition for treating Duchenne muscular dystrophy, which comprises the single-stranded nucleic acid or double-stranded nucleic acid or derivative thereof as an effective component, and further comprises pharmaceutically acceptable adjuvants such as solvents, fillers, etc.

[0016] The single-stranded nucleic acid or double-stranded nucleic acid of the present application can realize exon skipping, increase the expression of dystrophin, and promote the recovery of muscle strength of patients with Duchenne muscular dystrophy. In clinical application, the administration object is a mammal including human. The administration route in animal body is parenteral route such as intravenous administration, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intracerebroventricular injection, or enteral administration (oral administration, gastrointestinal nutrition tube, anal plug, etc.), and the dosage is 0.01-100 mg / kg / day, which can be repeated.

[0017] The double-stranded nucleic acid of the present application can better play the inhibitory effect (inhibit the following exon) of the double-stranded nucleic acid on the target gene when the main chain is specifically modified (including main chain gapmer modification) and the complementary strand is not methylated. When the base sequence or modification method is changed, the double-stranded nucleic acid cannot play the preset role (cannot play the role of exon skipping). Moreover, it is generally believed in the prior art that the effect is better after methylating modification of the complementary strand, but the present application unexpectedly finds that the effect is better when the sequence is not methylated. In addition, the present application finds through experimental research that when the complementary strand is DNA, the carrier alpha-tocopherol has the best effect when connected to the 5' end of the complementary strand (the effect is not good when connected to other positions, such as the 3' end of the sense strand, the 3' end of the complementary strand, the 5' end of the sense strand, and the methylated complementary strand).

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

[0019] (1) The technical difficulty of nucleic acid medicine is solved. The present application finds through research that the exon skipping effect and the expression amount of dystrophin can be obviously increased after intramuscular injection of the single-stranded nucleic acid or double-stranded nucleic acid of the present application in animal body (mice), which can be used as a potential therapeutic drug for treating Duchenne muscular dystrophy. (2) The problem of high cost of double-stranded nucleic acid is solved. The present application greatly reduces the cost of nucleic acid synthesis through heterologous double-stranded nucleic acid, and the second strand does not need to be methylated. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1: Exon 23 exon skipping electrophoresis detection result schematic diagram 1.

[0021] Figure 2: Exon 23 exon skipping electrophoresis detection result schematic diagram 2.

[0022] Figure 3: Exon skipping efficiency determination result schematic diagram (Example 2).

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

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

[0025] Figure 6: Schematic diagram 1 of the determination results of exon skipping efficiency (Example 5).

[0026] Figure 7: Schematic diagram 2 of the determination results of exon skipping efficiency (Example 5).

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

[0028] Figure 9: Schematic diagram of the expression level of dystrophin protein (Example 6).

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

[0030] Figure 11: Schematic diagram of the expression level of dystrophin protein (Example 7).

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

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

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

[0034] The application will be further described below in conjunction with examples. However, the scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

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

[0036] The single-stranded nucleic acid and double-stranded nucleic acid of the application can be completely or partially prepared and synthesized by chemical synthesis method (currently mainly using automatic synthesizer) or enzymatic reaction process (including but not limited to polymerase, ligase or restriction reaction).

[0037] The locked nucleic acid (LNA) mentioned in the present application is an oligonucleotide derivative, and the 2'-0 and 4'-C positions of β-D-ribofuranose in the structure are connected by condensation to form a rigid structure.

[0038] The nucleic acid (antisense nucleic acid chain, complementary chain) is prepared by using the conventional phosphoramidite triester synthesis method. The method is the most widely used oligonucleotide synthesis method and is also the synthesis method used by the mainstream synthesis instrument at home and abroad. The method has the characteristics of efficient and rapid coupling and relatively stable starting reactants. First, the RNA monomer and the DNA monomer are synthesized by a conventional method. The RNA methylation, DNA thio, and locked nucleic acid (bridged nucleic acid) used in the present application are all conventional methods known to those skilled in the art. The RNA or DNA chain is synthesized by fixing the RNA monomer and the DNA monomer or the locked nucleic acid on a solid support. The synthesis is extended from the 3' end to the 5' end of the primer to be synthesized. The adjacent nucleotides are connected by 3'→5' phosphodiester bonds. According to the predetermined base sequence, the corresponding phosphoramidite monomer and other necessary chemical reagents are added to the solid support with a pre-prepared surface modification through a liquid system to complete the synthesis of the specified oligonucleotide sequence. After the synthesis is completed, the product is cut off from the solid support by using ammonia gas or other alkaline conditions, and then collected, thereby obtaining the oligonucleotide of the target base sequence. The specific operation can be as follows:

[0039] Synthesis parameters: synthesis scale: 200 umol; coupling time: 12 min; ammonia solution conditions: 55℃, concentrated ammonia (25% ammonia solution).

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

[0041] Table 1

[0042] Reagent 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 reagent DTTT (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, and MOE-C (5Me) are all from Wuhu Huaren Technology Co., Ltd.

[0044] Instrument synthesis reaction steps:

[0045] The first step is deprotection, using trichloroacetic acid to remove the DMT on the nucleoside attached to the CPG to expose the 5' hydroxyl group for the next coupling. This step needs to be careful that TCA is a strong acid and can cause depurination, so the contact time of TCA with the oligonucleotide should not exceed 3 minutes.

[0046] The second step is activation, before coupling, the monomer is mixed with tetrazole and enters the synthesis column, at this time the tetrazole provides a proton to the N atom of the diisopropylamine group on the 3' phosphate, the protonated diisopropylamine is a good radical, and forms a phosphoramidite tetrazole active intermediate with tetrazole. The excess of 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 attached to the CPG, it undergoes nucleophilic reaction with its 5' hydroxyl group, coupling occurs and tetrazole is removed, and the synthesized oligonucleotide chain is extended by one. The excess of monomer relative to the 5'-hydroxyl group on the nucleotide attached to the CPG in this step ensures high efficiency of coupling.

[0048] The fourth step is capping, in order to prevent the unreacted 5' hydroxyl group attached to the CPG from being extended in the subsequent cycle, it needs to be closed after the coupling reaction is fully carried out, and acetylation is commonly used to close this hydroxyl group. A highly active acetylation reagent is formed by mixing acetic anhydride and N-methyl imidazole just before use, and an ester bond is formed with a small amount of 5' hydroxyl group that does not participate in the coupling reaction. Due to the small amount of hydroxyl group that needs to be closed and the high activity and sufficient excess of the acetylation reagent, the reaction speed is very fast, a few seconds are enough. Too long capping time may cause acetylation reaction at unintended positions, and increase the risk of acid attack on the newly formed phosphite ester bond by acetic anhydride and trace water.

[0049] The fifth step is thio, the newly added nucleotide after the coupling reaction is connected to the oligonucleotide chain on the CPG through a phosphite ester bond (phosphorus is trivalent), this phosphite ester bond is unstable and easily hydrolyzed by acid and alkali, so it 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] Repeat steps 1 to 5 above, and when the oligonucleotide chain is extended to the desired length, the synthesis is complete.

[0051] Cutting and deprotection: add concentrated ammonia (25% ammonia solution) to the carrier (PS), heat to 55°C and react overnight for ammoniaysis. Then filter the carrier, precipitate in ethanol, and then prepare.

[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] Desalting after ion preparation, freeze-drying to obtain the product.

[0055] Linking alpha tocopherol: using conventional methods to covalently link alpha tocopherol at the 5' end or 3' end of the complementary strand.

[0056] Preparation of double-stranded compounds: after synthesis of the main chain and the side chain, the main chain and the side chain are mixed in the same amount of substance, the mixture is heated at 95°C for 5 minutes, and then incubated at 37°C for 1 hour to allow the strands to anneal, and stored at 4°C.

[0057] The experimental mice mentioned in the present application are 7-week-old C57 / BL mice (male mice) unless otherwise specified, and the weight of the mice is about 20g. Unless otherwise specified, the number of mice used in each group is 3.

[0058] Example 1 Screening of single-stranded sequences

[0059] (I) Construction of Minigene model

[0060] (1) The full gene sequence of DMD was searched through NCBI and USCS databases, and then the genomic DNA of HEK293 cells was extracted, amplified by PCR, and the exons 21, 23, 24, 25 and their partial flanking introns of DMD gene were obtained. Since exon 23 on the minigene is detected subsequently, it is named Mini-DMD23.

[0061] (2) SLIC (sequence and ligase independent clonging) technology was used for cloning, and then PCR was used to add overlapping sequences (generally 25 bp in length) at both ends of the insert and the vector, and then T4 DNA polymerase was used for treatment to construct the recombinant vector.

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

[0063] (II) Screening of target nucleic acid sequences with better Exon 23 skipping effect

[0064] (1) Lipofectamin 2000 transfection.

[0065] (2) The hExon21-25 HEK293 in good condition is used for in vitro screening experiment, the medium is replaced after 4 hours, and the expression levels of exon 23 mRNA and protein are detected by qPCR and PCR electrophoresis respectively after 24 hours, so as to screen effective target nucleic acid sequences.

[0066] Results: Eight effective target nucleic acid sequences capable of realizing the skipping treatment of Exon 23 are screened out, as shown in Table 2. The electrophoresis detection results of the extracted mRNA reverse-transcribed DNA are shown in Figures 1 and 2 (the bands in Figures 1 and 2 are not the same batch). As can be seen from the figures, only DYS-4 has weak development, and the others have no development.

[0067] Table 2 Eight effective target nucleic acid sequences

[0068] No. DNA sequence No. in sequence 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 Influence of different numbers of locked nucleic acids on exon skipping effect

[0070] In the previous research, it has been proved that the modified DNA or RNA is stronger and more stable than the naked DNA or RNA. Therefore, the DNA molecule shown in DYS-4 is modified and reformed in the present application, and the influence of the nucleic acid molecules after different modifications and reforms on the exon skipping effect is compared.

[0071] Firstly, the deoxyribose bases are replaced by ribose bases to perform locked nucleic acid modification, and the numbers of locked nucleic acids are 4, 6 and 8 respectively. At the same time, the sulfation modification and methylation modification (including conventional methylation modification, i.e. 2'-0-methylation, and cytosine methylation modification, i.e. 5-methylcytosine) are performed, and a total of 6 kinds of nucleic acid molecules are obtained after modification and reform (as shown in Table 3), and the 6 kinds of nucleic acid molecules are artificially synthesized.

[0072] Table 3 Six kinds of nucleic acid molecules after modification and reform (direction 5'-3')

[0073] DYS-4-LNA4 tG(+) aaA(+) ttT(+) tcG(+) aa DYS-4-LNA6 tG(+) aaA(+) aT(+) ttT(+) tmC(+) gaA(+) a DYS-4-LNA8 tG(+) A(+) aaA(+) T(+) ttT(+) tcG(+) 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] wherein, capital letter represents RNA; small letter represents DNA; (+) represents locked nucleic acid modification (2'-0 linked with 4' carbon on ribose); (M) represents regular methylation modification (2'-0-methylation); m represents 5' methylation modification of cytosine (5'-0-methylation); ^ represents phosphorothioate bond modification.

[0075] The above six nucleic acid molecules were subjected to in vivo experiment in mice, and the administration mode was intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice were randomly divided into 7 groups: control group, experimental groups 1-6. 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 groups were intramuscularly injected (tibialis anterior muscle) with the above prepared single-stranded nucleic acid molecules, which were diluted with PBS buffer to a concentration of 1000 μM before injection. The injection dose was 2 nmol / one (referring to the dose of nucleic acid molecules) (calculated as 100 nmol / kg). The control group was only injected with the same amount of PBS buffer.

[0077] After 14 days of injection, the mice were anesthetized, and the blood was taken from the eyeball, and the serum was extracted and sent for blood biochemical detection; then the heart, muscle, liver and other tissues were extracted by PBS perfusion. RNA was extracted, and PCR quantitative detection was performed to calculate the exon skipping efficiency.

[0078] The determination results of exon skipping efficiency are shown in Figure 3 (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 experimental group 2 is the best, which is significantly better than that of other experimental groups, and the data is statistically significant. It can be seen that the effect of DYS-4-LNA6 (the number of locked nucleic acid modifications is 6 and the interval modification) is the best.

[0079] Example 3 Influence of different nucleic acid chain lengths on exon skipping effect

[0080] Although the target point of the design sequence is screened in Example 1, the length of the target point does not represent the best length. It is generally believed in the art that the best effect can be achieved within 7-35 mer. According to the previous research experience, the inventors of the present application believe that the best effect can be achieved when the length is 13, 17, 20, or 25 mer. Therefore, length modification is performed on the basis of DYS-4, and various modifications (including locked nucleic acid modification, methylation modification, sulfation modification, etc.) are performed to obtain four kinds of nucleic acid molecules (as shown in Table 4, wherein DYS-4-13 mer is DYS-4-LNA6 of Example 2), and the four kinds of nucleic acid molecules are artificially synthesized.

[0081] Table 4 Four kinds of nucleic acid molecules after length and modification modification (direction 5'-3')

[0082] 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-4-25 mer 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 kinds of nucleic acid molecules are subjected to in vivo experiments in mice, and the administration mode is intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice are randomly divided into 5 groups: a control group and experimental groups 1-4. Among them, the experimental group 1 is injected with DYS-4-13 mer, the experimental group 2 is injected with Dys-4-17 mer, the experimental group 3 is injected with Dys-4-20 mer, and the experimental group 4 is injected with Dys-4-25 mer.

[0084] The single-stranded nucleic acid molecules prepared above were injected into the muscles (tibialis anterior muscle) of the experimental group after being diluted with PBS buffer to a concentration of 1000 μM. The injection dose was 2 nmol per mouse (referring to the dose of nucleic acid molecules) (100 nmol / kg).

[0085] Fourteen days after the muscle injection, the mice were anesthetized, and the eyeballs were taken for blood serum extraction for blood biochemical detection. Then the mice were dissected, and the heart, muscle, liver and other tissues were extracted by PBS perfusion. RNA was extracted, and the exon skipping efficiency was calculated by PCR quantification detection.

[0086] The results of the determination of the exon skipping efficiency are shown in Figure 4 (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 experimental group 1 is the best, which is significantly better than that of the other experimental groups, and the data are statistically significant. It can be seen that the DYS-4-13 mer (nucleic acid chain length of 13 mer) has the best effect.

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

[0088] According to the screening of Examples 2 and 3, the effect of DYS-4-LNA6 is the best, so DYS-4-LNA6 is used as a single-stranded nucleic acid for mouse experiments. At the same time, the complementary chain (5'-UUCGAAAAUUUCA-3') of Dys-4 is modified (sulfated modification), and the modified complementary chain is 5'-U^U^C^GAAAAUU^U^C^A-3', which is artificially synthesized. A double-stranded nucleic acid compound is prepared by using DYS-4-LNA6 as the main chain and combining the modified complementary chain.

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

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

[0091] The single-stranded experimental groups are all injected intramuscularly (in the tibialis anterior muscle) with the above single-stranded nucleic acids, which are diluted with PBS buffer to a concentration of 1000 μM before injection. The dosages (referring to the dosages of the single-stranded nucleic acids) of the single-stranded experimental groups 1, 2 and 3 are 2 nmol / each, 4 nmol / each and 8 nmol / each (calculated as 100 nmol / kg, 200 nmol / kg and 400 nmol / kg) respectively.

[0092] The control group is only injected with an equal amount of PBS buffer.

[0093] Fourteen days after the intramuscular injection, the mice are anesthetized, and the eyeballs are taken out to extract serum for blood biochemical detection. Then the mice are dissected, and the heart, muscle, liver and other tissues are extracted by PBS perfusion. RNA is extracted, and PCR quantitative detection is performed to calculate the exon skipping efficiency.

[0094] The determination results of the exon skipping efficiency are shown in FIG. 5 (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 those of the other experimental groups, and the data are statistically significant. It can be seen that the effect of the double-stranded nucleic acid compound (the complementary strand without methylation modification) is obviously stronger than that of the single-stranded nucleic acid.

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

[0096] It is generally believed in the prior art that the effect is better after the complementary strand is modified by methylation, therefore, on the basis of Example 4, the complementary strand is further modified by methylation in the present application, and the modified complementary strand (referred to as complementary strand M) is 5'-U(M)^U(M)^C(M)^GAAAAUU^U(M)^C(M)^A(M)-3'. The double-stranded nucleic acid compound (referred to as double-stranded nucleic acid compound M) is prepared by combining the DYS-4-LNA6 as the main chain with the complementary strand M.

[0097] The above double-stranded nucleic acid compound M, as well as the single-stranded nucleic acid and the double-stranded nucleic acid compound of Example 4, are subjected to in vivo experiments on mice, and the administration mode is intramuscular injection (in the tibialis anterior muscle). 7-week-old C57 / BL mice are randomly divided into 8 groups: a control group, single-stranded groups, double-stranded nucleic acid compound groups 1-3 (i.e. double-stranded groups 1-3), double-stranded nucleic acid compound M groups 1-3 (i.e. double-stranded groups M1-M3).

[0098] Double-stranded nucleic acid compound group 2: The double-stranded nucleic acid compound prepared above was diluted with PBS buffer to a concentration of 1000 μM and then injected. The dose of the double-stranded nucleic acid compound group 2 (referring to the dose of the compound) was 4 nmol / each (calculated as 200 nmol / kg).

[0099] Double-stranded nucleic acid compound group M: The double-stranded nucleic acid compound M prepared above was diluted with PBS buffer to a concentration of 1000 μM and then injected. The dose of the double-stranded nucleic acid compound group M (referring to the dose of the compound) was 2 nmol / each, 4 nmol / each, and 8 nmol / each (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg, respectively).

[0100] Single-stranded group: The single-stranded nucleic acid was diluted with PBS buffer to a concentration of 1000 μM and then injected. The dose of the single-stranded group (referring to the dose of the single-stranded nucleic acid) was 4 nmol / each (calculated as 200 nmol / kg).

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

[0102] After 14 days of intramuscular injection, the mice were anesthetized, and the eyeballs were taken out to extract serum for blood biochemical detection. Then the mice were dissected, and the heart, muscle, liver, and other tissues were extracted by PBS perfusion. RNA was extracted, and the exon skipping efficiency was calculated by PCR quantification detection.

[0103] The results of the determination of the exon skipping efficiency are shown in FIG. 6 and FIG. 7 (the exon skipping efficiency of the control group is 0, so it is not shown in the figures) (in FIG. 6, double-stranded group M refers to double-stranded group M2, and double-stranded group refers to double-stranded group 2). The results of FIG. 6 show that, under the same dose conditions, the double-stranded nucleic acid compound (the complementary strand without methylation modification) is obviously stronger than the double-stranded nucleic acid compound M (the complementary strand containing methylation modification), and the results are statistically significant. The results of FIG. 7 show that, under different dose conditions, the double-stranded nucleic acid compound (the complementary strand without methylation modification) is obviously stronger than the double-stranded nucleic acid compound M (the complementary strand containing methylation modification), and has a dose-effect dependence, and the results are statistically significant. The research results of the present embodiment show that the effect is not good after the complementary strand is subjected to methylation modification, which is contrary to the conventional cognition in the prior art. It can be seen that the double-stranded nucleic acid compound for treating Duchenne muscular dystrophy has particularity.

[0104] Example 6 Effect of low-concentration nucleic acid intramuscular injection on expression of anti-muscle atrophy protein in animals

[0105] The single-stranded nucleic acid and double-stranded nucleic acid compounds prepared in Example 4 were used for mouse experiments. Seven-week-old Dystrophin muscular dystrophy model mice (20 g, 12) were randomly divided into three 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) were used as a 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, were bred in a laboratory at SPF level, were fed with high-fat (6%) feed, and were bred in a feeding room with a temperature of (20±2) °C and humidity of (55±5)%, with a 12 h light-dark cycle.

[0107] The double-stranded group was injected with double-stranded nucleic acid compounds by intramuscular injection (tibialis anterior muscle im), and the injection was diluted with PBS buffer to a concentration of 1000 μM. The dose was 2 nmol per mouse (100 nmol / kg).

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

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

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

[0111] Three days after intramuscular injection, the mice were anesthetized, and blood was taken from the retrobulbar, and serum was extracted for blood biochemical testing. Then, the heart, muscle, liver, and other tissues were extracted by PBS perfusion, and Western Blot was performed to detect the expression level of dystrophin protein.

[0112] The results of Western Blot detection are shown in Figure 8, and the expression level of dystrophin protein is shown in Figure 9 (“normal mouse (B10)” in Figures 8 and 9 refers to the control group). The results show that under the same dose conditions, both single-stranded nucleic acid and double-stranded nucleic acid compounds can significantly increase the expression of dystrophin protein in model mice, and the effect of double-stranded nucleic acid compounds is significantly stronger than that of single-stranded nucleic acid, and the results are statistically significant.

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

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

[0115] Three days after intramuscular injection, the mice were anesthetized and blood was taken from the retrobulbar, and serum was extracted for blood biochemical detection. Then the mice were dissected and the heart, muscle, liver and other tissues were extracted by PBS perfusion for Western Blot detection of the expression level of dystrophin.

[0116] The results of Western Blot detection are shown in Figure 10, and the expression level of dystrophin is shown in Figure 11 ("normal mice (B10)" in Figures 10 and 11 refer to the control group). The results show that under the same dose conditions, both the single-stranded nucleic acid and the double-stranded nucleic acid compound can significantly increase the expression of dystrophin in the model mice, and the effect of the double-stranded nucleic acid compound is significantly better than that of the single-stranded nucleic acid. At the same time, the high dose has a better effect than the low dose (compared with Example 6). The results are statistically significant.

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

[0118] Vitamin E is a fat-soluble compound, which includes tocopherols and tocotrienols, 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 functions of vitamin E also involve the regulation of enzyme activity, the regulation of gene expression, and neurobiological functions, etc. Vitamin E absorbed into the blood plasma through the digestive tract is mediated by receptors into hepatocytes, and the vitamin E entering the hepatocytes enters extrahepatic tissues under the transport of vitamin E binding protein. Therefore, α-tocopherol has the possibility of becoming a carrier of gene drugs (to achieve organ-targeted delivery of gene drugs). This embodiment verifies this possibility.

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

[0120] Table 5 Four kinds of complementary strands

[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 single-stranded nucleic acid and the double-stranded nucleic acid compound are subjected to in vivo experiments on mice, and the administration mode is intramuscular injection (tibialis anterior muscle). 7-week-old C57 / BL mice are randomly divided into 6 groups: a control group, a single-stranded group, a double-stranded group (5'M), a double-stranded group (3'M), a double-stranded group (5'), and a double-stranded group (3'). Among them, the double-stranded group (5'M) is injected with the double-stranded nucleic acid compound 5'M, the double-stranded group (3'M) is injected with the double-stranded nucleic acid compound 3'M, the double-stranded group (5') is injected with the double-stranded nucleic acid compound 5', and the double-stranded group (3') is injected with the double-stranded nucleic acid compound 3'.

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

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

[0125] The control group is only injected with an equal amount of PBS buffer.

[0126] After 14 days of intramuscular injection, the mice are anesthetized, and blood is taken from the eyeball for serum extraction for blood biochemical detection; then the heart, muscle, liver and other tissues are extracted by PBS perfusion. RNA is extracted, and PCR quantitative detection is performed to calculate the exon skipping efficiency.

[0127] The results of the determination of the exon skipping efficiency are shown in FIG. 12 (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 dose conditions, the double-stranded nucleic acid compound 5' (the complementary strand is a DNA strand, and there is no methylation modification, and α-tocopherol is connected to the 5' end of the complementary strand) 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 linked carrier 2

[0129] To further compare the difference of the exon skipping effect caused by the position of the linker, DYS-4-LNA6 was used as the main chain of the single-stranded nucleic acid and the double-stranded nucleic acid compound. Alpha-tocopherol was covalently linked to the 5' or 3' end of the main chain (as shown in Table 6), and combined with the modified complementary strand (5'-U^U^C^gaaaatt^U^C^A-3') of Example 8 to prepare two double-stranded nucleic acid compounds, which were named double-stranded nucleic acid compound 5' main and double-stranded nucleic acid compound 3' main, respectively.

[0130] Table 6 Alpha-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 strand 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 subjected to 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, 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 with the above-mentioned double-stranded nucleic acid compound in the tibialis anterior muscle, which was diluted with PBS buffer to a concentration of 1000 μM before injection. The dose (referring to the dose of the compound) was 4 nmol per mouse (calculated as 200 nmol / kg).

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

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

[0136] After 14 days of intramuscular injection, the mice were anesthetized, and blood was taken from the retrobulbar of the mice, and serum was extracted for blood biochemical detection; then the heart, muscle, liver and other tissues were extracted by PBS perfusion. RNA was extracted, and PCR quantitative detection was performed to calculate the exon skipping efficiency.

[0137] The results of the determination of the exon skipping efficiency are shown in Figure 13 (the exon skipping efficiency of the control group is 0, and thus is not shown in the figure). The results show that, under the same dosage conditions, the effect of the double-stranded nucleic acid compound 5' (the complementary strand is a DNA strand, and is not modified by methylation, and the α-tocopherol is connected to the 5' end of the complementary strand) is the best, and is significantly better than the other experimental groups (the exon skipping effects of the double-stranded nucleic acid compounds 5' in which the α-tocopherol is connected to the 5' or 3' end of the main chain, and the single-stranded nucleic acid are all not as good as the double-stranded nucleic acid compound 5'). The results are statistically significant.

[0138] Example 10 Dose-effect relationship of the exon skipping effect of the double-stranded nucleic acid connected carrier

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

[0140] The double-stranded groups 1-3 were all injected with the above-mentioned double-stranded nucleic acid compound 5' by intramuscular injection (in the tibialis anterior muscle), and the injection was performed after dilution with a PBS buffer to a concentration of 1000 μM. The doses (referring to the doses of the compounds) of the double-stranded groups 1-3 were 2 nmol per mouse, 4 nmol per mouse, and 8 nmol per mouse (calculated as 100 nmol / kg, 200 nmol / kg, and 400 nmol / kg) respectively.

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

[0142] After 14 days of intramuscular injection, the mice were anesthetized, and blood was taken from the eyeballs for extraction of serum for blood biochemical detection. Then, the mice were dissected, and the heart, muscle, liver, and other tissues were extracted by PBS perfusion. RNA was extracted, and PCR quantitative detection was performed to calculate the exon skipping efficiency.

[0143] The results of the determination of the exon skipping efficiency are shown in Figure 14 (the exon skipping efficiency of the control group is 0, and thus is not shown in the figure). The results show that, with the increase of the dose, the exon suppression effect of the double-stranded nucleic acid compound 5' also increases. The results are statistically significant.

[0144] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure disclosed herein. Modifications obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A double-stranded nucleic acid, characterized by: The double-stranded nucleic acid is composed of ① a modified antisense nucleic acid chain and ② a modified complementary chain, wherein 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; The modified antisense nucleic acid chain is: 5'-t^G(+)^a^A(+)^a^T(+)^t^T(+)^t^mC(+)^g^A(+)^a-3', wherein the capital letter represents RNA; the lowercase letter represents DNA; (+) represents a locked nucleic acid modification; m represents a 5' methylation modification of cytosine; ^ represents a phosphorothioate bond modification; The modified complementary chain is: 5'-U^U^C^GAAAATT^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', the complementary chain is connected with alpha-tocopherol, and the alpha-tocopherol is connected to the 5' end of the complementary chain.

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

3. A pharmaceutical composition for treating Duchenne Muscular Dystrophy, characterized by: The effective component is the double-stranded nucleic acid in claim 1.

4. The pharmaceutical composition for treating Duchenne muscular dystrophy according to claim 3, characterized by: It also includes commonly used solvents and fillers in pharmacy. The effective component is the double-stranded nucleic acid in claim 1. It also includes commonly used solvents and fillers in pharmacy.

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