siRNA, modified substance, drug conjugate and application for inhibiting DC624545

Abstract

The present invention provides an siRNA, a modified substance, a drug conjugate and an application for inhibiting DC624545, belonging to the technical field of biomedicine. The siRNA comprises a sense chain as shown in SEQ ID No.1 and an antisense chain as shown in SEQ ID No.2. The present invention improves the base combination and modification on the basis of the original siRNA, thereby greatly improving the target binding efficiency and the pertinence to the liver in practical applications, thereby achieving the effect of effectively lowering blood lipids. The drug conjugate has better efficacy and can reduce the number of injections, thereby reducing the time cost and money cost of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a siRNA, a modified substance, a drug conjugate and an application thereof for inhibiting DC624545. Background Art

[0002] Obesity can lead to a variety of metabolic complications, including an increased risk of type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. So far, hyperlipidemia is basically incurable, and existing drugs can only control the progression of the disease to a certain extent. Moreover, patients are very likely to have recurrent attacks after severe dietary control. At the same time, some oral drugs, such as atorvastatin calcium, may cause serious adverse reactions in patients, including adverse reactions in muscles and liver.

[0003] There is increasing evidence that lncRNA plays an important role in chronic non-infectious diseases. Related research reports have shown that genetic variation in PPP1R3B located on chromosome 8p23.1 is associated with a variety of metabolic characteristics in humans, mainly blood fatty acid metabolism and liver steatosis with genome-wide significance. According to public information, in the metabolic pathway of steatosis, it was found that Ppp1r3b deficiency and reduced sugar production transferred the storage of exogenous glucose to liver triglycerides (TG). When PPP1R3B expression increased, the SNP carriers with related SNPs had reduced plasma liver fat and lower blood lipids.

[0004] To exert its interference effect as a drug, siRNA needs to achieve cellular endocytosis, escape from lysosomes, and avoid degradation by nucleases, which requires high stability. However, existing siRNA targeting DC624545 has problems of insufficient stability and poor effect. N-acetylgalactosamine (GalNAc) is a high-affinity ligand of the hepatocyte-specific asialoglycoprotein receptor (ASGPR), which can participate in clathrin-mediated endocytosis, allowing siRNA to enter hepatocytes, and has great advantages in the field of liver targeted delivery.

[0005] In summary, it is very necessary to obtain a siRNA for inhibiting DC624545 that is highly safe, does not require auxiliary treatment, and can simultaneously promote the conversion of glucose into glycogen and lower blood lipids. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a siRNA, a modified product, a drug conjugate and an application for inhibiting DC624545, wherein the siRNA has high inhibitory activity against DC624545, and the drug conjugate has better efficacy and can reduce the number of injections, thereby reducing costs.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a siRNA for inhibiting DC624545, comprising a positive strand as shown in SEQ ID No.1 and an antisense strand as shown in SEQ ID No.2.

[0009] Preferably, the 3' ends of the sense strand and the antisense strand are both overhanged with dTdT.

[0010] The present invention also provides a modified product obtained by chemically modifying the siRNA, wherein the chemical modification includes fluorine modification, methoxy modification and phosphorothioate modification;

[0011] The fluorinated modified nucleotides are located in the sense strand and the antisense strand of the nucleotide sequence, in the direction from the 5' end to the 3' end, and the modification method comprises:

[0012] The nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 of the antisense strand are fluorinated modified nucleotides;

[0013] or

[0014] The nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 2, 6, 12, and 13 of the antisense strand are fluorinated modified nucleotides;

[0015] or

[0016] The nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 3, 5, 6, 7, 13, and 15 of the antisense strand are fluorinated modified nucleotides;

[0017] or

[0018] The nucleotides at positions 7, 8, 9, 10, and 11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 3, 5, 6, 7, 8, 9, 10, 11, 13, and 15 of the antisense strand are fluorinated modified nucleotides;

[0019] or

[0020] The nucleotides at positions 1, 2, 3, 4, and 5 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 3, 13, and 15 of the antisense strand are fluorinated modified nucleotides;

[0021] or

[0022] The nucleotides at positions 4, 8, 12, 16, and 20 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 13 and 15 of the antisense strand are fluorinated modified nucleotides;

[0023] The methoxy-modified nucleotides are located in the sense strand and the antisense strand of the nucleotide sequence, in the direction from the 5' end to the 3' end, and the modification method comprises:

[0024] The nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of the antisense strand are methoxy-modified nucleotides;

[0025] or

[0026] The nucleotides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0027] or

[0028] The nucleotides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 4, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0029] or

[0030] The nucleotides at positions 1, 2, 3, 4, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 4, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0031] or

[0032] The nucleotides at positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0033] or

[0034] The nucleotides at positions 1, 2, 3, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0035] The nucleotides linked by the thiophosphate groups are located in the sense strand and the antisense strand of the nucleotide sequence. In the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by the thiophosphate groups, and the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by the thiophosphate groups.

[0036] The present invention also provides a drug conjugate, comprising a ligand and the modified substance.

[0037] Preferably, the ligand is connected to the 3' end of the positive strand of the modified siRNA via a linker group.

[0038] Preferably, the ligand is GalNAc.

[0039] Preferably, the linking group is an acetyl group.

[0040] The present invention also provides the use of the modified substance or the drug conjugate in preparing a drug for inhibiting DC624545.

[0041] The present invention also provides the use of the modified substance or the drug conjugate in preparing a drug for treating hyperlipidemia.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The drug conjugate provided by the present invention has better efficacy and can reduce the number of injections, thereby reducing the time and money costs of patients; experimental results show that the silencing position of the drug conjugate is located upstream of the protein PPP1R3B that affects glycogen, and compared with the direct administration of chemical drugs, the side effects of the therapeutic effect achieved by reducing the gene expression amount are necessarily small; at the same time, the present invention improves the target binding efficiency and the specificity to the liver on the basis of the original siRNA in practical applications, and achieves the effect of effectively lowering blood lipids; the modified siRNA has an inhibitory effect of more than 80% on DC624545 in living animals at a dosage of 10 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The changes of triglycerides in hyperlipidemic monkeys after injection of high-dose drug conjugates;

[0045] Figure 2 The changes in triglycerides in hyperlipidemic monkeys after injection of low-dose drug conjugates;

[0046] Figure 3 The changes of high-density lipoprotein in hyperlipidemic monkeys after injection of high-dose drug conjugates;

[0047] Figure 4 The changes of high-density lipoprotein in hyperlipidemic monkeys after injection of low-dose drug conjugates;

[0048] Figure 5 The changes of blood glucose regulating protein PPP1R3B in hyperlipidemic cynomolgus monkeys after injection of different doses of drug conjugates, where "BLANK" is the control group;

[0049] Figure 6 The figure shows the changes in DC624545, an upstream regulatory gene of blood glucose regulating protein, in hyperlipidemic crab-eating macaques after injection of different doses of drug conjugates, where "BLANK" is the control group. DETAILED DESCRIPTION

[0050] The present invention provides a siRNA for inhibiting DC624545, comprising a positive strand as shown in SEQ ID No.1 and an antisense strand as shown in SEQ ID No.2.

[0051]

[0052] The present invention also provides a modified product obtained by chemically modifying the siRNA, wherein the chemical modification includes fluorine modification, methoxy modification and phosphorothioate modification;

[0053] The fluorinated modified nucleotides are located in the sense strand and the antisense strand of the nucleotide sequence, in the direction from the 5' end to the 3' end, and the modification method comprises:

[0054] The nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 of the antisense strand are fluorinated modified nucleotides;

[0055] or

[0056] The nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 2, 6, 12, and 13 of the antisense strand are fluorinated modified nucleotides;

[0057] or

[0058] The nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 3, 5, 6, 7, 13, and 15 of the antisense strand are fluorinated modified nucleotides;

[0059] or

[0060] The nucleotides at positions 7, 8, 9, 10, and 11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 3, 5, 6, 7, 8, 9, 10, 11, 13, and 15 of the antisense strand are fluorinated modified nucleotides;

[0061] or

[0062] The nucleotides at positions 1, 2, 3, 4, and 5 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 3, 13, and 15 of the antisense strand are fluorinated modified nucleotides;

[0063] or

[0064] The nucleotides at positions 4, 8, 12, 16, and 20 of the sense strand are fluorinated modified nucleotides, and the nucleotides at positions 13 and 15 of the antisense strand are fluorinated modified nucleotides;

[0065] The methoxy-modified nucleotides are located in the sense strand and the antisense strand of the nucleotide sequence, in the direction from the 5' end to the 3' end, and the modification method comprises:

[0066] The nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of the antisense strand are methoxy-modified nucleotides;

[0067] or

[0068] The nucleotides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0069] or

[0070] The nucleotides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 4, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0071] or

[0072] The nucleotides at positions 1, 2, 3, 4, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 4, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0073] or

[0074] The nucleotides at positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0075] or

[0076] The nucleotides at positions 1, 2, 3, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, and 21 of the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides;

[0077] The nucleotides connected by the thiophosphate group are located in the sense strand and the antisense strand of the nucleotide sequence, and in the direction from the 5' end to the 3' end, the nucleotides at the 1st and 2nd, and the 2nd and 3rd positions of the sense strand are connected by the thiophosphate group, and the nucleotides at the 1st and 2nd, the 2nd and 3rd, the 19th and 20th, and the 20th and 21st positions of the antisense strand are connected by the thiophosphate group. In the present invention, the nucleotide sequences of the sense strand and the antisense strand of the siRNA modification are shown in Table 1.

[0078] Table 1 Nucleotide sequences of siRNA and its modifications

[0079]

[0080]

[0081] Note: (s) indicates that the two nucleotides are connected by a thiophosphate backbone; fA, fG, fC, fU represent 2'-fluoro-modified ribonucleotides A, G, C, U, respectively; mA, mG, mC, mU represent 2'-methoxy-modified ribonucleotides A, G, C, U, respectively.

[0082] The present invention also provides a drug conjugate, comprising a ligand and the modified substance.

[0083] In the present invention, the ligand is preferably connected to the 3' end of the positive strand of the modified siRNA via a linking group; the ligand is preferably GalNAc, and the linking group is preferably an acetyl group.

[0084] The present invention also provides the use of the modified substance or the drug conjugate in preparing a drug for inhibiting DC624545.

[0085] The present invention also provides the use of the modified substance or the drug conjugate in preparing a drug for treating hyperlipidemia.

[0086] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0087] Example 1 Synthesis of siRNA

[0088] The gene sequence of cynomolgus monkey DC624545 was obtained from the Genebank database, siRNA was designed using siDirect siRNA design software, and then siRNA with a GC content of 50% was selected therefrom. The siRNA was then compared with the cynomolgus monkey DC624545 sequence included in the Genebank database using the NCBI BLAST function, and siRNA such as SEQ ID No. 1 was designed using siDirect version 2.0 siRNA design software.

[0089] The sense strand sequence is identical to the target sequence designed on the target gene (U replaces T), and the antisense strand sequence is completely complementary to the target sequence (U replaces T); at the same time, the 3' ends of both the sense strand and the antisense strand are overhanging with dTdT.

[0090] The specific contents of the above synthesis were obtained by entrusting Sangon Biotechnology (Shanghai) Co., Ltd.

[0091] Example 2 Preparation of siRNA Modifications

[0092] In order to enhance the double-stranded stability of siRNA, siRNA was chemically modified to obtain Modifier 1, Modifier 2, Modifier 3, Modifier 4, Modifier 5, and Modifier 6, respectively, and the specific nucleotide sequences are shown in Table 1. The preparation process of the modifiers was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0093] The obtained siRNA modifications were coupled with GalNAc ligands to obtain drug conjugates. The drug conjugates were commissioned to be synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0094] Experimental Example 1 Effect of drug conjugate on hyperlipidemia monkeys

[0095] 1.1 Animal experiments

[0096] The drug conjugates prepared by modifiers 1, 2 and 3 in Example 2 were formulated into a liquid medicine with a concentration of 1 mg / kg (low dose), and the drug conjugates prepared by modifiers 4, 5 and 6 in Example 2 were formulated into a liquid medicine with a concentration of 7 mg / kg (high dose), and subcutaneously injected into cynomolgus monkeys once, and the changes in fasting blood triglycerides were observed every day for 7 consecutive days after the injection. And 14 days after the injection, a part of the liver tissue block was taken by liver puncture, nucleic acid was extracted, and the expression of the target gene was verified. At the same time, a control group was set. The above content was entrusted to Guangzhou Huazhen Biotechnology Co., Ltd.

[0097] 1.2 qPCR detection of DC624545 expression

[0098] The liver tissue blocks obtained in part 1.1 were ground and homogenized using steel beads and Tissuelyser at 24 hours and 48 hours respectively, and RNA was extracted using the TRIZOL method. Reagents were added according to the instructions of the Trizol kit (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.), and cDNA was synthesized using the PCR conditions of 42°C, 15 min, and 85°C, 5 s.

[0099] The cDNA was diluted one-fold and then qPCR reaction was performed using TransStart Top Green qPCR SuperMix (purchased from TAKARA).

[0100] After the qPCR reaction, reagents were added according to the instructions of the reverse transcription kit (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.), and a two-step qPCR was set up.

[0101] The reaction conditions of the qPCR were: 94°C for 30 s, 94°C for 5 s, 60°C for 30 s, and 40 cycles; the reaction system was: 20 μL, 10 μL Mastermix (including enzymes and other substances), 10 nM primers (forward + reverse), 100 ng cDNA, and the rest was supplemented with ddH2O.

[0102] When performing qPCR, ACTIN (NM_001101.5) was used as an internal reference and the primers used were shown in Table 2.

[0103] Table 2 Primer sequences used in qPCR

[0104]

[0105] The results are as follows Figures 1 to 6 shown.

[0106] The results showed that after administration, triglycerides and high-density fatty acids showed a significant downward trend, which had a certain therapeutic effect on fatty liver. At the same time, the product has certain drugability in the future.

[0107] At present, the common atorvastatin, during clinical use, often causes side effects such as impotence in men. However, small nucleic acid molecule drugs regulate at the genetic level and have relatively fewer side effects.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A siRNA modified substance for inhibiting DC624545, characterized in that: The siRNA modifier is modifier 1, modifier 2, modifier 3, modifier 4, modifier 5 or modifier 6; the sequence of the siRNA modifier is as follows: Wherein: (s) indicates that the two nucleotides before and after are connected by a thiophosphate backbone; fA, fG, fC, fU represent ribonucleotides A, G, C, U modified by 2'-fluoro, respectively; mA, mG, mC, mU represent ribonucleotides A, G, C, U modified by 2'-methoxy, respectively.

2. The siRNA modification according to claim 1, characterized in that The 3' ends of both the sense strand and the antisense strand have overhangs of dTdT.

3. A drug conjugate, characterized in that: The invention comprises a ligand and the siRNA modification substance according to claim 1.

4. The drug conjugate according to claim 3, characterized in that The ligand is connected to the 3' end of the sense strand of the siRNA modification via a linker group.

5. The drug conjugate according to claim 3 or 4, characterized in that The ligand is GalNAc.

6. The drug conjugate according to claim 4, characterized in that The linking group is an acetyl group.

7. Use of the modified siRNA according to claim 1 or the drug conjugate according to any one of claims 3 to 6 in the preparation of an agent for inhibiting the expression of DC624545.

8. Use of the modified siRNA according to claim 1 or the drug conjugate according to any one of claims 3 to 6 in the preparation of a drug for treating hyperlipidemia.

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