Double-stranded nucleic acid-galactosamine-cholesterol compound and use thereof

Abstract

A double-stranded nucleic acid-galactosamine-cholesterol compound and a use thereof, relating to the technical field of nucleic acid drugs. The double-stranded nucleic acid-galactosamine-cholesterol compound is composed of a double-stranded nucleic acid, galactosamine, and cholesterol which are linked in sequence; the double-stranded nucleic acid is a double-stranded nucleic acid capable of inhibiting the transcription level and / or expression level of a target gene, and is composed of an antisense nucleic acid strand and a complementary strand; the galactosamine is linked to the 5' end of the complementary strand of the double-stranded nucleic acid. Also provided is a use of the double-stranded nucleic acid-galactosamine-cholesterol compound in the preparation of a drug having the effect of inhibiting the transcription level and / or expression level of a target gene and having a protective effect on the kidney. The double-stranded nucleic acid-galactosamine-cholesterol compound can cross the blood-brain barrier and can significantly reduce the renal burden, providing a technical platform for the treatment of refractory neurological diseases such as Alzheimer's disease and Parkinson's disease.

Description

Double-stranded nucleic acid-galactosamine-cholesterol compound and application thereof TECHNICAL FIELD

[0001] The present application relates to a double-stranded nucleic acid-galactosamine-cholesterol compound and application thereof, and belongs to the technical field of nucleic acid drugs. BACKGROUND

[0002] Nucleic acid drugs mainly refer to compounds containing nucleotides or deoxynucleotides with genetic properties and pharmacological activities, and are an important class of drugs that can be used for the treatment of tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. One of the great challenges of nucleic acid drugs is the specificity and effectiveness of in vivo delivery to target cells. Chemical modification, nanoparticle encapsulation and ligand-conjugated oligonucleotides can all improve their in vivo stability, and are strategies to improve pharmacokinetics and target specificity. Many studies have shown that targeted delivery technology can drive further development of oligonucleotide drug therapy.

[0003] Chinese patent CN 104126010 A discloses a double-stranded nucleic acid complex for inhibiting target gene expression by antisense action, comprising a first nucleic acid strand complexed with a second nucleic acid strand, wherein the first nucleic acid strand (i) comprises nucleotides and optionally nucleotide analogs, and the total number of nucleotides and nucleotide analogs in the first nucleic acid strand is 8 to 100, (ii) comprises at least 4 consecutive nucleotides recognized by RNase H when the first nucleic acid strand is hybridized to a transcription product, and (iii) the first nucleic acid strand is hybridized to the transcription product; and the second nucleic acid strand comprises nucleotides and optionally nucleotide analogs. The antisense nucleic acid can be delivered in the double-stranded complex and can selectively and very effectively inhibit the expression of the target gene or the level of the transcription product. The double-stranded complex can be delivered to the target site with high specificity and high efficiency by associating a delivery moiety with the complex. However, the double-stranded nucleic acid complex can increase the burden on the kidneys when a large dose is administered or multiple doses are administered (one of the reasons is that the double-stranded nucleic acid complex contains phosphorothioated nucleotides or phosphorothioated nucleotide analogs; if not phosphorothioated, the stability of the double-stranded nucleic acid is very poor), which greatly limits the drug user population.

[0004] In addition, double-stranded nucleic acid complexes cannot effectively cross the blood-brain barrier, and how to deliver sufficient levels of double-stranded nucleic acid complexes to the administration site is a difficult problem. According to a relevant report, researchers from Tokyo Medical and Dental University (TMDU), Ionis Pharmaceuticals, and Takeda Corporation cooperated to find a new method for subcutaneous or intravenous injection of ASO to cross the blood-brain barrier and be delivered to the central nervous system: coupling cholesterol (Cholesterol, CHS) on a heteroduplex oligonucleotide (heteroduplex oligonucleotides, HDOs) composed of DNA and RNA, which can effectively knock down the expression of target genes in the brain through subcutaneous or intravenous injection in mouse or rat experiments. The experimental results show that this cholesterol-coupled HDOs can be widely distributed in the brain, spine and peripheral tissues, and can inhibit the expression level of the four target genes evaluated in the central nervous system (CNS) by up to 90%. Moreover, the research team further proved that the ability of this HDOs to cross the blood-brain barrier did not cause a decrease in the integrity of the blood-brain barrier itself.

[0005] N-acetylgalactosamine (GalNAc), also known as 2-acetamido-2-deoxy-D-galactose, D-GalNAc, N-acetylgalactosamine, has the chemical formula C8H 15 N06. N-acetylgalactosamine is a commonly used liver-targeting carrier in the prior art, and N-acetylated galactosamine coupling modification is the most commonly used small nucleic acid drug delivery system: GalNAc is covalently conjugated to the 3' end of the sense strand of DNA or RNA of different sequences in a monovalent or trivalent manner to form polysaccharide DNA, polysaccharide RNA conjugates, thereby achieving specific delivery to hepatocytes. GalNAc is an asialoglycoprotein receptor (ASGPR) ligand that can bind to ASGPR with high affinity in the nM range, and then the receptor takes up GalNAc and nucleic acids into cells through clathrin-mediated endocytosis. The advantage of GalNAc coupling modification is that the coupled nucleic acid has a small molecular weight, and the effect lasts for several months. However, due to the specific high expression of ASGPR only in hepatocytes, there is a tissue limitation. SUMMARY

[0006] In view of the above prior art, the present application provides a double-stranded nucleic acid-galactosamine-cholesterol compound and its application. The double-stranded nucleic acid-galactosamine-cholesterol compound of the present application can penetrate the blood-brain barrier and significantly reduce the burden on the kidney. The compound can be used to prepare a drug that has the effect of inhibiting the transcription level and / or expression level of a target gene and has a protective effect on the kidney, has great application potential, and has a broad application prospect.

[0007] The present application is realized by the following technical solutions:

[0008] A double-stranded nucleic acid-galactosamine-cholesterol compound, consisting of a double-stranded nucleic acid, a galactosamine and a cholesterol connected in sequence; wherein the double-stranded nucleic acid is a double-stranded nucleic acid having the function of inhibiting the transcription level and / or expression level of a target gene, consisting of an antisense nucleic acid strand (also known as sense strand, template strand, parent strand, main strand) and a complementary strand (also known as vice strand); the galactosamine is connected to the 5' end of the complementary strand of the double-stranded nucleic acid.

[0009] Further, the type of the antisense nucleic acid strand is selected from any one or a combination of two or more of circular RNA, small activating RNA (SaRNA), antisense RNA (asRNA), antisense DNA (asDNA) and microRNA (miRNA).

[0010] Further, the antisense nucleic acid strand consists of nucleotides and / or nucleotide analogs, has a length of 7-50 bp, has 2-3 bridged nucleotides or nucleotide analogs at the 3' end, has 2-3 bridged nucleotides or nucleotide analogs at the 5' end, and the number of bridged nucleotides or nucleotide analogs accounts for 10%-70% of the total number of nucleotides in the strand; the bridged nucleotides are selected from LNA, cEt-BNA, amide BNA (AmNA), cMOE-BNA or any other bridged nucleotide; the nucleotides and nucleotide analogs are all phosphorylated.

[0011] Further, the nucleic acid type of the complementary strand is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and / or peptide nucleic acid (PNA).

[0012] Further, the complementary strand consists of nucleotides and / or nucleotide analogs, has 2-3 methylated modified (2-0-methylated) nucleotides or nucleotide analogs at the 3' end, has 2-3 methylated modified nucleotides or nucleotide analogs at the 5' end, and the nucleotides and nucleotide analogs are connected by phosphodiester bonds.

[0013] Further, the galactosamine is selected from N-acetyl galactosamine or N-acetyl glucosamine (GlcNAc).

[0014] Further, the connection mode of the galactosamine and the cholesterol can be TEG-link (a conventional connection mode in the prior art, the cholesterol is connected to the TEG-link by an amide bond, and the TEG is connected to the GalNAc by a phosphodiester bond), PEG-link or directly connected by a phosphodiester bond, or other known connection modes in the prior art.

[0015] The double-stranded nucleic acid-galactosamine-cholesterol compound is used as or prepared into a drug having the function of inhibiting the transcription level and / or expression level of a target gene and having a protective effect on the kidney.

[0016] A medicine with inhibiting transcription level and / or expression level efficacy of target gene and protecting kidney, its effective component is the above-mentioned double-stranded nucleic acid-galactosamine-cholesterol compound, and it can also include pharmaceutically common adjuvants, such as solvents, fillers, etc.

[0017] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present application is administered to mammals including humans. The animal in-vivo experiment or clinical administration route is parenteral route such as intravenous administration, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrathecal injection, intracerebroventricular injection, or gastrointestinal administration (oral administration, gastrointestinal nutrition tube, anal plug, etc.), wherein the dosage is 0.01-100 mg / kg / day, and the administration can be repeated.

[0018] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present application is composed of double-stranded nucleic acid, galactosamine and cholesterol, wherein the cholesterol carries the double-stranded nucleic acid to pass through the blood-brain barrier into neurons, the double-stranded nucleic acid has the pharmacodynamic efficacy of inhibiting the transcription level and / or expression level of target gene, and the galactosamine can bind to ASGPR on the surface of liver parenchymal cells, thereby bringing the excess double-stranded nucleic acid into hepatocytes for metabolism and reducing the burden on the kidney. The present application solves the following technical problems and achieves the following technical effects:

[0019] (1) The problem that double-stranded nucleic acid cannot pass through the blood-brain barrier is solved. The present application finds through research that, after the compound of the present application is injected into the tail vein of animals (mice) in-vivo experiment, the amount of double-stranded nucleic acid passing through the blood-brain barrier into brain neuron cells can be obviously increased, which provides a technical platform for treating refractory diseases of nervous system such as Alzheimer's disease and Parkinson's disease.

[0020] (2) The problem of acute and chronic kidney injury after increasing the dosage or multiple administrations of double-stranded nucleic acid is solved. At present, in order to improve the delivery effectiveness and stability of double-stranded nucleic acid, a variety of modification techniques and carriers are usually combined. The kidney is a metabolic organ for some drugs, and the kidney burden of some patients can be increased after long-term drug use. The present application binds N-acetylgalactosamine to ASGPR on the surface of liver parenchymal cells, enters the cells by endocytosis, and forms endosomes, thereby bringing the excess amount of double-stranded nucleic acid into hepatocytes for decomposition; therefore, multiple injections do not affect the effectiveness of the drug while having obvious protective effect on the kidney, and the use population of double-stranded nucleic acid is expanded (including the population with kidney injury).

[0021] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present application has the following mechanisms of action: ① antisense DNA combines with genomic DNA in the form of base complementary pairing in the nucleus to form a triple-stranded nucleic acid structure or combines with single-stranded DNA to form a double-stranded structure, thereby preventing replication and transcription of the target gene; ② antisense RNA combines with mRNA to form a complementary double strand, thereby blocking the binding of ribosomes to mRNA and inhibiting the process of ribosome-mediated translation of mRNA into protein; ③ antisense nucleic acid, after combining with mRNA in the nucleus, can inhibit the export of mRNA from the nucleus; and ④ antisense nucleic acid combines with mRNA to form a hybrid double-stranded molecule, thereby inducing RNase H to degrade RNA, thus shortening the half-life of mRNA and reducing the translation of target protein.

[0022] The double-stranded nucleic acid-galactosamine-cholesterol compound of the present application, through experimental research, it is found that only when galactosamine-cholesterol is connected to the 5' end of the complementary strand of double-stranded nucleic acid, the inhibitory effect of double-stranded nucleic acid on the target gene can be exerted. When galactosamine-cholesterol is connected to other positions (3' end of the sense strand, 3' end of the complementary strand, 5' end of the sense strand), double-stranded nucleic acid does not show an inhibitory effect on the target gene. Moreover, the present application, through experimental research, has also found that the connection order of double-stranded nucleic acid, galactosamine and cholesterol also has a significant impact on the effect of the compound. Only when the order of double-stranded nucleic acid-galactosamine-cholesterol is connected, the double-stranded nucleic acid can exert a significant inhibitory effect. When the connection order is changed to double-stranded-cholesterol-galactosamine, the double-stranded nucleic acid does not show an inhibitory effect on the target gene. In addition, it is generally believed in the prior art that the smaller the molecular weight or volume of the carrier is, the easier it is to be absorbed by cells. However, the present application, through experimental research, shows that there is no obvious negative impact on the inhibitory effect when the nucleic acid chain is less than 100 bp, the carrier is extended (TEG-link), the molecular weight (double or multiple carriers) is increased or the volume is increased.

[0023] The various terms and phrases used in the present application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1: Structure diagram of double-stranded nucleic acid-galactosamine-cholesterol compound.

[0025] Figure 2: Schematic diagram of the connection of cholesterol and galactosamine.

[0026] Figure 3: Mass spectrum of galactosamine-cholesterol.

[0027] Figure 4: Schematic diagram of the relative SNCA mRNA level in the cerebral cortex of mice in Example 2, wherein the mRNA level of the control group is 1.

[0028] Figure 5: Schematic representation of the relative MALAT1 mRNA levels in the cortex of mice in Example 3, wherein the mRNA level of the control group is 1.

[0029] Figure 6: Schematic representation of the relative MALAT1 mRNA levels in the cortex of mice in Example 4, wherein the mRNA level of the control group is 1.

[0030] Figure 7: Schematic representation of the relative SRB1 mRNA levels in the substantia nigra of mice in Example 5, wherein the mRNA level of the control group is 1

[0031] Figure 8: Schematic representation of the relative MALAT1 mRNA levels in the cortex of mice in Example 6, wherein the mRNA level of the control group is 1.

[0032] Figure 9: Schematic representation of the relative MALAT1 mRNA levels in the hippocampus of mice in Example 6, wherein the mRNA level of the control group is 1.

[0033] Figure 10: Schematic representation of the relative MALAT1 mRNA levels in the striatum of mice in Example 6, wherein the mRNA level of the control group is 1.

[0034] Figure 11: Schematic representation of the relative OAT3 mRNA levels in the vascular endothelial cells of mice in Example 7, wherein the mRNA level of the control group is 1. DETAILED DESCRIPTION

[0035] The application will be further described below with reference to the following 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.

[0036] The instruments, reagents, materials involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are conventional experimental methods, detection methods already existing in the prior art.

[0037] The 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 by enzymatic reaction process (including but not limited to polymerase, ligase or restriction reaction).

[0038] The present application does not particularly limit the chain length of the nucleic acid strand (template strand), but the chain length is usually 7 to 50 bases. The chain length can be up to 120 bases. The length is generally selected depending on the balance between the effect of antisense action and the specificity of the targeted nucleic acid strand, and other factors such as cost, synthesis yield, etc. are also influential.

[0039] The DNA referred to in the present application means a naturally occurring DNA deoxynucleotide, or a DNA deoxynucleotide partially having a modified base, sugar or phosphate bond subunit. The modified base, sugar or phosphate bond subunit means mainly that kind in which a single substituent has been added or substituted in the subunit, such as 5-methylation, phosphorylation, 2'-0-methylation, etc. The connection between the DNA deoxynucleotides is mainly thiophosphorylation.

[0040] The locked nucleic acid (LNA) referred to in the present application is an oligonucleotide-like derivative having a rigid structure in which the 2'-0, 4'-C positions of β-D-ribofuranose are connected by a contraction.

[0041] The nucleotide chain of the present application is prepared by using a conventional phosphoramidite triester synthesis method, which is the most widely used oligonucleotide synthesis method and is also the synthesis method used by the mainstream synthesis instrument at home and abroad. This method has the characteristics of efficient and rapid coupling and relatively stable starting reactants. First, RNA monomers and DNA monomers 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 synthesis of RNA or DNA chain is completed by fixing RNA monomers, DNA monomers or 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, and the adjacent nucleotides are connected by 3'→5' phosphodiester bond. The steps include four steps of deprotection, coupling, capping and oxidation. According to the predetermined base sequence, the corresponding phosphoramidite monomer and other necessary chemical reagents are added in turn on the solid support which has been surface modified in advance through the liquid system, so as 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:

[0042] (1) Synthesis: crude synthesis is carried out by a synthesizer, mainly including deprotection, condensation, blocking, oxidation and other processes.

[0043] (1) The first step of deprotection: the chemical active group pre-connected on the solid support is placed in the synthesizer and reacted with TCA added into the synthesizer from the reagent bottle pipe;

[0044] (2) Second step condensation: tetrazole is added from the reagent bottle to the synthesizer through a pipe, mixed with the monomer dissolved in acetonitrile to obtain an activated intermediate to undergo condensation reaction;

[0045] (3) Third step closure: reagents CapA and CapB are input into the synthesizer from the reagent bottle through a pump to terminate the condensation reaction and perform subsequent reactions on a small number of substances that do not participate in the reaction;

[0046] (4) Fourth step oxidation: tetrahydrofuran reagent with iodine is added from the reagent bottle using a pump to perform an oxidation reaction on the condensed group to convert the phosphine into a more stable phosphotriester;

[0047] (5) Repeat the above four steps until all the required synthesized groups are attached to obtain a crude product.

[0048] (II) Ammonolysis: first, ammonolysis pretreatment is performed to remove impurities generated during synthesis, then washing is performed to further purify the crude product. After the pretreatment is completed, the crude product is transferred to the ammonolysis chamber and added to the ammonolysis instrument for ammonolysis deprotection, and finally the sample to be eluted is obtained.

[0049] (III) HPLC purification: the sample after ammonolysis is purified using an HPLC purification instrument.

[0050] The mice referred to in the present application are all 4 to 6 week old C57 / BL mice (both male and female), and the weight of the mice is about 20 g. Unless otherwise specified, the number of mice used in each group of experiments is 3 or 4.

[0051] Example 1 Preparation of double-stranded nucleic acid-galactosamine-cholesterol compound

[0052] For the target gene alpha-synuclein (SNCA) gene, a double-stranded nucleic acid-galactosamine-cholesterol compound (referred to as compound S in the present application) is prepared.

[0053] Compound S is composed of double-stranded nucleic acid, galactosamine and cholesterol connected in sequence, and the structural diagram is shown in Figure 1; wherein the galactosamine is connected to the 5' end of the complementary strand of the double-stranded nucleic acid; the galactosamine and the cholesterol are connected through TEG-link (cholesterol is connected to TEG-link through an amide bond, and TEG is connected to GalNAc through a phosphodiester bond), as shown in Figure 2.

[0054] The steps are as follows:

[0055] (1) Synthesis of single-stranded nucleic acid and double-stranded nucleic acid

[0056] The double-stranded nucleic acid targeting α-synuclein is designed, wherein the primary designed main chain is DNA, and the nucleotide sequence is 5'-aatgacattctta-3', as shown in SEQ ID NO. 1.

[0057] The primary designed secondary chain is RNA, and the nucleotide sequence is 5'-TAAGAATGTCATT-3', as shown in SEQ ID NO. 2.

[0058] Then, without changing the nucleotide sequence, some bases are modified and modified (the purpose of modification is to enhance the therapeutic effect or make it more stable or have other positive effects), and the modification is selected from any one or more of the following: locked nucleic acid modification, methylation modification (including conventional 2' methylation modification, and 5' methylation modification of cytosine), phosphorothioate bond modification, DNA nucleic acid chain chimeric RNA modification, etc.

[0059] The modified main chain is as follows: 5'-A(+)^A(+)^t^g^a^c^a^t^t^c^T(+)^T(+)^A(+)-3'.

[0060] The modified secondary chain is as follows: 5'-T(M)^A(M)^A(M)^GAATGTCA^T(M)^T(M)-3'.

[0061] Among them, the capital letter represents RNA; the lowercase letter represents DNA; the capital letter (+): represents the locked nucleic acid modification (2'-0 and 4' carbon on ribose are linked together); (M) represents methylation modification (2'-0-methylation); ^ represents phosphorothioate bond modification.

[0062] After synthesizing the modified main chain and secondary chain by conventional methods, the main chain and the secondary chain are mixed in the same amount of substance, the mixture is heated at 95°C for 5 minutes, and then is placed at a constant temperature of 37°C for 1 hour to allow the chain to anneal, and is stored in a 4°C environment.

[0063] (2) Preparation of double-stranded nucleic acid-galactosamine-cholesterol compound

[0064] (a) Cholesterol is connected with TEG-linker, the hydroxyl group on the cholesterol is acylated by condensing agent and reacts with the activated fat of TEG to form an amide bond (this method is a conventional technical means), the specific method is as follows: 300 grams of cholesterol, 350 grams of TEG-linker are dissolved in 50 milliliters of acetone, 0.5 grams of condensing agent acylase is added, and placed in an air bath constant temperature oscillator (23°C, 250 r·min -1 ) for oscillation for 24 hours; after the reaction is completed, the enzyme is filtered out, and the solvent is recovered, to obtain cholesterol-TEG;

[0065] (b) Synthesis of galactosamine-cholesterol by enzymatic method (which is a routine technique), the specific method is as follows: 100 g hydrogenated soybean phospholipid (HSPC), 300 g cholesterols, 20 g distearoylphosphatidylglycerol sodium (DSPG-Na), 330 g cholesterols-TEG and 300 g N-acetylgalactosamine are mixed, dissolved in 500 ml chloroform, placed in a 37℃ water bath rotary evaporator, and the chloroform is evaporated to form a lipid film on the inner wall of the bottle, which is then dried in a vacuum dryer for 12 hours. Then add 500 ml ultrapure water to hydrate, stir slowly at 55℃, avoid light incubation for 2 hours, then pass through 0.1 μm and 0.05 μm polycarbonate membranes 10 times with a high-pressure extruder, and galactosamine-cholesterol is obtained. The product is purified by flash silica gel column chromatography, and the purified product is identified by ESI, HNMR and CNMR. The results are shown in Figure 3, which confirms that the structure of the product is the structure shown in Figure 2, and one cholesterols molecule is connected to three N-acetylgalactosamine molecules.

[0066] (c) Synthesis of complementary strand by routine method (synthesized by 3'-5', and the 5' end protection group DMT is removed after synthesis to expose the hydroxyl group); the complementary strand and galactosamine-cholesterol are placed in L96 CPG at a molar ratio of 1:3, and an L96 carrier containing an oligonucleotide sequence with a protection group is synthesized by solid-phase synthesis technology. Then, the deprotection (3% DCA, 0.3M BMT, 0.5% NMI in ACN) is carried out to obtain the crude oligonucleotide from the solid-phase carrier. The galactosamine-cholesterol couples with the exposed hydroxyl group and forms a phosphodiester bond through oxidation to conjugate to the 5' end of the complementary strand, forming a complementary strand-galactosamine-cholesterol;

[0067] (d) Mix the main chain and the complementary strand-galactosamine-cholesterol at the same amount of substance, heat the mixture at 95℃ for 5 minutes, and stand at 37℃ constant temperature for 1 hour to allow the strands to renature, and obtain the double-stranded nucleic acid-galactosamine-cholesterol compound, which is stored at 4℃ environment.

[0068] The raw materials used are as follows:

[0069] Cholesterol (Japan Seiyu K.K., batch number: A90719, mass fraction > 98.5%);

[0070] N-acetylgalactosamine (Shanghai Aladdin Bio-Chem Technology Co., Ltd., mass fraction > 98.0%);

[0071] TEG-linker (Shanghai Zhiwei Technology Development Co., Ltd., CB96327030);

[0072] Lipozyme TLIM lipase (Novozymes Denmark, enzyme activity > 250 IUN·g -1 );

[0073] Ultrafiltration centrifuge tube (PALL Corporation, USA, 1 mL, 100 KD);

[0074] MD34 dialysis bag (Shanghai Baishai Biotechnology Co., Ltd., cut-off relative molecular mass 0.8×10 4 ~1.4×10 4 );

[0075] Other reagents are commercially available analytical pure.

[0076] Example 2 Application 1 of double-stranded nucleic acid-galactosamine-cholesterol compound

[0077] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Excessive aggregation of intracellular alpha-synuclein is a characteristic pathological change of Parkinson's disease. Its early pathological changes are limited to dopaminergic neurons in the substantia nigra compacta, so local gene delivery is likely to be an effective treatment measure.

[0078] The mice were randomly divided into 5 groups: control group, single-stranded experimental group 1, single-stranded experimental group 2, compound experimental group 1, and compound experimental group 2.

[0079] Compound experimental group 1 and compound experimental group 2 were both injected with the double-stranded nucleic acid-galactosamine-cholesterol compound prepared in Example 1 via the tail vein, diluted with PBS buffer to a concentration of 1200 μM, and then injected. The dose of compound experimental group 1 (referring to the dose of compound) was 50 mg / kg, and the dose of compound experimental group 2 was 25 mg / kg, injected once a week, for a total of 4 weeks, for a total of 4 times.

[0080] Single-stranded experimental group 1 and single-stranded experimental group 2 were both injected with single-stranded nucleic acid (i.e. the backbone of Example 1) via the tail vein, diluted with PBS buffer to a concentration of 1200 μM, and then injected. The dose of single-stranded experimental group 1 (referring to the dose of single-stranded nucleic acid) was 50 mg / kg, and the dose of single-stranded experimental group 2 was 25 mg / kg, injected once a week, for a total of 4 weeks, for a total of 4 times.

[0081] The control group was only injected with an equal amount of PBS buffer.

[0082] Three days after tail vein injection, the mice were anesthetized, and blood was taken from the retrobulbar, and serum was extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver, and other tissues were extracted by PBS perfusion.

[0083] Figure 4 shows the relative SNCA mRNA levels in the mouse cerebral cortex. As shown in Figure 4, after tail vein injection of single-stranded nucleic acid, double-stranded nucleic acid-galactosamine-cholesterol compound, the SNCA mRNA levels were significantly decreased, and the effect of double-stranded nucleic acid-galactosamine-cholesterol compound was better than that of single-stranded nucleic acid. Meanwhile, the high dose had better effect than the low dose.

[0084] Table 1 shows the biochemical indicators of mouse serum. The lower the value, the smaller the side effect. As shown in Table 1, the liver function (AST, ALT) and kidney function (BUN, Cre) of the compound experimental group were significantly lower than those of the single-stranded experimental group, and the double-stranded nucleic acid-galactosamine-cholesterol compound had no obvious side effect.

[0085] Table 1 Biochemical indicators of mouse serum

[0086] T-Bil (mg / dl) BUN (mg / dl) Cre (mg / dl) AST (U / l) ALT (U / l) LD (U / l) ALP (U / l) PBS 0.067±0.009 33.033±1.250.110±0.005 8101±15 37±11 312±62 427±35 Single-stranded experimental group 20.963±0.111 28.133±1.430.113±0.012 0933±768 21±198 02±439 49±7 Single-stranded experimental group 11.953±0.120 31.500±1.950.110±0.005 81519±761 967±901 998±122 1971±296 Compound experimental group 20.803±0.093 24.767±3.110.103±0.008 8183±281 35±334 3±165 15±25 Compound experimental group 11.007±0.038 18.300±2.950.090±0.010 0317±252 87±153 29±477 36±9

[0087] Example 3 Application 2 of double-stranded nucleic acid-galactosamine-cholesterol compound

[0088] This example is directed to the target gene MALAT1, and single-stranded nucleic acid, double-stranded nucleic acid are designed and prepared, and galactosamine, cholesterol, and galactosamine-cholesterol are connected to the 5' end of the side chain of the double-stranded nucleic acid, respectively (the connection method is a conventional technical means), to prepare double-stranded nucleic acid-galactosamine compound, double-stranded nucleic acid-cholesterol compound, and double-stranded nucleic acid-galactosamine-cholesterol compound (referred to as compound MA in the present application).

[0089] Specifically, the double-stranded nucleic acid is composed of a sense strand (primary strand) and a complementary strand (secondary strand), wherein the primary strand of the initial design is DNA, and the nucleotide sequence is 5'-gaactcaaaatatatt-3', as shown in SEQ ID NO. 3.

[0090] The secondary strand of the initial design is RNA, and the nucleotide sequence is 5'-AATATATTTTGAGTTC-3', as shown in SEQ ID NO. 4.

[0091] Then, without changing the nucleotide sequence, some bases are modified and reformed, and the modified and reformed primary strand is as follows: 5'-G(+)^A(+)^A(+)^c^t^c^a^a^a^a^t^a^t^A(+)^T(+)^T(+)-3'.

[0092] The modified and reformed secondary strand is as follows: 5'-A(M)^A(M)^T(M)^ATATTTTGAGT(M)^T(M)^C(M)-3'.

[0093] The single-stranded nucleic acid refers to the sense strand described above.

[0094] The effects of the three compounds are compared, and the experimental process is as follows:

[0095] The mice are randomly divided into 5 groups: a control group, a single-stranded experimental group, a double-stranded experimental group, a galactosamine double-stranded experimental group, a cholesterol double-stranded experimental group, and a cholesterol-galactosamine double-stranded experimental group. Among them, the single-stranded experimental group is injected with the above-mentioned single-stranded nucleic acid through the tail vein, the double-stranded experimental group is injected with the above-mentioned double-stranded nucleic acid through the tail vein, the galactosamine double-stranded experimental group is injected with the above-mentioned double-stranded nucleic acid-galactosamine compound through the tail vein, the cholesterol double-stranded experimental group is injected with the above-mentioned double-stranded nucleic acid-cholesterol compound through the tail vein, and the cholesterol-galactosamine double-stranded experimental group is injected with the above-mentioned compound MA through the tail vein. The control group is only injected with an equal amount of PBS buffer. After dilution with PBS buffer to a concentration of 1200 µM, injection is performed, and the dose is 50 mg / kg. Injection is performed once a week for 4 weeks, for a total of 4 times.

[0096] Three days after tail vein injection, the mouse eyeball is taken after anesthesia, and serum is extracted for blood biochemical detection; then, the substantia nigra, cerebral cortex, striatum, hippocampus, liver and other tissues are extracted by PBS perfusion.

[0097] Figure 5 shows the relative MALAT1 mRNA levels in the mouse cerebral cortex. As can be seen from Figure 5, after tail vein injection of single-stranded nucleic acid, double-stranded nucleic acid, double-stranded nucleic acid-galactosamine compound, double-stranded nucleic acid-cholesterol compound, and compound MA, the MALAT1 mRNA levels all decreased, and the decrease was significant after injection of compound MA, which was significantly better than the double-stranded nucleic acid-galactosamine compound and the double-stranded nucleic acid-cholesterol compound. This shows that only connecting galactosamine or cholesterol on the double-stranded nucleic acid cannot achieve the desired inhibitory effect; when both galactosamine and cholesterol are connected on the double-stranded nucleic acid, a significant inhibitory effect can be achieved.

[0098] Example 4 Application 3 of double-stranded nucleic acid-galactosamine-cholesterol compound

[0099] This example is directed to the target gene MALAT1, and a double-stranded nucleic acid (as in Example 3) is prepared, and galactosamine-cholesterol is connected to the 3' end of the sense strand, the 3' end of the complementary strand, the 5' end of the sense strand, and the 5' end of the complementary strand, respectively, by conventional coupling and condensation reactions, to prepare four compounds, which are referred to as sense strand 3' compound A, complementary strand 3' compound A, sense strand 5' compound A, and complementary strand 5' compound A (this is compound MA of the present application).

[0100] The effects of these four compounds are compared, and the experimental process is as follows:

[0101] The mice are randomly divided into five groups: a control group, a compound main chain 3' end A group, a compound main chain 5' end A group, a compound side chain 3' end A group, and a compound side chain 5' end A group. Among them, the compound main chain 3' end A group is injected with the above-mentioned sense strand 3' compound A via the tail vein, the compound main chain 5' end A group is injected with the above-mentioned sense strand 5' compound A via the tail vein, the compound side chain 3' end A group is injected with the above-mentioned complementary strand 3' compound A via the tail vein, and the compound side chain 5' end A group is injected with the above-mentioned compound MA via the tail vein. The control group is only injected with an equal amount of PBS buffer. After dilution with PBS buffer to a concentration of 1200 µM, injection is performed, and the dose is 12.5 mg / kg. Injection is performed once a week for four consecutive weeks, for a total of four times.

[0102] Three days after tail vein injection, the mice are anesthetized, and blood is taken from the retrobulbar, and serum is extracted for blood biochemical testing; then the substantia nigra, cerebral cortex, striatum, hippocampus, liver, and other tissues are extracted by PBS perfusion.

[0103] Figure 6 shows the relative MALAT1 mRNA levels in the mouse cerebral cortex. As shown in Figure 6, after tail vein injection of sense strand 3' compound A, sense strand 5' compound A, the MALAT1 mRNA levels are significantly increased; after injection of complementary strand 3' compound A, the MALAT1 mRNA level has no significant change; after injection of compound MA, the MALAT1 mRNA level is significantly decreased. This shows that only compound MA can achieve an inhibitory effect, i.e. when galactosamine-cholesterol is connected to the 5' end of the complementary strand of double-stranded nucleic acid, it can play an obvious inhibitory effect, and when it is connected to other positions, the double-stranded nucleic acid cannot play the preset role.

[0104] Example 5 Application 4 of double-stranded nucleic acid-galactosamine-cholesterol compound

[0105] This example is directed to target gene SRB1, and a double-stranded nucleic acid is designed and prepared, and galactosamine-cholesterol is connected to the 3' end of the sense strand, the 3' end of the complementary strand, the 5' end of the sense strand, and the 5' end of the complementary strand, respectively, by conventional coupling and condensation reaction, to prepare four compounds, which are called sense strand 3' compound B, complementary strand 3' compound B, sense strand 5' compound B, and complementary strand 5' compound B (this is the double-stranded nucleic acid-galactosamine-cholesterol compound of the present application, referred to as compound SR).

[0106] Specifically, the double-stranded nucleic acid is composed of a sense strand (main strand) and a complementary strand (secondary strand), wherein the initially designed main strand is DNA, and the nucleotide sequence is 5'-ttcagtcatgact-3', as shown in SEQ ID NO. 5.

[0107] The initially designed secondary strand is RNA, and the nucleotide sequence is 5'-AGTCATGACTGAA-3', as shown in SEQ ID NO. 6.

[0108] Then, without changing the nucleotide sequence, some bases are modified and modified, and the modified main strand is as follows: 5'-T(+)^T(+)^c^a^g^t^c^a^t^g^A(+)^C(+)(5M)^T(+)-3', wherein 5M represents 5' methylation modification of cytosine (5'-0-methylation).

[0109] The nucleotide sequence of the modified secondary strand is as follows: 5'-A(M)^G(M)^T(M)^CATGACTGA(M)^A(M)-3'.

[0110] The effects of the four compounds are compared, and the experimental process is as follows:

[0111] The mice are randomly divided into 5 groups: a control group, a compound main chain 3' end B group, a compound main chain 5' end B group, a compound side chain 3' end B group, and a compound side chain 5' end B group. The compound main chain 3' end B group is injected with the above sense chain 3' compound B via the tail vein, the compound main chain 5' end B group is injected with the above sense chain 5' compound B via the tail vein, the compound side chain 3' end B group is injected with the above complementary chain 3' compound B via the tail vein, and the compound side chain 5' end B group is injected with the above compound SR via the tail vein. The compound is injected via the tail vein in each experimental group, and the compound is diluted with PBS buffer to a concentration of 1200 µM before injection. The dose is 12.5 mg / kg. The control group is only injected with an equal amount of PBS buffer.

[0112] Three days after the tail vein injection, the mice are anesthetized, and blood is taken from the eyeball posterior to extract serum for blood biochemical testing. Then, the mice are dissected and perfused with PBS to extract the substantia nigra, cerebral cortex, striatum, hippocampus, liver, and other tissues.

[0113] Figure 7 shows the relative SRB1 mRNA level in the substantia nigra of mice. As can be seen from Figure 7, after the injection of the sense chain 3' compound B, the sense chain 5' compound B, and the complementary chain 3' compound B via the tail vein, the SRB1 mRNA level does not change significantly. After the injection of the compound SR, the SRB1 mRNA level decreases significantly. This indicates that only the compound SR can achieve an inhibitory effect, i.e., galactosamine-cholesterol connected to the 5' end of the complementary chain of double-stranded nucleic acid can play a significant inhibitory role, and when connected to other positions, the double-stranded nucleic acid cannot play the intended role.

[0114] Example 6 Application of double-stranded nucleic acid-galactosamine-cholesterol compound 5

[0115] This example is directed to the target gene MALAT1, and a double-stranded nucleic acid (same as in Example 3) is prepared. Cholesterol is connected to the oligonucleotide on the solid support through conventional coupling and condensation reactions, and galactosamine is connected through oxidation. The double-stranded nucleic acid-cholesterol-galactosamine compound is prepared by linking the side chain 5' end through a phosphate bond. The experimental process is compared with the double-stranded nucleic acid-galactosamine-cholesterol compound (this is the compound MA of the present application) of the present application as follows:

[0116] The mice are randomly divided into 3 groups: a control group, a double-stranded-cholesterol-galactosamine compound group, and a double-stranded nucleic acid-galactosamine-cholesterol compound group. The double-stranded-cholesterol-galactosamine compound group is injected with the above double-stranded nucleic acid-cholesterol-galactosamine compound via the tail vein, and the double-stranded nucleic acid-galactosamine-cholesterol compound group is injected with the above compound MA via the tail vein. The compound is diluted with PBS buffer to a concentration of 1200 µM before injection. The dose is 25 mg / kg. The control group is only injected with an equal amount of PBS buffer.

[0117] Three days after the tail vein injection, the mice were anesthetized and the eyeballs were taken out to extract serum for blood biochemical test. Then the mice were dissected and the tissues of substantia nigra, cerebral cortex, striatum, hippocampus, liver, etc. were extracted by PBS perfusion.

[0118] Figure 8 shows the relative MALAT1 mRNA level in the cerebral cortex of mice. As can be seen from Figure 7, after the tail vein injection of double-stranded-cholesterol-galactosamine compound, the MALAT1 mRNA level did not change significantly; after the injection of compound MA, the MALAT1 mRNA level decreased significantly.

[0119] Figure 9 shows the relative MALAT1 mRNA level in the hippocampus of mice. As can be seen from Figure 8, after the tail vein injection of double-stranded-cholesterol-galactosamine compound, the MALAT1 mRNA level did not change significantly; after the injection of compound MA, the MALAT1 mRNA level decreased significantly.

[0120] Figure 10 shows the relative MALAT1 mRNA level in the striatum of mice. As can be seen from Figure 9, after the tail vein injection of double-stranded-cholesterol-galactosamine compound, the MALAT1 mRNA level increased significantly; after the injection of compound MA, the MALAT1 mRNA level decreased significantly.

[0121] The above results show that when the double-stranded nucleic acid is connected in the order of double-stranded nucleic acid-galactosamine-cholesterol, the double-stranded nucleic acid can play a significant inhibitory effect. When the connection order is changed to double-stranded-cholesterol-galactosamine, the double-stranded nucleic acid cannot play the preset role (cannot effectively penetrate the blood-brain barrier and cannot reduce the burden on the kidney).

[0122] Example 7 Application 6 of double-stranded nucleic acid-galactosamine-cholesterol compound

[0123] This example is directed to the target gene OAT3, and single-stranded nucleic acid and double-stranded nucleic acid are designed and prepared, and galactosamine, cholesterols and galactosamine-cholesterol are connected to the 5' end of the secondary strand of the double-stranded nucleic acid, respectively (the connection method is a conventional technical means), to prepare a double-stranded nucleic acid-galactosamine-cholesterol compound (referred to as compound M in the present application).

[0124] Specifically, the double-stranded nucleic acid is composed of a sense strand (primary strand) and a complementary strand (secondary strand), wherein the initially designed primary strand is DNA, and the nucleotide sequence is 5'-cggccaaacctgtc-3', as shown in SEQ ID NO. 7.

[0125] The initially designed secondary strand is RNA, and the nucleotide sequence is 5'-GACAGGTTTGGCCG-3', as shown in SEQ ID NO. 8.

[0126] Then, without changing the nucleotide sequence, part of the bases are modified and reformed, the modified and reformed main chains have 5, which are named M-4, M-6, M-8, M-10, M-12, corresponding to the number of locked nucleic acids in the main chain, which are 4, 6, 8, 10, 12 respectively, as shown in Table 2 (direction 5'-3').

[0127] Table 2

[0128] M-4 C (+) (5M) ^G (+) ^g ^c ^c ^a ^a ^a ^c ^c ^t ^g ^T (+) ^C (+) (5M) M-6 C (+) (5M) ^G (+) ^G (+) ^c ^c ^a ^a ^a ^c ^c ^t ^G (+) ^T (+) ^C (+) (5M) M-8 C (+) (5M) ^G (+) ^G (+) ^C (+) (5M) ^c ^a ^a ^a ^c ^c ^T (+) ^G (+) ^T (+) ^C (+) (5M) M-10 C (+) (5M) ^G (+) ^G (+) ^C (+) (5M) ^C (+) (5M) ^a ^a ^a ^c ^C (+) (5M) ^T (+) ^G (+) ^T (+) ^C (+) (5M) M-12 C (+) (5M) ^G (+) ^G (+) ^C (+) (5M) ^C (+) (5M) ^A (+) ^a ^a ^C (+) (5M) ^C (+) (5M) ^T (+) ^G (+) ^T (+) ^C (+) (5M)

[0129] The modified and reformed side chain is as follows: 5'-G(M) ^A(M) ^C(M) ^AGGTTTGGC(M) ^C(M) ^G(M)-3'.

[0130] Wherein, the capital letter represents RNA; the lowercase letter represents DNA; the capital letter (+) represents locked nucleic acid modification (2'-0 and 4' carbon on ribose are connected together); (M) represents methylation modification (2'-0-methylation); ^ represents phosphorothioate bond modification, (5M) represents 5'methylation modification of cytosine (5'-0-methylation).

[0131] The single-stranded nucleic acid refers to the main chain described above.

[0132] The effects of the five compounds are compared, and the experimental process is as follows:

[0133] The mice were randomly divided into 6 groups: control group, cholesterol-galactosamine double-stranded experimental group (compound M-4), cholesterol-galactosamine double-stranded experimental group (compound M-6), cholesterol-galactosamine double-stranded experimental group (compound M-8), cholesterol-galactosamine double-stranded experimental group (compound M-10), and cholesterol-galactosamine double-stranded experimental group (compound M-12). The cholesterol-galactosamine double-stranded experimental group (compound M-4) was injected with the above compound M-4 via the tail vein, the cholesterol-galactosamine double-stranded experimental group (compound M-6) was injected with the above compound M-6 via the tail vein, the cholesterol-galactosamine double-stranded experimental group (compound M-8) was injected with the above compound M-8 via the tail vein, the cholesterol-galactosamine double-stranded experimental group (compound M-10) was injected with the above compound M-10 via the tail vein, and the cholesterol-galactosamine double-stranded experimental group (compound M-12) was injected with the above compound M-12 via the tail vein. The control group was only injected with an equal amount of PBS buffer. After dilution with PBS buffer to a concentration of 1200 μM, the injection dose was 50 mg / kg.

[0134] Three days after tail vein injection, the mice were anesthetized, and blood was taken from the retrobulbar, and serum was extracted for blood biochemical testing. Then the mice were dissected and perfused with PBS to extract the substantia nigra, cerebral cortex, striatum, hippocampus, liver, and other tissues.

[0135] Figure 11 shows the relative OAT3 mRNA levels in mouse vascular endothelial cells. As can be seen from Figure 11, after tail vein injection of the cholesterol-galactosamine double-stranded experimental group (compound M-4), the cholesterol-galactosamine double-stranded experimental group (compound M-6), the cholesterol-galactosamine double-stranded experimental group (compound M-8), the cholesterol-galactosamine double-stranded experimental group (compound M-10), and the cholesterol-galactosamine double-stranded experimental group (compound M-12), the OAT3 mRNA levels were all decreased, and the decrease was significant after injection of the compound cholesterol-galactosamine double-stranded experimental group (compound M-6), which was significantly better than the other compounds. This indicates that too much or too little lock nucleic acid in the main chain cannot achieve the ideal inhibitory effect; when the lock nucleic acid accounts for 10% to 70% of the double-stranded chain, it can achieve a significant inhibitory effect.

[0136] The above results show that too much or too little lock nucleic acid in the main chain cannot achieve the ideal inhibitory effect; when the lock nucleic acid accounts for 10% to 70% of the double-stranded chain, it can achieve a significant inhibitory effect.

[0137] 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. Double-stranded nucleic acid-galactosamine-cholesterol compound, characterized in that: consists of a double-stranded nucleic acid, a galactosamine and a cholesterol connected in sequence; wherein the double-stranded nucleic acid is a double-stranded nucleic acid having inhibitory effect on the transcription level and / or expression level of a target gene, and consists of an antisense nucleic acid strand and a complementary strand; the galactosamine is connected to the 5' end of the complementary strand of the double-stranded nucleic acid; the galactosamine is selected from N-acetylgalactosamine; the galactosamine is connected to the cholesterol in a TEG-link or PEG-link manner; the target gene of the double-stranded nucleic acid is alpha-synuclein gene, and the double-stranded nucleic acid is a modified antisense nucleic acid strand and a complementary strand, wherein the modified antisense nucleic acid strand is 5'-A(+)^A(+)^t^g^a^c^a^t^t^c^T(+)^T(+)^A(+)-3', and the modified complementary strand is 5'-T(M)^A(M)^A(M)^GAATGTCA^T(M)^T(M)-3'; or: the target gene of the double-stranded nucleic acid is MALAT1, and the double-stranded nucleic acid is a modified antisense nucleic acid strand and a complementary strand, wherein the modified antisense nucleic acid strand is 5'-G(+)^A(+)^A(+)^c^t^c^a^a^a^a^t^a^t^A(+)^T(+)^T(+)-3', and the modified complementary strand is 5'-A(M)^A(M)^T(M)^ATATTTTGAGT(M)^T(M)^C(M)-3'; or: the target gene of the double-stranded nucleic acid is SRB1, and the double-stranded nucleic acid is a modified antisense nucleic acid strand and a complementary strand, wherein the modified antisense nucleic acid strand is 5'-T(+)^T(+)^c^a^g^t^c^a^t^g^A(+)^C(+)(5M)^T(+)-3', and the modified complementary strand is 5'-A(M)^G(M)^T(M)^CATGACTGA(M)^A(M)-3'; or: the target gene of the double-stranded nucleic acid is OAT3, and the double-stranded nucleic acid is a modified antisense nucleic acid strand and a complementary strand, wherein the modified antisense nucleic acid strand is 5'-C(+)(5M)^G(+)^G(+)^c^c^a^a^a^c^c^t^G(+)^T(+)^C(+)(5M)-3', and the modified complementary strand is 5'-G(M)^A(M)^C(M)^AGGTTTGGC(M)^C(M)^G(M)-3'; wherein, the capital letter represents RNA; the lowercase letter represents DNA; the capital letter (+) represents locked nucleic acid modification; (M) represents methylation modification; (5M) represents 5' methylation modification of cytosine; and ^ represents phosphorothioate bond modification.

2. Use of the double-stranded nucleic acid-galactosamine-cholesterol compound of claim 1 in the preparation of a drug having inhibitory effect on the transcription level and / or expression level of a target gene and protective effect on the kidney.

3. A medicament having an effect of inhibiting the transcription level and / or expression level of a target gene and having a protective effect on the kidney, characterized by: The effective component of the drug is the double-stranded nucleic acid-galactosamine-cholesterol compound of claim 1.

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