Oligonucleotide conjugate, exosome conjugate containing oligonucleotide conjugate and application of oligonucleotide conjugate and exosome conjugate

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an oligonucleotide conjugate, an exosome conjugate containing the oligonucleotide conjugate and application of the oligonucleotide conjugate and the exosome conjugate. The invention provides an oligonucleotide conjugate. The oligonucleotide conjugate comprises R1-L-R2, or an isomer or a pharmaceutically acceptable salt thereof; wherein R1 is a targeting group; r2 is an oligonucleotide; and L is a linker (linker). The invention further provides an exosome conjugate containing the oligonucleotide conjugate. Compared with the prior art, the exosome conjugate has the advantages that the siRNA loading capacity, loading efficiency and loading stability of the exosome are remarkably improved, the treatment effect is superior to that of the conjugate prepared by the existing TEG linker in the aspect of long-term effectiveness, and the application prospect in clinical medicine is wide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an oligonucleotide conjugate, an exosome conjugate containing the same, and applications thereof. Background Art

[0002] Exosomes are extracellular vesicles secreted by cells, with a diameter of 30 - 150 nm. Due to their characteristics such as low immunogenicity, low toxicity, tissue targeting, and the ability to be engineered, they have become potential drug delivery carriers. There have been a large number of related studies on exosome drug loading.

[0003] Currently, all drug - loading studies can classify the drug - loading methods of exosomes into exogenous drug loading (after exosome isolation) and endogenous drug loading (during exosome biogenesis). Among them, exogenous exosome loading can be divided into active loading and passive loading. Passive loading includes the co - incubation method, which is rapid, simple, and has no impact on exosome integrity, but has low efficiency and is more effective for hydrophobic molecules; active loading includes electroporation, extrusion, sonication, freeze - thaw cycling, membrane penetration, and dialysis. These methods also have disadvantages such as affecting exosome quality, low loading efficiency, and easy aggregation of exosomes. For example, Chinese Patent Application No. 202310657477.6 discloses a method for constructing an exosome drug - loading system targeting damaged kidney tissue, in which the prepared exosomes and PINK1 are mixed in equal proportion in an electroporation buffer to prepare an electroporation mixture for preparing an exosome drug - loading system targeting damaged kidney tissue; Chinese Patent Application No. 202310679982.0 discloses a blood tumor - targeted exosome delivery carrier and its application, in which this application uses sonication to load protein polypeptides into exosomes and incubates the qualified exosomes in the recombinant EGFP protein to construct a blood tumor - targeted exosome delivery carrier.

[0004] In summary, current various exosome drug - loading methods all have certain limitations. Therefore, the efficient and stable loading of drugs into exosomes is also one of the bottlenecks for the wide clinical application of exosomes as carriers. Therefore, finding an efficient exosome drug - loading method will greatly expand the clinical application scope of exosomes and thus solve the current clinical application problems. Summary of the Invention

[0005] Chemical conjugation of exosomes is to install various functional molecules, such as polypeptides, antibodies, proteins, lipids, aptamers, small molecules, polymers, through "lipid chemical reactions" or "membrane protein chemical reactions" for controllable and adjustable multifunctional modification on exosomes.

[0006] To achieve the above technical objectives, the present invention hereby provides the following technical solutions:

[0007] In one aspect, the present invention provides an oligonucleotide conjugate, which comprises:

[0008] R1-L-R2 (I), or an isomer or a pharmaceutically acceptable salt thereof;

[0009] wherein R1 is a targeting group;

[0010] R2 is an oligonucleotide;

[0011] L is a linker.

[0012] In some embodiments, the L is selected from:

[0013]

[0014]

[0015] In some embodiments, the targeting group is a lipophilic targeting group.

[0016] In some embodiments, the targeting group includes, but is not limited to, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O-3-(oleoyl) lithocholic acid, O-3-(oleoyl) cholenoic acid, dimethoxytribenzyl or phenoxazine.

[0017] In some preferred embodiments, the targeting group may be cholesterol.

[0018] In some embodiments, the oligonucleotide includes, but is not limited to, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamer, antisense oligonucleotide, exon-skipping oligonucleotide, siRNA oligonucleotide, triple-helix-forming oligonucleotide, ribozyme, or a chemically modified form thereof or a combination thereof.

[0019] In some preferred embodiments, the oligonucleotide may be an siRNA oligonucleotide.

[0020] In some preferred embodiments, the oligonucleotide may be an siRNA oligonucleotide that inhibits the expression of the hPCSK9 gene.

[0021] In another aspect, the present invention provides an exosome conjugate, and the exosome conjugate comprises any one of the foregoing oligonucleotide conjugates and exosomes.

[0022] In some embodiments, the exosome conjugate is represented by formula (II):

[0023] R3 - formula (I); or

[0024] R3-R1-L-R2 (II);

[0025] Wherein R3 is an exosome.

[0026] In some embodiments, the exosomes include, but are not limited to, exosomes secreted from endothelial cells, immune cells, 293 cells, red blood cells, iPSC cells, or MSC cells.

[0027] In some embodiments, the 293 cells include, but are not limited to, 293T cells, 293A cells, 293H cells, 293E cells, 293F cells, or 293F cells.

[0028] In some embodiments, the exosomes are isolated from body fluids.

[0029] In some preferred embodiments, the exosomes can be engineered exosomes.

[0030] In another aspect, the present invention provides a pharmaceutical composition comprising any one of the aforementioned exosome conjugates and a pharmaceutically acceptable carrier.

[0031] In some embodiments, the pharmaceutically acceptable carrier is selected from solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweetening agents, ion exchangers, mold release agents, coating agents, flavor correctors, or antioxidants.

[0032] In yet another aspect, the present invention provides a method for preparing an exosome conjugate, the method comprising the following steps:

[0033] (1) Mix the exosomes with any one of the aforementioned oligonucleotide conjugates uniformly and incubate in the dark at room temperature to obtain a crude exosome conjugate;

[0034] (2) Add the crude exosome conjugate to a size exclusion chromatography column (SEC) for molecular sieve chromatography to obtain a purified exosome conjugate.

[0035] In some embodiments, the incubation time in step (1) can be 30 min.

[0036] In some embodiments, the size exclusion chromatography column (SEC) in step (2) can be ExoSpin TM mini, EX03.

[0037] In another aspect, the present invention provides the use of any of the foregoing exosome conjugates, any of the foregoing pharmaceutical compositions, and the exosome conjugate obtained by the preparation method in the preparation of a medicament for preventing and / or treating hyperlipidemia.

[0038] In some embodiments, R2 in the exosome conjugate is an oligonucleotide that inhibits the expression of the hPCSK9 gene.

[0039] In some embodiments, R2 in the exosome conjugate is an siRNA oligonucleotide that inhibits the expression of the hPCSK9 gene.

[0040] In another aspect, the present invention provides a method for preventing and / or treating hyperlipidemia, the method comprising:

[0041] administering to a subject a therapeutically effective amount of any of the foregoing exosome conjugates;

[0042] wherein, R2 in the exosome conjugate is an oligonucleotide that inhibits the expression of the hPCSK9 gene.

[0043] In some preferred embodiments, R2 in the exosome conjugate is an siRNA oligonucleotide that inhibits the expression of the hPCSK9 gene.

[0044] The exosome conjugate provided by the present invention has at least the following beneficial effects compared with the existing Linker technology and electroporation technology:

[0045] (1) Significantly improved the siRNA loading amount of exosomes, and the copy number of siRNA loaded in each exosome can reach 40,000 - 50,000;

[0046] (2) Significantly improved the siRNA loading efficiency of exosomes, and the loading efficiency of exosomes for siRNA can be maintained above 90%;

[0047] (3) Significantly improved the loading stability, and the loading efficiency and loading amount can be maintained for about 22 hours

[0048] (4) The therapeutic effect is superior to the conjugate prepared by the existing TEG linker in terms of long-term efficacy. [[ID=_{35}]]BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Shows the electron microscopy identification result of exosomes, and the scale bar is 500 nm.

[0050] Figure 2 Shows the particle size identification result of exosomes.

[0051] Figure 3 and Figure 4The positive rates of the nano-flow cytometry-detected siRNA-loaded exosome groups S0 and S3 are shown respectively.

[0052] Figure 5 The change in the exosome loading efficiency within 22 hours is shown.

[0053] Figure 6 The change in the exosome loading amount within 22 hours is shown.

[0054] Figure 7 The change in the mouse serum hPCSK9 levels of different treatment groups within the 1st to 56th days after administration is shown. Detailed implementation manners

[0055] Unless otherwise defined, all technical terms and scientific and technological terms used in the present invention have the same meanings as those commonly used in the field to which the present invention pertains. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form and vice versa.

[0056] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents. For example, reference to "a cell" includes multiple such cells and equivalents known to those skilled in the art and the like.

[0057] The term "about" used herein indicates a range of ±20% of the value thereafter. In some embodiments, the term "about" indicates a range of ±10% of the value thereafter. In some embodiments, the term "about" indicates a range of ±5% of the value thereafter.

[0058] The numerical ranges used herein should be understood to have enumerated all the numbers within that range. For example, the range from 1 to 20 should be understood to include any number, combination of numbers, or sub-range from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0059] The term "comprises" or "comprising" used herein means "including but not limited to". This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0060] As used herein, the terms "optionally", "any one", "any", or "any item" mean that the subsequently described event or circumstance may or may not occur, and this description includes the case where the event or circumstance occurs or does not occur. As used in the present invention, "a" and "an" are used in the present invention to refer to one or more than one grammatical object.

[0061] As used herein, the term "and / or" should be understood to mean any one of the alternatives or any combination of two or more of the alternatives.

[0062] As used herein, the term "conjugate" (sometimes also referred to as conjugate, conjugate, conjugate, conjugate, and sometimes also referred to as conjugate in the literature) corresponds to "conjugate" or "conjugates" in English. A conjugate refers to a new compound formed by covalently linking (coupling) the molecules of two or more compounds through a divalent or polyvalent compound molecule with a linking function. A conjugate can also be formed by directly coupling or condensing two molecules.

[0063] As used herein, the term "siRNA" refers to a molecule containing siRNA as defined herein, and this molecule mediates the targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. Via a process known as RNA interference (RNAi), iRNA guides the sequence-specific degradation of mRNA. Generally, most of the nucleotides of each strand of siRNA are ribonucleotides, but as detailed herein, each or both of the two strands can also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used in this specification, "siRNA" can include ribonucleotides with chemical modifications; siRNA can include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions, or removals of, for example, a functional group or atom in the internucleotide linkage, sugar moiety, or nucleobase. Modifications applicable to the present invention include all types of modifications disclosed herein or known in the art.

[0064] As used herein, the term "coupling" refers to a chemical process in which two or more compound molecules undergo a reaction to form a new chemical bond and a new molecule. In a certain context, "coupling" can be used interchangeably with or substituted for "linking".

[0065] The terms "exosome" and "ectosome" as used herein are used interchangeably and refer to vesicles with a lipid bilayer membrane structure, having a diameter of 30 - 150 nm, produced by cells. Exosomes are secreted by living cells and naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and milk. They are released extracellularly from cells by the fusion of late endosomes in the cellular endocytic system with the cell membrane or directly through the cell membrane, and play an important role in intercellular signal transduction.

[0066] As used herein, the term "pharmaceutically acceptable" means that these structures, materials, compositions, and / or dosage forms are suitable for contact with human and animal tissues within the scope of reasonable medical judgment, commensurate with a reasonable benefit / risk ratio, and without excessive toxicity, irritation, allergic response, or other problems or complications.

[0067] As used herein, the term "pharmaceutically acceptable carrier, excipient, and / or diluent" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining agents, absorption-delaying agents, preservatives. For example, pH regulators include but are not limited to phosphate buffer solutions. Surfactants include but are not limited to cationic, anionic, or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure-maintaining agents include but are not limited to sugars, NaCl, and their analogs. Absorption-delaying agents include but are not limited to monostearates and gelatin. Diluents include but are not limited to water, aqueous buffer solutions (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include but are not limited to various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or their degradation products (such as lactalbumin hydrolysate), etc.

[0068] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.

[0069] As used herein, the terms "therapeutically effective amount", "therapeutically effective dose", and "effective amount" refer to the amount of the exosome conjugate of the present invention that, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, is effective in preventing or ameliorating the symptoms of one or more diseases or conditions or the development of such disease or condition. A therapeutically effective dose also refers to the amount of the exosome conjugate sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates the relevant medical condition or increases the rate of treatment, cure, prevention, or amelioration of such condition. When the active ingredient is administered alone to an individual, the therapeutically effective dose refers only to that ingredient. When administered in combination, the therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0070] As used herein, the terms "prevent" or "preventing", when used in connection with a disease, disorder, or condition that would benefit from a reduction in the expression of the hPCSK9 gene, mean reducing the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition.

[0071] The Linker and siRNA used in the present invention were synthesized by Chengdu Lead Discovery Co., Ltd. and Suzhou GenePharma Co., Ltd.

[0072] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with examples. Those not specifying specific conditions in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer were conventional products that could be purchased commercially. To better illustrate the present invention, numerous specific details are given in the following detailed description. The specific examples described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning: A Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Press), Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc., and Wiley-Interscience.

[0073] Preparation of the conjugate of Formula (I) in Example 1

[0074] In this example, taking Linker4 as an example, the synthesis route of the conjugate of Formula (I) is illustrated as follows:

[0075]

[0076] Step 1: Dissolve Compound 1 (12.9 g, 100.00 mmol) in methanol (100 mL) and water (100 mL), add sodium thiosulfate (19.0 g, 120.00 mmol), reflux at 90 °C for 16 hours, then add an iodine methanol solution (60.00 mmol, 10 mL), and continue to reflux at 90 °C for 16 hours. After the reaction is completed, concentrate under reduced pressure to remove methanol. The aqueous solution is alkalized with sodium hydroxide solution (1 N, 100 mL). The system is extracted with dichloromethane (50 mL × 3), and the organic phase is washed with saturated brine (50 mL), dried over anhydrous sodium sulfate to obtain Compound 2 (7.0 g, 38.89 mmol, yield: 38.9%). Calculated value of MS-ESI [M+H]+181.1, measured value 181.2.

[0077] Step 2: Add cholesterol formyl chloride (5.22 g, 11.64 mmol) to dichloromethane (60 mL), and dropwise add a dichloromethane solution of Compound 2 (23.3 mL, 1 M, 23.30 mmol). After reacting at room temperature for 1 hour, concentrate under reduced pressure to remove dichloromethane, and perform column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain Compound 3 (1.2 g, 2.02 mmol, yield: 17.4%). Calculated value of MS-ESI [M+H]+593.4, measured value 593.7.

[0078] Step 3: Dissolve Compound 3 (1.2 g, 2.02 mmol) in tetrahydrofuran (10 mL), add N,N'-carbonyldiimidazole (490 mg, 3.03 mmol), and react at room temperature for 1 hour. After the reaction is completed, add Compound 4 (1.7 g, 4.04 mmol), and react at room temperature for 16 hours. Concentrate under reduced pressure to remove dichloromethane, and perform column chromatography (petroleum ether:ethyl acetate = 15:1) to obtain Compound 5 (0.4 g, 0.38 mmol, yield: 18.8%).

[0079] 1H NMR (400 MHz, DMSO-d6) δ 7.37 - 7.25 (m, 4H), 7.24 - 7.16 (m, 5H), 7.12 (t, J = 5.6 Hz, 1H), 6.93 - 6.81 (m, 4H), 6.20 (t, J = 5.5 Hz, 1H), 5.32 (d, J = 4.9 Hz, 1H), 4.89 (d, J = 4.2 Hz, 1H), 4.32 (d, J = 4.9 Hz, 2H), 4.06 (dd, J = 8.4, 4.3 Hz, 1H), 3.73 (s, 6H), 3.40 (dd, J = 10.4, 5.2 Hz, 1H), 3.21 (d, J = 9.5 Hz, 1H), 3.10 (d, J = 3.8 Hz, 1H), 3.04 (dq, J = 12.8, 6.2 Hz, 4H), 2.91 (dd, J = 9.1, 3.6 Hz, 1H), 2.66 (q, J = 7.0 Hz, 4H), 2.35 - 2.13 (m, 2H), 2.03 - 1.87 (m, 3H), 1.86 - 1.76 (m, 4H), 1.71 (q, J = 7.1 Hz, 4H), 1.57 - 0.98 (m, 20H), 0.95 (s, 3H), 0.90 (d, J = 6.5 Hz, 4H), 0.84 (dd, J = 6.6, 1.9 Hz, 6H), 0.65 (s, 3H).

[0080] Step 4: Dissolve compound 5 (36 mg, 347 μmol) in dichloromethane (10 mL), add triethylamine (105 mg, 1.04 mmol), succinic anhydride (87 mg, 867 μmol) and 4-dimethylaminopyridine (42 mg, 347 μmol), and react at room temperature for 2 hours. After the reaction, quench with water (20 mL), extract with dichloromethane (10 mL), combine the organic phases, wash 3 times with saturated sodium chloride (20 mL), dry over anhydrous sodium sulfate, and concentrate to obtain the crude product of compound 6. Dissolve the crude product of compound 6 in N,N-dimethylformamide (10 mL), add 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (278 mg, 867 μmol) and N,N-diisopropylethylamine (179 mg, 1.39 mmol), stir at room temperature for 5 minutes, and add controlled pore glass (360 mg, 120 μmol / g). Shake the system at room temperature for 16 hours. After the reaction, filter the system to remove the liquid, and wash the solid 3 times with dichloromethane (10 mL) and N,N-dimethylformamide (10 mL). Obtain the immobilized product 7 (350 mg, 60.8 μmol / g, yield: 6.1%).

[0081] Example 2 siRNA Sequence Synthesis

[0082] The solid support obtained in Example 1 was directly used in traditional solid-phase RNA synthesis, and finally a copolymer of a small molecule and RNA was obtained, as shown in formula (III).

[0083]

[0084] Briefly summarized as follows: The oligonucleotide raw material used in the synthesis is a structural unit of nucleoside phosphoramidite. Through a four-step cycle, one nucleotide is added in each cycle. In step S1 "detritylation", the 5'-DMT protecting group is removed from the nucleoside linked to the first solid support. In step S2 "coupling", the free 5'-OH of the nucleoside linked to the first solid support attacks the phosphorus of the incoming second nucleoside, displacing its diisopropylamino group. In step S3 "oxidation", the unstable phosphite triester is converted into a stable phosphate triester, which allows the start of the next cycle, and the detritylation of the second nucleoside is carried out in step 1. However, before entering the next cycle, in step S4 "capping", the nucleoside linked to the solid support with unreacted 5'-OH is acetylated, thereby preventing the extension of the sequence with deletion mutations. After synthesizing the single-stranded nucleic acid according to the desired sequence through this cycle, all protecting groups are removed. And its complementary strand is synthesized in the same way. Then the two sequences are annealed to form double-stranded siRNA.

[0085] Preparation of Exosome Conjugate in Example 3

[0086] The exosomes in this example were isolated from 293 cells and were independently purified by our company. The exosome identification was entrusted to Xiamen Life Interconnect. The exosome identification results showed that the morphology (cup-shaped, with complete structure) and particle size of the exosomes purified in this example both met the standards ( Figure 1 and Figure 2 ).

[0087] The siRNA sequence and modification used in this example are the same as those of Novartis' marketed drug Inclisiran (see

[0088] (Inclisiran) injection instruction manual,

[0089] https: / / www.accessdata.fda.gov / drugsatfda_docs / nda / 2022 / 214012Orig1s000lbl.pdf).

[0090] · Linking of Exosomes and siRNA

[0091] Add about 1×10 purified exosomes to a 1.5 mL centrifuge tube 6Individuals were added to a Cy5-labeled siRNA mixture in a PBS system solution at a ratio of 1:100,000 and incubated in the dark at room temperature for 30 min.

[0092] · Purification after exosome-siRNA conjugation

[0093] After incubation, the resulting conjugate was purified through an SEC column (ExoSpin TM mini, EX03) to purify exosomes conjugated with siRNA.

[0094] (1) Use a pipette to aspirate and discard the preservative solution from the top of the purification column, immediately add 250 μL of PBS buffer, and let the liquid enter the column matrix under the action of gravity, then discard the outflowing buffer;

[0095] (2) Add 100 μL of exosome resuspension to the purification column, and then place the column on the waste liquid tube;

[0096] (3) Let the liquid enter the column matrix under the action of gravity, and discard the outflowing liquid;

[0097] (4) Place the purification column in a new 1.5 ml centrifuge tube, and add 180 μl of PBS to the top of the purification column;

[0098] (5) Remove the Exo-spinTM purification column from the sample collection tube, and briefly centrifuge the sample collection tube containing the isolated exosomes at 100 × g for 30 s to collect all the liquid at the bottom of the tube.

[0099] Example 4 Detection of siRNA loading efficiency in exosome conjugates

[0100] The calculation method of the exosome loading efficiency (LE) in this example is as follows:

[0101] Loading efficiency (LE) = exosomes carrying siRNA / total exosomes (unit: %).

[0102] The calculation method of the exosome loading capacity (LC) in this example is as follows:

[0103] Loading capacity (LC) = siRNA copy number / exosome particle number (unit: Copy / EV);

[0104] Where Copy is the siRNA copy number and EV is the exosome particle number.

[0105] A1: In this example, the nanoparticle flow cytometry was used to detect the exosome particle number and particle size in Example 2, and the exosome loading efficiency was detected simultaneously

[0106] A2: The fluorescence intensity of the purified exosome sample was detected by reading with an enzyme-linked immunosorbent assay reader

[0107] · Fabricate a fluorescence-concentration standard curve using siRNA standards

[0108] · Detect the fluorescence intensity of the purified exosome sample, substitute it into the standard curve, and calculate the siRNA concentration in the purified exosome sample

[0109] · Divide the calculated concentration by the number of exosome particles measured by nanoscale flow cytometry to obtain the loading capacity data of exosomes

[0110] A3: Repeat steps A1 and A2 at the 0th hour (T = 0), 6th hour (T = 6), and 22nd hour (T = 22) after incubation and purification respectively, measure the experimental data, and calculate the loading capacity of exosomes at different times

[0111] The results showed that compared with the exosome sample S0 without siRNA loading, the positive rate of exosomes S3 after siRNA loading was higher ( Figure 3 and Figure 4 ); within 22 hours, the loading efficiency of siRNA by exosomes could be maintained above 90% ( Figure 5 ); within 22 hours, the copy number of siRNA loaded by each exosome could reach 40,000 - 50,000 ( Figure 6 ).

[0112] While using the conventional electroporation method to load siRNA into exosomes derived from the same 293 cells, the encapsulation efficiency was low, only about 10 - 20% (doi:10.3791 / 58814). The loading efficiency of electroporation was low, and the need to add a large amount of siRNA would cause greater raw material consumption

[0113] Example 5 Application of exosome conjugates in the treatment of hyperlipidemia

[0114] 5.1 Model mouse construction and experimental grouping

[0115] In this example, 6 - 8-week-old B6-hPCSK9-UTR male mice (purchased from Jicui Yakang) were selected, and the mice were fed a Western diet for 5 - 6 weeks to construct a hyperlipidemia model

[0116] The B6-hPCSK9-UTR mice were fed a Western diet for 5 - 6 weeks (the Western diet feed was purchased from ResearchDiets, product number D12079B) to construct a hyperlipidemia model. Blood samples were collected during the modeling period (D - 3) to detect LDL-C (detected by a biochemical analyzer, Hitachi 7020) and hPCSK9 levels (ELISA kit, Invitrogen, product number EH384RB). Using LDL-C as the main indicator and hPCSK9 as the auxiliary, the groups were divided to ensure that the mean value and SEM of each group were basically the same

[0117] On the modeling period (D-4), blood was collected to detect the levels of LDL-C and hPCSK9. Using hPCSK9 as the main index and LDL-C as the auxiliary index, the mice were divided into groups of 6 each, with a total of 9 groups:

[0118] Group 1 (G1) was the blank control group;

[0119] G2 was the group administered with exosome conjugate (TEG linker);

[0120] G3 was the group administered with exosome conjugate (Linker 1);

[0121] G4 was the group administered with exosome conjugate (Linker 2);

[0122] G5 was the group administered with exosome conjugate (Linker 3);

[0123] G6 was the group administered with exosome conjugate (Linker 4);

[0124] G7 was the group administered with exosome conjugate (Linker 5);

[0125] G8 was the group administered with exosome conjugate (Linker 6);

[0126] G9 was the group administered with exosome conjugate (Linker 7).

[0127] The day of grouping was counted as D0. On the day of D0, the mice were administered drugs according to different groups. The volume of drug administration (exosome conjugate) for the mice was: 5 μL / g.

[0128] On D-4, D14, D28, D42, and D56, blood was collected, and plasma was separated to detect the expression level of hPCSK9 in the mice.

[0129] 5.2 Treatment of hyperlipidemia with exosome conjugate

[0130] The preparation of the exosome conjugates (exosome conjugate (Linker 1), exosome conjugate (Linker 2), exosome conjugate (Linker 3), exosome conjugate (Linker 5), exosome conjugate (Linker 6), and exosome conjugate (Linker 7)) in this example was the same as that of the exosome conjugate (Linker 4) in Example 2.

[0131] The control used in this example was the commonly used TEG structure linker, and its structure is as follows:

[0132]

[0133] Among them, R1 is cholesterol and R2 is siRNA.

[0134] The results of detecting the changing levels of mouse hPCSK9 showed that after administration, hPCSK9 in the mice of the TEG and Linker1-7 groups was significantly inhibited. Starting from 28 days after administration, the level of hPCSK9 in the TEG group began to rise, while the level of hPCSK9 in the mice of the Linker1-7 group was significantly lower than that of the TEG group, showing a better long-acting performance ( Figure 7 ).

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An oligonucleotide conjugate, characterized in that, It includes: R1-L-R2 (Ⅰ), or its isomer, pharmaceutically acceptable salt; wherein R1 is a targeting group; R2 is an oligonucleotide; L is a linker.

2. The oligonucleotide conjugate according to claim 1, wherein The L is selected from:

3. The oligonucleotide conjugate according to claim 1 or 2, characterized in that, The targeting group is a lipophilic targeting group, including cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O-3-(oleoyl) lithocholic acid, O-3-(oleoyl) cholenoic acid, dimethoxytribenzyl or phenoxazine.

4. The oligonucleotide conjugate according to claim 3, wherein The targeting group is cholesterol.

5. The oligonucleotide conjugate according to claim 4, wherein The oligonucleotide includes single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, aptamer, antisense oligonucleotide, exon-skipping oligonucleotide, siRNA oligonucleotide, triple helix-forming oligonucleotide, ribozyme, or its chemically modified form or its combination.

6. The oligonucleotide conjugate according to claim 5, wherein The oligonucleotide is an siRNA oligonucleotide.

7. An exosome conjugate, characterized in that, The exosome conjugate contains the oligonucleotide conjugate according to any one of claims 1-6 and exosomes.

8. The exosome conjugate according to claim 7, wherein The exosome conjugate is shown as formula (Ⅱ): R3-R1-L-R2 (Ⅱ); wherein, R3 is an exosome.

9. The exosome conjugate according to claim 8, wherein The exosomes include exosomes secreted from endothelial cells, immune cells, 293 cells, red blood cells, iPSC cells or MSC cells.

10. The exosome conjugate according to claim 8, wherein, The exosomes are isolated from body fluids.

11. The exosome conjugate according to any one of claims 7-9, characterized in that, The R3 is an engineered exosome.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the exosome conjugate according to any one of claims 7-11 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, characterized in that, The pharmaceutically acceptable carrier is selected from solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, mold release agents, coating agents, flavor correctors or antioxidants.

14. A method for preparing an exosome conjugate according to any one of claims 7-11, characterized in that, The preparation method includes the following steps: (1) After uniformly mixing exosomes and the oligonucleotide conjugate, incubate in the dark at room temperature to obtain a crude exosome conjugate; (2) Add the crude exosome conjugate to a size exclusion chromatography column (SEC) for molecular sieve chromatography to obtain a purified exosome conjugate.

15. Use of the exosome conjugate according to any one of claims 7-11, the pharmaceutical composition according to any one of claims 12-13, or the exosome conjugate obtained by the preparation method according to claim 14 in the preparation of a drug for preventing and / or treating hyperlipidemia.

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