WO2024131403A1 - 基于核酸适体的脾脏及其亚细胞的mRNA靶向递送*** - Google Patents

基于核酸适体的脾脏及其亚细胞的mRNA靶向递送*** Download PDF

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WO2024131403A1
WO2024131403A1 PCT/CN2023/132420 CN2023132420W WO2024131403A1 WO 2024131403 A1 WO2024131403 A1 WO 2024131403A1 CN 2023132420 W CN2023132420 W CN 2023132420W WO 2024131403 A1 WO2024131403 A1 WO 2024131403A1
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nucleic acid
cells
preparation
short
lipid material
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French (fr)
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谭蔚泓
唐鹤鸣
谢斯滔
甘绍举
左超
刘湘圣
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中国科学院基础医学与肿瘤研究所(筹)
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Publication of WO2024131403A1 publication Critical patent/WO2024131403A1/zh

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  • the invention belongs to the technical field of biomedicine and nanomedicine, and in particular relates to an mRNA targeted delivery system for the spleen and its subcells based on nucleic acid aptamers.
  • LNPs lipid nanoparticles
  • targeting molecules such as antibodies
  • the existing targeting molecule modified LNP delivery system can improve the specific mRNA expression efficiency of immune cells, but it is difficult to achieve highly specific mRNA expression in the spleen.
  • selective organ targeting SORT-LNP
  • SORT-LNP selective organ targeting
  • ssDNA negatively charged linear DNA
  • Apt aptamer
  • the spleen As the main immune organ of the body, the spleen is rich in various immune cells and their different subtypes, such as T cells, dendritic cells, macrophages, and B cells. Different immune cells have completely different immune functions. For this reason, the specific mRNA delivery of different immune cells in the spleen is of great significance for immunotherapy.
  • the above passive targeting system has achieved highly specific spleen-targeted mRNA expression, the high cost and large amount of screening work required for system development are still its defects that cannot be ignored.
  • the screening of different subcellular targeting systems in the spleen requires further in vitro sorting of different immune cells and quantitative measurement of mRNA expression in different cells. The test is more difficult, requires more work, is more expensive, and takes longer. For this reason, current passive targeting systems are generally limited to screening at the organ level, making it difficult to further conduct large-scale screening of subcellular tissues.
  • the active targeting system modifies molecules with targeting effects on the surface of LNP or other nanomaterials, and achieves targeting through the specific binding of targeting molecules to targets.
  • liver-targeted delivery systems based on GalNAc for example, tumor-targeted delivery systems based on glycosaminoglycans, and T-cell targeted delivery systems based on anti-CD5 antibodies, etc.
  • the targeting mechanism of active targeting systems is clearer.
  • the above-mentioned targeted delivery systems show certain effects, they still face a series of problems, the most important of which is that the efficiency of binding of targeting molecules to targets is limited and the targets of targeting molecules are not only expressed in a single tissue.
  • the active targeting delivery system is mainly achieved by modifying antibodies on the surface proteins of immune cell membranes on LNP, including CD5 antibodies, CD4 antibodies, CD3 antibodies, and CD8 antibodies.
  • the active targeting system improves the targeting effect of immune cells, it is difficult to significantly improve the spleen targeting effect due to reasons such as off-target and the target not only being expressed in the spleen.
  • mRNA can be delivered to the spleen by adding negatively charged lipids to the standard four-component (cationic lipid, auxiliary lipid, cholesterol and PEG lipid) lipid nanoparticle formulation.
  • the linear DNA (ssDNA) fragments with a charge not only have the properties of traditional nucleic acids, but also have the ability to specifically bind to targets due to their special three-dimensional structure. Therefore, the modification of nucleic acid aptamers on the surface of LNP is expected to increase the negative charge of the LNP system, thereby achieving spleen-targeted delivery of mRNA.
  • nucleic acid aptamers Since nucleic acid aptamers also have target-specific binding ability, nucleic acid aptamers modified on the surface of LNP are expected to further target immune cells in the spleen.
  • PEG lipid material As the outermost lipid material of LNP, PEG lipid material is often used to modify antibodies and other targeting molecules. Therefore, in this patent, an mRNA delivery system of nucleic acid aptamer-modified LNP is constructed for PEG lipid material.
  • nucleic acid properties of nucleic acid aptamers are beneficial to the change of the pKa of the modified LNP, thereby achieving spleen-specific mRNA delivery; using the characteristics of its three-dimensional structure that can specifically bind to the target, LNP is further targeted to the corresponding immune subcells in the spleen.
  • the nucleic acid aptamer-modified LNP has the advantages of both passive targeting system and active targeting system, thereby achieving mRNA-specific delivery to the spleen organ and its different immune subcells at the same time (Figure 1).
  • the present invention provides a targeted preparation, composition and application thereof for delivering nucleic acid drugs.
  • the present invention provides a preparation for delivering nucleic acid drugs, characterized in that the surface of the preparation is modified with short-chain nucleic acids and loaded with nucleic acid drugs selected from siRNA, aiRNA, miRNA, dsRNA, aRNA, lncRNA, mRNA, and DNA.
  • the nucleic acid drug is mRNA.
  • the mRNA encodes pathogen antigens, tumor-associated antigens, tumor-specific antigens, chimeric antigen receptors CARs, cytokines and other proteins or antibodies, and any combination thereof.
  • the mRNA encodes a chimeric antigen CAR, preferably an anti-CD33 CAR.
  • the preparation is nanoliposome LNP, liposome, polymer nanoparticle, inorganic nanoparticle or a combination of the above preparations.
  • the preparation is nanoliposome LNP.
  • the nanoliposome LNP includes a lipid material and a nucleic acid drug encapsulated by the lipid material.
  • the lipid material includes cationic lipid material, auxiliary lipid material, structural lipid, and PEG lipid material.
  • the cationic lipid material is selected from SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA; preferably, the cationic lipid material is SM102; the structural formula of SM-102 is as follows:
  • the auxiliary lipid material is selected from DSPC, DOTAP, DOPE, DOPC, DOPG or DOPS; preferably, the auxiliary lipid material is DSPC.
  • the structured lipid is cholesterol or a cholesterol derivative; preferably, the structured lipid is cholesterol.
  • the PEG lipid material is selected from DMG-PEG2000, DSPE-PEG2000, DTDA-PEG2000, and TPGS; preferably, the PEG lipid material is selected from DMG-PEG2000 and DSPE-PEG2000.
  • the usage ratio of cationic lipids, auxiliary lipids, structural lipids, and PEG lipids is 50%:10%:38.5%:1.5%; preferably, the usage ratio of SM102, DSPC, cholesterol, and PEG lipids is 50%:10%:38.5%:1.5%.
  • the short-chain nucleic acid is a random sequence nucleic acid or a nucleic acid aptamer; preferably, the nucleic acid aptamer is an RNA nucleic acid aptamer or a DNA nucleic acid aptamer; more preferably, the DNA nucleic acid aptamer is a ssDNA nucleic acid aptamer.
  • the preparation is a targeted preparation.
  • the preparation can target the spleen and various immune cells therein.
  • the target of the nucleic acid aptamer or preparation is selected from: immune cells, antigen presenting cells, T cells, resident T cells, B cells, natural killer (NK) cells, cancer cells, cells associated with a disease or condition, tissues associated with a disease or condition, brain tissue, central nervous system tissue, lung tissue, apical surface tissue, epithelial cells, endothelial cells, liver tissue, intestinal tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, macrophages, spleen tissue, muscle tissue, joint tissue, tumor cells, diseased tissue, lymph node tissue, lymphatic circulation and the group consisting of any combination thereof.
  • immune cells antigen presenting cells
  • T cells resident T cells
  • B cells natural killer cells
  • cancer cells cells associated with a disease or condition
  • tissues associated with a disease or condition brain tissue, central nervous system tissue, lung tissue, apical surface tissue, epithelial cells, endothelial cells, liver tissue, intestinal tissue, colon tissue,
  • the target of the nucleic acid aptamer or preparation is selected from spleen tissue and its subcells; preferably, the spleen subcells are spleen CD8 + T cells, CD4 + T cells, macrophages, and B cells.
  • sequence of the ssDNA nucleic acid aptamer is selected from SEQ ID NO.1-7; preferably, the sequence of the ssDNA nucleic acid aptamer is selected from SEQ ID NO.5-7.
  • the short-chain nucleic acid is CD4 Apt shown in SEQ ID NO.5, it targets splenic CD4 + T cells.
  • the short-chain nucleic acid when the short-chain nucleic acid is CD8 Apt shown in SEQ ID NO.6, it targets splenic CD8 + T cells.
  • the short-chain nucleic acid when the short-chain nucleic acid is DC-SIGN shown in SEQ ID NO.7, it targets splenic macrophages.
  • the surface of the preparation is modified with short-chain nucleic acids
  • the modification methods used include but are not limited to physical connection modification methods and chemical connection modification methods.
  • the above-mentioned physical connection modification methods include: electrostatic adsorption connection (for example, electrostatic adsorption between a positively charged preparation (for example, a lipid material of a positively charged preparation, exemplified by a positively charged PEG lipid material, a cationic lipid material, an auxiliary lipid material, a structural lipid) and a negatively charged short-chain nucleic acid), hydrophobic insertion connection (for example, a short-chain nucleic acid is inserted into the preparation through hydrophobic insertion, exemplified by a lipid material inserted into the preparation), etc.
  • electrostatic adsorption connection for example, electrostatic adsorption between a positively charged preparation (for example, a lipid material of a positively charged preparation, exemplified by a positively charged PEG lipid material, a cationic
  • the above-mentioned chemical connection modification method is modification through chemical connection reaction (for example, the short-chain nucleic acid and the preparation are modified through chemical connection reaction); further preferably, the chemical connection reaction includes but is not limited to: azide-alkynyl coupling reaction without catalyst, azide-alkynyl coupling reaction with catalyst, tetraazine-trans-cyclooctene connection reaction, maleimide-thiol coupling reaction, etc.
  • the above-mentioned chemical ligation modification method requires modification of the short-chain nucleic acid
  • the modification methods include but are not limited to: azidation modification, alkynyl modification (such as diphenylcyclooctyne modification), phosphorylation modification, methylation modification, amination modification, thiol modification, biotin modification, fluorescent marker modification, radioisotope modification, etc.
  • the above-mentioned chemical ligation modification method requires modification of the lipid material (such as PEG lipid material, cationic lipid material, auxiliary lipid material, structural lipid), and the modification methods include but are not limited to: azide modification, diphenylcyclooctyne modification, phosphorylation modification, methylation modification, amino modification, thiol modification, biotin modification, fluorescent marker modification, radioisotope modification, etc.
  • the lipid material such as PEG lipid material, cationic lipid material, auxiliary lipid material, structural lipid
  • the modification methods include but are not limited to: azide modification, diphenylcyclooctyne modification, phosphorylation modification, methylation modification, amino modification, thiol modification, biotin modification, fluorescent marker modification, radioisotope modification, etc.
  • the short-chain nucleic acid is modified by modifying the short-chain nucleic acid on the nanoliposome, preferably on the lipid material of the nanoliposome, such as PEG lipid material, cationic lipid material, auxiliary lipid material, and structural lipid.
  • the present invention also provides a method for preparing the above-mentioned preparation for delivering nucleic acid drugs, wherein the preparation method is selected from any one of the following methods:
  • Method 1) preparing a preparation using a preparation component modified with a short-chain nucleic acid to obtain the preparation modified with a short-chain nucleic acid;
  • Method 2 using a preparation component with a reactive group to prepare a preparation to obtain a preparation with a reactive group on the surface; then, under appropriate conditions, a short-chain nucleic acid with another reactive group is reacted with the preparation with a reactive group on the surface to obtain a preparation modified with a short-chain nucleic acid;
  • Method 3 mixing the short-chain nucleic acid with the preparation to obtain the preparation modified with the short-chain nucleic acid; preferably, the short-chain nucleic acid and the preparation are mixed by physical connection to obtain the preparation modified with the short-chain nucleic acid; the physical connection is for example electrostatic adsorption connection and hydrophobic membrane insertion connection.
  • the preparation component modified with short-chain nucleic acids is a lipid material modified with short-chain nucleic acids, such as a PEG lipid material modified with short-chain nucleic acids, a cationic lipid material modified with short-chain nucleic acids, an auxiliary lipid material modified with short-chain nucleic acids, and a structural lipid modified with short-chain nucleic acids.
  • a lipid material modified with short-chain nucleic acids such as a PEG lipid material modified with short-chain nucleic acids, a cationic lipid material modified with short-chain nucleic acids, an auxiliary lipid material modified with short-chain nucleic acids, and a structural lipid modified with short-chain nucleic acids.
  • the preparation modified with short-chain nucleic acid is prepared using method 2).
  • the reactive group and another reactive group are groups that undergo click chemistry reaction.
  • the reaction between the reactive group and another reactive group is a chemical connection reaction, including but not limited to: an azide-alkynyl coupling reaction without a catalyst, an azide-alkynyl coupling reaction with a catalyst, a tetrazine-trans-cyclooctene connection reaction, a maleimide-thiol coupling reaction, etc.
  • the reactive group and/or another reactive group is selected from: an azide group, an alkynyl group (e.g., a diphenylcyclooctyne group), a phosphorylation group, a methylation group, an amino group, a sulfhydrylation group, a biotin group, a fluorescent marker group, a radioisotope group, etc.
  • an alkynyl group e.g., a diphenylcyclooctyne group
  • a phosphorylation group e.g., a diphenylcyclooctyne group
  • a phosphorylation group e.g., a methylation group
  • an amino group e.g., a sulfhydrylation group
  • biotin group e.g., a fluorescent marker group
  • a radioisotope group e.g., a radioisotope group
  • the formulation is nanoliposome LNP.
  • the formulation component with reactive groups is a lipid material with reactive groups, such as PEG lipid material with reactive groups, cationic lipid material with reactive groups, auxiliary lipid material with reactive groups, and structural lipid with reactive groups.
  • the above preparation method comprises the following steps:
  • PEG lipids, auxiliary lipids, structural lipids, cationic lipids and nucleic acid drugs are used to synthesize nanoliposomes, which are then subjected to click chemistry reactions with short-chain nucleic acids to form short-chain nucleic acid-modified nanoliposomes.
  • the above preparation method comprises the following steps:
  • Nanoliposomes are synthesized by azide-modified PEG lipids, auxiliary lipids, structural lipids, cationic lipids and nucleic acid drugs, and then the nanoliposomes undergo click chemistry reaction with short-chain nucleic acids modified with azacyclooctyne DBCO to form short-chain nucleic acid-modified nanoliposomes.
  • the present invention also provides a composition, which comprises the above-mentioned preparation for delivering nucleic acid drugs.
  • the composition is a vaccine composition or a pharmaceutical composition; preferably, the vaccine is selected from RNA vaccine and DNA vaccine; more preferably, the vaccine is an mRNA vaccine.
  • the above composition further includes other therapeutic or preventive agents; preferably, the other therapeutic or preventive agents are selected from any at least one of nucleic acid drugs, small molecule drugs, protein drugs, and pharmaceutically active molecules.
  • the present invention also provides the use of the above preparations and compositions in the preparation of vaccines.
  • the vaccine is selected from RNA vaccine and DNA vaccine; preferably, the vaccine is an mRNA vaccine.
  • the preparation, composition, and vaccine can target the spleen and various types of immune cells therein.
  • the preparation, composition, or vaccine delivers the nucleic acid drug to a target (eg, a target cell and/or a target organ).
  • a target eg, a target cell and/or a target organ.
  • the target is selected from: immune cells, antigen presenting cells, T cells, resident T cells, B cells, natural killer (NK) cells, cancer cells, cells associated with a disease or condition, tissues associated with a disease or condition,
  • the group consisting of brain tissue, central nervous system tissue, lung tissue, apical surface tissue, epithelial cells, endothelial cells, liver tissue, intestinal tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, macrophages, spleen tissue, muscle tissue, joint tissue, tumor cells, diseased tissue, lymph node tissue, lymphatic circulation and any combination thereof.
  • the target is selected from spleen tissue and its subcells; preferably, the spleen subcells are spleen CD8 + T cells, CD4 + T cells, macrophages, and B cells.
  • the preparation, composition, vaccine is used to prevent and/or treat a disease or condition.
  • the present invention also provides the use of the above-mentioned preparations, compositions and vaccines in the preparation of drugs for preventing and/or treating diseases or symptoms.
  • the present invention also provides a method for delivering a nucleic acid drug to a subject in need thereof, the method comprising administering a therapeutically effective amount of the above-mentioned preparation, composition, or vaccine to the subject.
  • the present invention also provides a method for preventing and/or treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the above-mentioned preparation, composition, or vaccine to the subject.
  • the disease or condition is selected from at least one of tumors, autoimmune diseases, metabolic diseases, viral infections, bacterial infections, fungal infections, parasitic infections, influenza infections, cancer, arthritis, heart disease, cardiovascular diseases, neurological disorders or diseases, genetic diseases, fetal diseases, genetic diseases affecting fetal development, or any combination thereof.
  • the present invention also provides a short-chain nucleic acid modified PEG lipid material, the structure of which is short-chain nucleic acid-A-PEG;
  • A is any chemical linking group or linking arm; preferably, A is a linking structure generated by click chemistry.
  • A is a connection structure generated by an azide-alkyne coupling reaction, a tetrazine-trans-cyclooctene connection reaction, a maleimide-thiol coupling reaction, or the like.
  • the PEG lipid material is selected from DMG-PEG2000, DSPE-PEG2000, DTDA-PEG2000, and TPGS; preferably, the PEG lipid material is selected from DMG-PEG2000 and DSPE-PEG2000.
  • the short-chain nucleic acid is a random sequence nucleic acid or a nucleic acid aptamer; preferably, the nucleic acid aptamer is an RNA nucleic acid aptamer or a DNA nucleic acid aptamer; more preferably, the DNA nucleic acid aptamer is a ssDNA nucleic acid aptamer.
  • sequence of the ssDNA nucleic acid aptamer is selected from SEQ ID NO.1-7; preferably, the sequence of the ssDNA nucleic acid aptamer is selected from SEQ ID NO.5-7.
  • the short-chain nucleic acid-modified PEG lipid is generated by click chemistry of an azide-modified PEG lipid and an azacyclooctyne DBCO-modified short-chain nucleic acid.
  • the present invention also provides a method for preparing the above-mentioned short-chain nucleic acid-modified PEG lipid material, the preparation method comprising:
  • the PEG lipid material with a reactive group reacts with a short-chain nucleic acid with another reactive group to obtain a PEG lipid material modified with a short-chain nucleic acid.
  • the reaction that occurs is a chemical connection reaction, including but not limited to: azide-alkynyl coupling reaction without catalyst, azide-alkynyl coupling reaction with catalyst, tetrazine-trans-cyclooctene connection reaction, maleimide-thiol coupling reaction, etc.
  • the reactive group and/or another reactive group is selected from: an azide group, an alkynyl group (e.g., a diphenylcyclooctyne group), a phosphorylation group, a methylation group, an amino group, a thiol group, a biotin group, a fluorescent marker group, a radioisotope group, etc.
  • the present invention utilizes the advantages of nucleic acid aptamers that have both nucleic acid properties and target binding ability properties, and uses nucleic acid aptamers to modify LNP, thereby integrating the advantages of traditional passive targeted mRNA delivery systems and traditional active targeted mRNA delivery systems, and constructing a nucleic acid delivery system that not only has spleen targeted delivery capabilities but also has immune subcellular targeted delivery capabilities within the spleen.
  • the mRNA expression efficiency of B cells can be increased to 15.4%
  • the expression efficiency of CD3T cells can be increased to 7.2%
  • the expression efficiency of CD8T cells can be increased to 23.2%
  • the expression efficiency of CD4T cells can be increased to 20.4%
  • the expression efficiency of macrophages can be increased to 15.7%.
  • the first mRNA organ and immune cell targeted delivery system based on nucleic acid aptamers.
  • the present invention has broad application prospects in mRNA-based immune cell therapy. Its CD8 apt-LNP system can efficiently express CAR genes in spleen CD8 + T cells in vivo and construct CD33 CAR-T. Among them, the CAR gene expression in spleen CD8 T cells is as high as 26.5%, which is better than the level of similar systems in the literature.
  • Figure 1 Schematic diagram of Apt-LNP achieving targeted mRNA delivery to the spleen and its immune subcells.
  • Figure 3 Schematic diagram of the ssDNA-LNP spleen targeting system and in vivo imaging of the organ;
  • Figure a is an in vivo imaging of Balb/c mice,
  • Figure b is the in vivo imaging of C57BL/6J mice.
  • FIG. 5 (a) Targeting effect of CD8 aptamer on Jurkat (T lymphocyte line) and splenic T cells, Lib is a scrambled sequence; (b) Targeting effect of DC-SIGN aptamer on DC 2.4 (dendritic cell line) and RAW267.4 (macrophage line), Lib is a scrambled sequence; (c) EGFP mRNA expression efficiency of LNP without aptamer modification, Lib sequence modification and DC-SIGN Apt modification in DC2.4 cells; (d) EGFP mRNA expression efficiency of LNP with control aptamer sequence modification (sequence 8 in Table 1), Lib sequence modification and DC-SIGN Apt modification in Jurkat cells.
  • Figure 6 (a) In vivo imaging effect of Apt-LNP; (b) Luciferase activity of T cells, B cells, and macrophages in the spleen; (c) The percentage of different cell brightness in the spleen to the total brightness of B cells, macrophages, and T cells; (d) EGFP positivity rate of B cells, macrophages, and T cells in different systems; (e) EGFP positivity rate of different subtypes of T cells in the CD8 Apt-LNP system; (f) The proportion of EFGP-positive cells in different systems.
  • Figure 8 (a) Schematic diagram of the mouse anti-CD33 CAR sequence; (b) CD33 CAR mRNA expression in vitro; (c) CD33 CAR mRNA expression on different T cells in the spleen in vivo.
  • An alcohol phase solution comprising DSPC lipid material, SM102 lipid material, cholesterol, and PEG lipid material.
  • the PEG lipid material of different LNPs can be one of DMG-PEG2000, DSPE-PEG2000, and N3-DSPE-PEG2000.
  • the molar ratios of SM102, DSPC, cholesterol, and PEG lipid material are 50%, 10%, 38.5%, and 1.5%, respectively.
  • N/P nitrogen-phosphorus ratio
  • LNPs were synthesized using N3-DSPE-PEG2000 as the PEG lipid material according to the above method.
  • ssDNA modified with azacyclooctyne (DBCO) was added and incubated at room temperature for 0.5-2 h. Free ssDNA was then removed using a 100K ultrafiltration tube.
  • the ssDNA was selected from sequences 1-7 in Table 1.
  • the particle size and potential were tested by nanoparticle size and potential analyzer.
  • the pKa was tested by TNS method as follows: mRNA preparation (60 ⁇ M total lipid) and TNS probe (2 ⁇ M) were incubated with a series of buffers containing 10 mM Hepes, 10 mM MES (4-morpholineethanesulfonic acid), 10 mM ammonium acetate and 130 mM NaCl (pH range 2.5 to 11) for 5 minutes.
  • Example 1 The LNP synthesized in Example 1 was resuspended in 50-300 ⁇ L PBS.
  • mice were selected and LNP solution was injected into the tail vein.
  • the injection volume for each mouse corresponded to 2-20 ⁇ g mRNA.
  • mice were shaved 6 hours after tail vein injection and D-luciferin sodium salt (15 mg/ml) was injected intraperitoneally, 100 ⁇ l per mouse, and the bioluminescence brightness was tested using a small animal live imaging instrument.
  • D-luciferin sodium salt 15 mg/ml
  • the heart, liver, spleen, lung, kidney, and lymph node of the mice were then dissected and the bioluminescence brightness of the organs was tested using a small animal live imaging instrument.
  • ssDNA-LNP The mRNA expressing luciferase (Luci mRNA) is used as a model, which catalyzes luciferin to produce bioluminescence and is often used Detection of in vivo mRNA expression efficiency.
  • ssDNA-LNP was injected into the body by intravenous injection, and it was found that compared with other commercial LNP systems (including four-component systems with DMG-PEG, DSPE-PEG and N3-DSPE-PEG as PEG lipid materials), ssDNA-LNP has a significant spleen-specific mRNA expression effect (Figure 3a). And this phenomenon is not limited to Balb/c mice, but also applies to C57BL/6J mice ( Figure 3b). In addition, after changing the ssDNA sequence, it was found that the LNP systems modified by the selected 7 ssDNA sequences (sequences 1-7 in Table 1) can achieve spleen-specific targeting ( Figures 4a-c).
  • BB solution preparation 0.1 mg/ml yeast tRNA and 1 mg/ml bovine serum albumin dissolved in WB solution.
  • EGFP mRNA as the model mRNA, lib-LNP, control Apt-LNP, CD8 Apt-LNP and DC-SIGN Apt-LNP were synthesized according to the route in Example 1 and diluted with PBS.
  • the amount of mRNA per 300,000 cells was 2-4 ⁇ g; for DC2.4 cells, the amount of mRNA per 300,000 cells was 0.5-2 ⁇ g.
  • lib in lib-LNP is sequence 4 in Table 1.
  • mice The spleen of mice was dissected and extracted 6 h after tail vein injection, ground in PBS solution, and passed through a 70-mesh sieve to remove tissue chunks. The spleen was then centrifuged and resuspended in 1-6 ml PBS.
  • the resuspended spleen cells were divided into three equal parts, and the corresponding immune cells were negatively sorted according to the CD3 T cell magnetic bead sorting kit, B cell magnetic bead sorting kit, and pDC cell magnetic bead sorting kit.
  • lib-LNP lib-LNP, CD4 Apt-LNP, CD8 Apt-LNP and DC-SIGN Apt-LNP were synthesized according to the route in Example 1. Immunocompetent mice were selected and LNP solution was injected into the tail vein. The injection amount of each mouse corresponded to 2-30 ⁇ g mRNA. Lib is sequence 4 in Table 1.
  • mice The spleen of mice was dissected and extracted 24 hours after tail vein injection, ground in PBS solution, and passed through a 70-mesh sieve to remove tissue chunks. The spleen was then centrifuged and resuspended in 1-6 ml PBS.
  • the spleen and its immune subcellular mRNA delivery system are expected to be used in the fields of in vivo immune cell reprogramming, gene therapy, and mRNA vaccines.
  • CD8 Apt-LNP which can significantly increase the mRNA expression of CD8T cells in the spleen
  • CAR-T is an antigen chimeric receptor T cell, which can recognize tumor cells and activate T cells through CAR to achieve tumor killing.
  • CAR-T treatment is mostly obtained by extracting T cells from the patient's blood in vitro, transforming them, and then re-implanting them into the body.
  • CAR-T cell therapy Due to cumbersome operations, low cell acquisition rates, and immunogenicity of allogeneic cells, the price of CAR-T cell therapy has remained high. In recent years, the construction of living CAR-T has made it possible to significantly reduce the cost of CAR-T treatment. CAR gene therapy based on mRNA is expected to reduce the impact of toxic side effects and cytokine syndrome.
  • Example 5 In vitro and in vivo expression of anti-CD33 CAR mRNA
  • CD8 Apt-LNP was synthesized according to the route in Example 1. 300,000 Jurkat cells were seeded in 500 ⁇ L complete medium in a 24-well plate and cultured overnight.
  • the suspended Jurkat cells were directly collected, centrifuged and resuspended in 200 ⁇ L PBS.
  • 3.6 ⁇ L FITC fluorescently labeled CD33 protein was added to each 200 ⁇ L PBS and incubated at room temperature for 15 minutes. 1-2 ml PBS was added to terminate the reaction.
  • the cells were then collected by centrifugation and resuspended in 300 microliters of PBS. The obtained cells were detected by flow cytometry for different FITC green fluorescence positivity rates to characterize the CAR expression efficiency of the cells.
  • mice were selected and LNP solution was injected into the tail vein.
  • the injection volume for each mouse corresponded to 20 ⁇ g mRNA.
  • the spleen was dissected and extracted, ground in PBS solution, and passed through a 70-mesh sieve to remove tissue chunks. The spleen was then centrifuged and resuspended in 1-6 ml PBS.
  • the sorted immune cells were resuspended in PBS. Resuspend in 200 ⁇ L PBS. Add 3.6 ⁇ L FITC fluorescently labeled CD33 protein to every 200 ⁇ L PBS and incubate at room temperature for 15 minutes. Add 1-2 ml PBS to terminate the reaction. Then collect the cells by centrifugation and resuspend in 300 ⁇ L PBS. The obtained cells were detected by flow cytometry for different FITC green fluorescence positivity rates to characterize the CAR expression efficiency of the cells.

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Abstract

本发明涉及一种核酸适体的脾脏及其亚细胞的mRNA靶向递送***，利用核酸适体同时拥有核酸特性以及与靶标结合能力特性的优势，用核酸适配体修饰LNP，从而集成了传统被动靶向mRNA递送体系和传统主动靶向mRNA递送体系的优势，构建出不仅有脾脏靶向递送能力还有脾脏内免疫亚细胞靶向递送能力的核酸递送体系。

Description

基于核酸适体的脾脏及其亚细胞的mRNA靶向递送***
本申请要求享有2022年12月19日向中国国家知识产权局提交的，专利申请号为202211635004.8，发明名称为“基于核酸适体的脾脏及其亚细胞的mRNA靶向递送***”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物医药和纳米医学技术领域，尤其是涉及一种基于核酸适体的脾脏及其亚细胞的mRNA靶向递送***。
背景技术
免疫治疗通过调控机体自身免疫***治疗疾病，是肿瘤、自身免疫疾病及传染病的有效治疗方法。mRNA被报道可作为疫苗、蛋白质替代疗法和基因治疗工具，调节机体免疫***，实现难治性疾病的预防和免疫治疗。例如，新冠肺炎的mRNA疫苗已被FDA批准紧急使用；基于mRNA的在体CAR-T被报道可用于血液肿瘤及心脏病治疗；基于mRNA的肿瘤免疫治疗已被广泛研究并正进行临床试验。这些mRNA疗法的应用在很大程度上依赖于开发一种可将mRNA安全、有效地靶向递送到免疫器官(脾脏或***)及其亚细胞的体系。
作为最先进的mRNA递送载体，脂质纳米颗粒(LNPs)可在外周血中有效维持mRNA稳定性，实现溶酶体逃逸，具有优异的递送和mRNA表达效率。然而，LNPs在肝脏的特异性富集严重限制了其在免疫治疗当中的应用。为了基于LNPs实现免疫器官的mRNA递送，已有研究引入靶向分子(如抗体)修饰LNP的体系，通过靶向分子靶向细胞膜表面蛋白，从而来提升免疫细胞的mRNA靶向递送效果[1]。然而，由于靶向分子与靶标结合能力有限且很多靶标并不仅在脾脏组织中表达，因此现有靶向分子修饰LNP递送体系虽可提升免疫细胞特异性mRNA表达效率，但难以实现高特异性的脾脏mRNA表达。最近，选择性器官靶向(SORT-LNP)引入带负电的SORT分子，实现mRNA的脾脏特异性递送[2]。此外，有研究致力于改变可电离脂材，实现脾脏特异性mRNA递送[3，4]。尽管如此，上述sort分子及可电离脂材的脾脏靶向机理并不明确，虽然实现了脾脏高特异性mRNA表达，却难以进一步高效调控脾脏内不同免疫亚细胞的mRNA表达效果。综上所述，同时实现脾脏器官及其不同免疫亚细胞的mRNA特异性递送调控仍然具有挑战。
在前期研究中，SORT-LNP提示带负电LNP更有利于实现脾脏特异性mRNA表达。而核酸适体(Aptamer，Apt)作为带有负电荷的线性DNA(ssDNA)片段，不仅拥有传统核酸性质，还因特殊三维结构具有与靶标特异性结合的能力。
脾脏作为机体主要的免疫器官，其富含T细胞、树突状细胞、巨噬细胞、B细胞等多种免疫细胞及其不同亚型。不同免疫细胞拥有截然不同的免疫功能。正因如此，脾脏内不同免疫细胞的特异性mRNA递送对免疫治疗具有重要意义。
目前用于脾脏及其免疫细胞靶向mRNA递送体系主要有两大类，分别是被动靶向体系和主动靶向体系。具体来说，被动靶向体系可通过改变脂材结构、改变脂材配比或加入新脂材改变LNP的理化性质，从而影响LNP的pKa及在外周血中黏附的蛋白，最终实现脾脏靶向。例如引入第五种脂材SORT分子构建的LNP可靶向脾脏；基于两性离子阳离子脂材构建的LNP可靶向脾脏；用PS脂材替换标准辅助脂质DOPE构建的LNP可靶向脾脏等等。尽管上述被动靶向体系已经实现了高特异性的脾脏靶向mRNA表达，但体系开发所必需的高成本及大量筛选工作仍是其难以忽略的缺陷。而相较于脾脏靶向体系的筛选，脾脏内不同亚细胞靶向体系的筛选需要进一步体外分选不同的免疫细胞，并对不同细胞内mRNA表达进行定量测量，测试难度更大、所需工作量更大、成本更高、耗时更长。正因如此，目前被动靶向体系普遍局限于器官水平的筛选，难以进一步对亚细胞进行大量筛选。
另一方面，主动靶向体系在LNP或其它纳米材料表面修饰具有靶向作用的分子，通过靶向分子与靶标的特异性结合实现靶向。例如，基于GalNAc的肝脏靶向递送体系、基于糖胺聚糖的肿瘤靶向递送体系、以及基于anti-CD5抗体的T细胞靶向递送体系等等。相比于被动靶向体系，主动靶向体系的靶向机制更加明确。然而，尽管上述靶向递送体系展现出一定的效果，但仍面临一系列问题，其中最主要的问题在于靶向分子与靶标结合效率有限、以及靶向分子的靶标不仅在单一组织中表达。在脾脏靶向递送体系中，主动靶向的递送体系主要通过在LNP上修饰免疫细胞膜表面蛋白的抗体来实现，包括CD5抗体、CD4抗体、CD3抗体以及CD8抗体等。可令人遗憾的是，主动靶向体系虽提升了免疫细胞的靶向效果，但由于脱靶以及靶标不仅在脾脏表达等原因，使得脾脏靶向效果难以得到显著提升。
综上所述，尽管已有的主动靶向及被动靶向体系分别具有利于免疫细胞靶向及脾脏靶向的优势，但没有一个体系能完美整合主动靶向及被动靶向体系的优势。因此，现有技术难以在实现高效脾脏靶向mRNA表达的同时有效调控其不同免疫亚细胞内的mRNA表达。
最近研究表明，在标准的四组分(阳离子脂材、辅助脂材、胆固醇和PEG脂材)脂质纳米颗粒制剂中添加带负电的脂质后，可以将mRNA递送至脾脏。而核酸适体作为带有负电 荷的线性DNA(ssDNA)片段，不仅拥有传统核酸性质，还因特殊三维结构具有与靶标特异性结合的能力。因此，核酸适体修饰在LNP表面有望能增加LNP体系负电荷，从而实现mRNA的脾脏靶向递送。由于核酸适体还具有靶标特异性结合能力，因此LNP表面修修饰的核酸适体有望在脾脏进一步靶向免疫细胞。而PEG脂材作为LNP的最外层脂材，其常被用于抗体及其它靶向分子的修饰。因此，本专利中，针对PEG脂材构建核酸适体修饰LNP的mRNA递送体系。利于核酸适体的核酸特性使其修饰后LNP的pKa改变，从而实现脾脏特异性mRNA递送；利用其3维结构可特异性结合靶标的特性，在脾脏中使LNP进一步靶向相应的免疫亚细胞。最终使核酸适体修饰的LNP得以同时拥有被动靶向***和主动靶向***的优势，从而同时实现脾脏器官及其不同免疫亚细胞的mRNA特异性递送(图1)。
(文献1：Science，2022，375，91-96；文献2：NatNanotechnol，2020，4，15，313-320；文献3：J.Am.Chem.Soc.2021，143，50，21321-21330；文献4：NatMater，2021，20，5，701-710)。
发明内容
为了改善上述技术问题，本发明提供一种递送核酸药物的靶向制剂、组合物及其应用。
本发明提供一种递送核酸药物的制剂，其特征在于，制剂的表面修饰有短链核酸，并装载有任选自siRNA、aiRNA、miRNA、dsRNA、aRNA、lncRNA、mRNA、DNA的核酸药物。
在一些技术方案中，核酸药物为mRNA。
在一些技术方案中，mRNA编码病原体抗原、肿瘤相关抗原、肿瘤特异性抗原、嵌合抗原受体CAR、细胞因子及其它蛋白或抗体、及其任意组合。
在一些技术方案中，mRNA编码嵌合抗原CAR，优选为编码anti-CD33 CAR。
在一些技术方案中，制剂为纳米脂质体LNP、脂质体、聚合物纳米颗粒、无机纳米颗粒或上述制剂的组合。
在一些技术方案中，制剂为纳米脂质体LNP。
在一些技术方案中，纳米脂质体LNP包括脂材和由脂材包载的核酸药物。
在一些技术方案中，脂材包括阳离子脂材、辅助脂材、结构脂质、PEG脂材。
在一些技术方案中，所述阳离子脂材选自SM-102、ALC-0315、ALC-0519、Dlin-MC3-DMA、DODMA、C12-200、DlinDMA；优选地，所述阳离子脂材为SM102；SM-102的结构式如下所示：
在一些技术方案中，所述辅助脂材选自DSPC、DOTAP、DOPE、DOPC、DOPG或DOPS；优选地，所述辅助脂材为DSPC。
在一些技术方案中，所述结构脂质为胆固醇或胆固醇衍生物；优选地，所述结构脂质为胆固醇。
在一些技术方案中，PEG脂材选自DMG-PEG2000、DSPE-PEG2000、DTDA-PEG2000、TPGS；优选地，PEG脂材选自DMG-PEG2000、DSPE-PEG2000。
在一些技术方案中，阳离子脂材、辅助脂材、结构脂质、PEG脂材的用量配比为50％∶10％∶38.5％∶1.5％；优选地，SM102、DSPC、胆固醇、PEG脂材的用量配比为50％∶10％∶38.5％∶1.5％。
在一些技术方案中，短链核酸为随机序列核酸或者核酸适体；优选地，核酸适体为RNA核酸适体或DNA核酸适体；更优选地，DNA核酸适体为ssDNA核酸适体。
在一些技术方案中，所述制剂为靶向制剂。优选地，所述制剂可靶向脾脏及其内各类免疫细胞。
在一些技术方案中，核酸适体或制剂的靶标选自：免疫细胞、抗原呈递细胞、T细胞、驻留T细胞、B细胞、自然杀伤(NK)细胞、癌细胞、与疾病或病症相关的细胞、与疾病或病症相关的组织、脑组织、中枢神经***组织、肺组织、顶端表面组织、上皮细胞、内皮细胞、肝组织、肠组织、结肠组织、小肠组织、大肠组织、粪便、骨髓、巨噬细胞、脾脏组织、肌肉组织、关节组织、肿瘤细胞、病变组织、***组织、淋巴循环和它们的任意组合组成的组。
在一些技术方案中，核酸适体或制剂的靶标选自脾脏组织及其亚细胞；优选地，所述脾脏亚细胞为脾脏CD8+T细胞、CD4+T细胞、巨噬细胞、B细胞。
在一些技术方案中，ssDNA核酸适体的序列选自SEQ ID NO.1-7；优选地，ssDNA核酸适体序列选自SEQ ID NO.5-7。
在一些技术方案中，短链核酸为SEQ ID NO.5所示的CD4 Apt时，靶向脾脏CD4+T细胞。
在一些技术方案中，短链核酸为SEQ ID NO.6所示的CD8 Apt时，靶向脾脏CD8+T细胞。
在一些技术方案中，短链核酸为SEQ ID NO.7所示的DC-SIGN时，靶向脾脏巨噬细胞。
在一些技术方案中，制剂的表面修饰有短链核酸所用的修饰方法包括但不限于物理连接修饰方法及化学连接修饰方法。优选地，上述物理连接修饰方法包括：静电吸附连接(例如带正电的制剂(例如带正电的制剂的脂材，示例性为带正电的PEG脂材、阳离子脂材、辅助脂材、结构脂质)与带负电的短链核酸之间的静电吸附)、疏水插膜连接(例如短链核酸通过疏水***制剂，示例性为***制剂的脂材)等。优选地，上述化学连接修饰方法为通过化学连接反应进行修饰(例如短链核酸与制剂通过化学连接反应进行修饰)；进一步优选地，化学连接反应包括但不限于：无催化剂的叠氮-炔基偶合反应、有催化剂的叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等。
在一些技术方案中，上述化学连接修饰方法需要对短链核酸进行修饰，修饰方法包括但不限于：叠氮化修饰、炔基修饰(例如二苯基环辛炔修饰)、磷酸化修饰、甲基化修饰、氨基化修饰、巯基化修饰、生物素修饰、荧光标记物修饰、放射性同位素修饰等。
在一些技术方案中，上述化学连接修饰方法需要对脂材(例如PEG脂材、阳离子脂材、辅助脂材、结构脂质)进行修饰，修饰方法包括但不限于：叠氮修饰、二苯基环辛炔修饰、磷酸化修饰、甲基化修饰、氨基化修饰、巯基化修饰、生物素修饰、荧光标记物修饰、放射性同位素修饰等。
在一些技术方案中，所述短链核酸修饰为将短链核酸修饰在纳米脂质体上，优选地修饰在纳米脂质体的脂材上，例如PEG脂材上、阳离子脂材上、辅助脂材上、结构脂质上。
本发明还提供上述递送核酸药物的制剂的制备方法，所述制备方法选自以下任一方法：
方法1)：使用修饰有短链核酸的制剂成分制备制剂，得到所述修饰有短链核酸的制剂；
方法2)：使用带有反应性基团的制剂成分制备制剂，得到表面具有反应性基团的制剂；然后，在合适条件下，使得带有另一反应性基团的短链核酸与表面具有反应性基团的制剂反应，得到修饰有短链核酸的制剂；
方法3)：将短链核酸与制剂混合，得到修饰有短链核酸的制剂；优选地，短链核酸与制剂通过物理连接作用混合，得到修饰有短链核酸的制剂；物理连接作用例如静电吸附连接、疏水插膜连接。
在一些技术方案中，方法1)中，修饰有短链核酸的制剂成分为修饰有短链核酸的脂材，例如修饰有短链核酸的PEG脂材、修饰有短链核酸的阳离子脂材、修饰有短链核酸的辅助脂材、修饰有短链核酸的结构脂质。
优选的，使用方法2)制备所述修饰有短链核酸的制剂。
优选的，方法2)中，所述反应性基团与另一反应基团为发生点击化学反应的基团。
在一些技术方案中，方法2)中，所述反应性基团与另一反应基团发生的反应为化学连接反应，包括但不限于：无催化剂的叠氮-炔基偶合反应、有催化剂的叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等。
在一些技术方案中，方法2)中，反应性基团和/或另一反应基团选自：叠氮基团、炔基基团(例如二苯基环辛炔基团)、磷酸化基团、甲基化基团、氨基化基团、巯基化基团、生物素基团、荧光标记物基团、放射性同位素基团等。
在一些技术方案中，制剂是纳米脂质体LNP。
在一些技术方案中，带有反应性基团的制剂成分为带有反应性基团的脂材，例如带有反应性基团的PEG脂材、带有反应性基团的阳离子脂材、带有反应性基团的辅助脂材、带有反应性基团的结构脂质。
在一些技术方案中，上述制备方法包括以下步骤：
PEG脂材、辅助脂材、结构脂质、阳离子脂材和核酸药物合成纳米脂质体，随后纳米脂质体与短链核酸发生点击化学反应，形成短链核酸修饰的纳米脂质体。
在一些技术方案中，上述制备方法包括以下步骤：
叠氮修饰的PEG脂材、辅助脂材、结构脂质、阳离子脂材和核酸药物合成纳米脂质体，随后纳米脂质体与氮杂环辛炔DBCO修饰的短链核酸发生点击化学反应，形成短链核酸修饰的纳米脂质体。
本发明还提供一种组合物，所述组合物包括上述递送核酸药物的制剂。
在一些技术方案中，所述组合物是疫苗组合物或药物组合物；优选地，疫苗选自RNA疫苗、DNA疫苗；更优选地，疫苗是mRNA疫苗。
在一些技术方案中，上述组合物中进一步包括其他治疗或预防剂；优选地，其他治疗或预防剂选自核酸药物、小分子药物、蛋白质药物、药物活性分子中的任意至少一种。本发明还提供上述制剂、组合物在制备疫苗中的应用。
在一些技术方案中，疫苗选自RNA疫苗、DNA疫苗；优选地，疫苗是mRNA疫苗。
在一些技术方案中，所述制剂、组合物、疫苗可靶向脾脏及其内各类免疫细胞。
在一些技术方案中，所述制剂、组合物、疫苗将核酸药物递送至靶标(例如靶细胞和/或靶器官)。
在一些技术方案中，所述靶标选自：免疫细胞、抗原呈递细胞、T细胞、驻留T细胞、B细胞、自然杀伤(NK)细胞、癌细胞、与疾病或病症相关的细胞、与疾病或病症相关的组织、 脑组织、中枢神经***组织、肺组织、顶端表面组织、上皮细胞、内皮细胞、肝组织、肠组织、结肠组织、小肠组织、大肠组织、粪便、骨髓、巨噬细胞、脾脏组织、肌肉组织、关节组织、肿瘤细胞、病变组织、***组织、淋巴循环和它们的任意组合组成的组。
在一些技术方案中，所述靶标选自脾脏组织及其亚细胞；优选地，所述脾脏亚细胞为脾脏CD8+T细胞、CD4+T细胞、巨噬细胞、B细胞。
在一些技术方案中，所述制剂、组合物、疫苗用于预防和/或治疗疾病或病症。
本发明还提供上述制剂、组合物、疫苗在制备预防和/或治疗疾病或病症的药物中的应用。
本发明还提供一种向有需要的受试者递送核酸药物的方法，所述方法包含向所述受试者施用治疗有效量的上述制剂、组合物、疫苗。
本发明还提供一种在有需要的受试者中预防和/或治疗疾病或病症的方法，包括向受试者施用治疗有效量的上述制剂、组合物、疫苗。
在一些技术方案中，所述疾病或病症选自肿瘤、自身免疫病、代谢类疾病、病毒感染、细菌感染、真菌感染、寄生虫感染、流感感染、癌症、关节炎、心脏病、心血管疾病、神经病症或疾病、遗传病、胎儿疾病、影响胎儿发育的遗传病或它们的任意组合中选择的至少一种。
本发明还提供一种短链核酸修饰的PEG脂材，结构为短链核酸-A-PEG；
其中，A为任意的化学连接基团或连接臂；优选地，A为点击化学生成的连接结构。
在一些技术方案中，A为叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等所生成的连接结构。
在一些技术方案中，PEG脂材选自DMG-PEG2000、DSPE-PEG2000、DTDA-PEG2000、TPGS；优选地，PEG脂材选自DMG-PEG2000、DSPE-PEG2000。
在一些技术方案中，短链核酸为随机序列核酸或者核酸适体；优选地，核酸适体为RNA核酸适体或DNA核酸适体；更优选地，DNA核酸适体为ssDNA核酸适体。
在一些技术方案中，ssDNA核酸适体的序列选自SEQ ID NO.1-7；优选地，ssDNA核酸适体序列选自SEQ ID NO.5-7。
在一些技术方案中，短链核酸修饰的PEG脂材由叠氮修饰的PEG脂材与氮杂环辛炔DBCO修饰的短链核酸通过点击化学生成。
本发明还提供上述短链核酸修饰的PEG脂材的制备方法，所述制备方法包括：
带有反应性基团的PEG脂材，与带有另一反应性基团的短链核酸发生反应，得到短链核酸修饰的PEG脂材。
在一些技术方案中，发生的反应为化学连接反应，包括但不限于：无催化剂的叠氮-炔基偶合反应、有催化剂的叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等。
在一些技术方案中，反应性基团和/或另一反应基团选自：叠氮基团、炔基基团(例如二苯基环辛炔基团)、磷酸化基团、甲基化基团、氨基化基团、巯基化基团、生物素基团、荧光标记物基团、放射性同位素基团等。
本发明利用核酸适体同时拥有核酸特性以及与靶标结合能力特性的优势，用核酸适配体修饰LNP，从而集成了传统被动靶向mRNA递送体系和传统主动靶向mRNA递送体系的优势，构建出不仅有脾脏靶向递送能力还有脾脏内免疫亚细胞靶向递送能力的核酸递送体系。
本发明的优点：
1.不仅可实现高特异性的脾脏靶向mRNA表达，还可调控脾脏内不同免疫亚细胞的mRNA表达。优选方案中可将B细胞的mRNA表达效率提升至15.4％，CD3T细胞表达效率提升至7.2％，CD8T细胞的23.2％，CD4T细胞的20.4％，巨噬细胞的表达效率提升至15.7％。
2.巧妙集成了主动靶向和被动靶向核酸递送体系。
3.首个基于核酸适体的mRNA器官及免疫细胞靶向递送体系。
4.本发明在基于mRNA的免疫细胞治疗中有广泛的应用前景。其CD8 apt-LNP体系可高效在体内使脾脏CD8+T细胞表达CAR基因，构建CD33 CAR-T。其中脾脏CD8T细胞中CAR基因表达高达26.5％，效果优于文献中相近体系水平。
本发明的技术方案有益效果在于：
1.通过ssDNA修饰LNP实现了高特异性的脾脏靶向mRNA递送和表达。
2.通过核酸适体修饰LNP实现了脾脏内不同免疫亚细胞的特异性mRNA递送和表达，包括B淋巴细胞、多种亚型的T淋巴细胞以及巨噬细胞。
通过本发明中CD8 Apt-LNP体系可实现体内CD33 CAR在CD8+T细胞上的特异性高表达，从而在体内构建CAR-T。
附图说明
图1.Apt-LNP实现脾脏及其免疫亚细胞靶向mRNA递送示意图。
图2.不同PEG脂材形成的LNP粒径及zeta电位表征。
图3.ssDNA-LNP脾脏靶向体系示意图及器官活体成像图；图a为Balb/c小鼠活体成像图， 图b为C57BL/6J小鼠活体成像图。
图4.(a)不同碱基序列ssDNA修饰LNP的脾脏靶向mRNA递送效果；(b)不同器官平均辐射亮度图；(c)不同器官mRNA表达生物发光分布图；(d)不同LNP的pKa值。
图5.(a)CD8核酸适体对Jurkat(T淋巴细胞系)和脾脏T细胞的靶向效果，Lib为乱序序列；(b)DC-SIGN核酸适体对DC 2.4(树突状细胞系)和RAW267.4(巨噬细胞系)的靶向效果，Lib为乱序序列；(c)DC2.4细胞中无核酸适体修饰、Lib序列修饰以及DC-SIGN Apt修饰LNP的EGFP mRNA表达效率；(d)Jurkat细胞中对照核酸适体序列修饰(表1中序列8)、Lib序列修饰以及DC-SIGN Apt修饰LNP的EGFP mRNA表达效率。
图6.(a)Apt-LNP的活体成像效果；(b)脾脏内T细胞、B细胞、巨噬细胞的荧光素酶活性；(c)脾脏内不同细胞亮度所占B细胞、巨噬细胞和T细胞总亮度的百分比；(d)B细胞、巨噬细胞和T细胞在不同体系中的EGFP阳性率；(e)CD8 Apt-LNP体系中，不同亚型T细胞的EGFP阳性率；(f)不同体系中EFGP阳性细胞占比。
图7.CD4 Apt-LNP体系中，不同亚型T细胞的EGFP阳性率；图7中，从左至右依次为Blank、CD3+T、CD4+T。
图8.(a)鼠源抗(anti)CD33 CAR序列示意图；(b)体外CD33 CAR mRNA表达；(c)体内脾脏不同T细胞的CD33 CAR mRNA表达。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1：LNP的合成
(1)配置醇相溶液，包含DSPC脂材、SM102脂材、胆固醇、PEG脂材。其中不同的LNP的PEG脂材可以分别是DMG-PEG2000、DSPE-PEG2000、N3-DSPE-PEG2000中的一种。其中的脂材配比SM102、DSPC、胆固醇、PEG脂材的摩尔比例分别为50％、10％、38.5％以及1.5％。
(2)配置包含mRNA的水相溶液(以20mM柠檬酸缓冲液为溶剂)。
(3)固定氮磷比(N/P)为6，将醇相与水相以1∶2的比例混合制备LNP，并调节pH，最后通过超滤离心管纯化浓缩。
(4)ssDNA-LNP合成：按上述方法以N3-DSPE-PEG2000为PEG脂材合成LNP。加入氮杂环辛炔(DBCO)修饰的ssDNA，室温孵育0.5-2h。随后用100K超滤管去除游离的ssDNA。其中，ssDNA选自表1中的序列1-7。
通过纳米粒度及电位分析仪测试粒径及电位。通过TNS法测试pKa，具体方法如下：mRNA制剂(60μM总脂质)和TNS探针(2μM)与一系列含有10mM Hepes、10mM MES(4-吗啉乙磺酸)、10mM乙酸铵和130mM NaCl(pH范围为2.5至11)的缓冲液孵育5分钟。每个孔(黑色底部96孔板)的平均荧光强度由Tecan板阅读器测量，λEx＝321nm，λEm＝445nm，数据归一化为pH 2.5。
通过叠氮修饰的PEG脂材(N3-DSPE-PEG2000)、DMG-PEG2000、DSPE-PEG2000，分别与辅助脂材(例如DSPC)、胆固醇、商用阳离子脂材(例如SM102)、mRNA制备成N3-LNP、DMG-LNP、DSPE-LNP(分别标记为表2-3中的DSPE-N3、DMG、DSPE)，随后N3-LNP与DBCO修饰的ssDNA/核酸适体发生点击化学反应，形成ssDNA-LNP(分别标记为表2-3中的序列1-7)。合成的ssDNA-LNP与其它商用四组分合成的LNP的粒径见图2及表2，其中zeta电位显著偏负(图2及表2)，相应的pKa如下表3。其中，图2中所用的ssDNA为序列6。
表1.ssDNA序列列表
表2.不同LNP的粒径和zeta电位
表3.不同ssDNA-LNP的pKa值
实施例2：涉及LNP体系的器官水平mRNA表达分析
(1)实施例1中合成的LNP用50-300μL PBS重悬。
(2)选用免疫健全小鼠，尾静脉注射LNP溶液，每只鼠的注射量对应2-20μg mRNA。
(3)对于luci mRNA，小鼠尾静脉注射6h后褪毛，腹腔注射D-荧光素钠盐(15mg/m1)，100微升每只鼠，之后通过小动物活体成像仪器测试生物发光亮度；随后对小鼠的心脏、肝脏、脾脏、肺脏、肾脏、***解剖，通过小动物活体成像仪器测试器官生物发光亮度。
以表达荧光素酶的mRNA(Luci mRNA)为模型，其会催化荧光素产生生物发光，常被用于 检测活体mRNA表达效率。通过静脉注射将ssDNA-LNP打入体内，发现相比于其它商用LNP体系(包括以DMG-PEG、DSPE-PEG以及N3-DSPE-PEG为PEG脂材的四组分体系)，ssDNA-LNP具有显著的脾脏特异性mRNA表达效果(图3a)。并且这一现象并不局限于Balb/c小鼠，在C57BL/6J小鼠上同样适用(图3b)。此外，在改变ssDNA序列后发现，所选择的7种ssDNA序列(表1中序列1-7)修饰的LNP体系均可实现脾脏特异性靶向(图4a-c)。
值得注意的是，对比于肝靶向LNP体系及肝加脾LNP体系普遍大于5的解离常数(pKa)，脾脏特异性靶向ssDNA-LNP体系的pKa值普遍小于5。证明了ssDNA修饰使得LNP的pKa值下降，从而赋予ssDNA-LNP体系脾脏靶向功能(图4d，表3)。
实施例3：涉及细胞水平核酸适体靶向和Apt-LNP特异性表达表征
1.核酸适体靶向性测试：
(1)WB溶液配置：4.5g/L葡萄糖、5mM MgCl2溶在PBS中；
BB溶液配置：0.1mg/ml酵母tRNA、1mg/ml牛血清白蛋白溶在WB溶液中。
(2)Jurkat、DC2.4、RAW267.4、脾脏T细胞分别50-100万重悬于250μL BB溶液，每孔加250nM带FITC荧光的核酸适体或Library序列，孵育1-1.5小时。
(3)离心溶液中游离的核酸适体，用WB溶液反复洗涤两次。
(4)以不加核酸的空白对照以及加Library序列的组别为对照，通过流式细胞仪测试FITC荧光，表征核酸适体的靶向性。
2.Apt-LNP特异性表达：
(1)在24孔板中种Jurkat、DC2.4细胞，30万细胞种在500μL完全培养基中，过夜培养。
(2)以EGFP mRNA为模型mRNA，按实施例1中路线分别合成的lib-LNP、对照Apt-LNP、CD8 Apt-LNP以及DC-SIGN Apt-LNP并用PBS稀释。对于Jurkat细胞，每30万细胞加样量对应2-4微克mRNA；对于DC2.4细胞，每30万细胞加样量对应0.5-2微克mRNA。其中，lib-LNP中的lib为表1中的序列4。
(3)LNP与细胞孵育过夜。
(4)悬浮的Jurkat细胞直接收集，离心后用300μL PBS重悬；贴壁的DC2.4细胞用胰蛋白酶消化收集，离心后用300μL PBS重悬。上述收集的样品通过流式细胞仪测试EGFP绿色荧光强度表征mRNA表达效率。以Lib-LNP为对照，如若APT-LNP对应细胞的EGFP强度提升则具有特异性表达效果。
选择了被报道分别具有CD8及DC-SIGN(分别在CD8T细胞、DC细胞及巨噬细胞表面高表达)靶向性的核酸适体(图5a-b)，并将其修饰在LNP表面构建Apt-LNP。以表达绿色荧光蛋 白的mRNA(EGFP mRNA)为模型mRNA，在体外实验中，CD8核酸适体(CD8 Apt)及DC-SIGN核酸适体(DC-SIGN Apt)修饰的LNP针对CD8高表达的Jurkat细胞及DC-SIGN高表达的DC 2.4细胞分别具有特异性mRNA递送效果(图5c-d)。
实施例4：mRNA在脾脏亚细胞内表达分析
1.对于Luci mRNA
(1)按实施例1中路线分别合成的lib-LNP、CD8 Apt-LNP以及DC-SIGN Apt-LNP。选用免疫健全小鼠，尾静脉注射LNP溶液，每只鼠的注射量对应20μg mRNA。其中，Lib为表1中序列4。
(2)小鼠尾静脉注射6h解剖提取脾脏器官，在PBS溶液中研磨，过70目筛网去除组织块。随后离心，加1-6ml PBS重悬。
(3)重悬的脾脏悬浮细胞等分为3等分，分别按CD3T细胞磁珠分选试剂盒、B细胞磁珠分选试剂盒、pDC细胞磁珠分选试剂盒中操作阴性分选对应的免疫细胞。
(4)分选出的免疫细胞重悬于D-荧光素钠盐溶液中，通过小动物活体检测相应孔板中生物发光强度。
(5)用该免疫细胞亮度强度除以三种细胞亮度总和，得到该细胞所占辐射亮度百分比。
2.对于EGFP mRNA
(1)按实施例1中路线分别合成的lib-LNP、CD4 Apt-LNP、CD8 Apt-LNP以及DC-SIGN Apt-LNP。选用免疫健全小鼠，尾静脉注射LNP溶液，每只鼠的注射量对应2-30μg mRNA。其中，Lib为表1中序列4。
(2)小鼠尾静脉注射24h解剖提取脾脏器官，在PBS溶液中研磨，过70目筛网去除组织块。随后离心，加1-6ml PBS重悬。
(3)取一定量脾脏细胞，分别按CD3T细胞磁珠分选试剂盒、CD8T细胞磁珠分选试剂盒、CD4T细胞磁珠分选试剂盒、B细胞磁珠分选试剂盒、巨噬细胞细胞磁珠分选试剂盒中操作分选对应的免疫细胞。
(4)分选出的免疫细胞用PBS重悬。通过流式细胞仪检测不同免疫细胞的EGFP绿色荧光蛋白阳性率，从而表征脾脏内不同免疫细胞的mRNA表达效率。
以Luci mRNA为模型mRNA，碱基乱序序列(Library，Lib)为对照，分别构建CD8 Apt-LNP以及DC-SIGN Apt-LNP体系。上述三种ssDNA-LNP被证明皆可有效实现脾脏特异性mRNA表 达(图6a)。在脾脏亚细胞分析中，Library序列修饰的LNP被证明Luci-mRNA表达的荧光主要分布在B细胞中；DC-SIGN Apt-LNP被证明Luci-mRNA表达的荧光主要分布在巨噬细胞中；CD8 Apt-LNP被证明可将T细胞内荧光分布从0％提升至10％左右(图6b-c)。
进一步，以EGFP mRNA为模型，在免疫亚细胞转染效率测定中证明，Library-LNP中EGFP mRNA在脾脏CD3+T、CD8+T、B、巨噬细胞中表达效率分别为3.47％、3.01％、16.53％和4.53％，其中B细胞的表达效率最高；而当序列变换成CD8 Apt后，EGFP-mRNA在脾脏CD3+T、CD8+T、B、巨噬细胞中表达效率分别为6.47％、22.8％、5.03％和7.96％，在CD8T细胞表达效率最高；进一步的，当序列变换成DC-SIGN Apt后，巨噬细胞中表达效率可进一步提升至15.7％。进一步的，CD4 Apt-LNP使mRNA在脾脏CD4+T中有特异性表达效率(图7)。上述结果证明了Aptamer-LNP可在靶向脾脏递送mRNA的基础上，进一步实现脾脏内免疫亚细胞的靶向mRNA递送及表达(图6d-f)。
上述脾脏及其免疫亚细胞mRNA递送体系有望应用于体内免疫细胞重编程、基因治疗、以及mRNA疫苗等领域中。以上述可显著提升脾脏内CD8T细胞mRNA表达的CD8 Apt-LNP为例，其在体内CAR-T、TCR-T治疗中都具有广阔的应用前景。其中，CAR-T是抗原嵌合受体T细胞，可通过CAR识别肿瘤细胞并激活T细胞，实现肿瘤杀伤。目前CAR-T治疗大多通过患者血液体外提取T细胞，进行改造，随后重新植入体内的方式获取。由于操作繁琐，细胞获取率低，异体细胞具有免疫原性等因素，使得CAR-T细胞治疗价格一直居高不下。近年来，活体CAR-T的构建为大幅降低CAR-T治疗费用带来了可能。而基于mRNA的CAR基因治疗则有望降低毒副作用和细胞因子综合症的影响。
实施例5：anti CD33 CAR mRNA的体外和体内表达
1.体外anti CD33 CAR mRNA
(1)按实施例1中路线合成CD8 Apt-LNP。在24孔板中将30万Jurkat细胞种在500μL完全培养基中，过夜培养。
(2)每30万细胞加样量对应1-6微克anti CD33 CAR mRNA。
(3)CD8 Apt-LNP与细胞孵育过夜。
(4)悬浮的Jurkat细胞直接收集，离心后用200μL PBS重悬。每200μL PBS加3.6μL FITC荧光标记的CD33蛋白，室温孵育15分钟。加1-2ml PBS终止反应。随后离心收集细胞，用300微升PBS重悬。所得细胞通过流式细胞仪检测不同FITC绿色荧光阳性率，从而表征细胞的CAR表达效率。
2.体内anti CD33 CAR mRNA
(1)按实施例1中路线合成CD8 Apt-LNP。
(2)选用Balb/c小鼠，尾静脉注射LNP溶液，每只鼠的注射量对应20μg mRNA。
(3)小鼠尾静脉注射6h后，解剖提取脾脏器官，在PBS溶液中研磨，过70目筛网去除组织块。随后离心，加1-6ml PBS重悬。
(4)取一定量脾脏细胞，分别按CD3T细胞磁珠分选试剂盒、CD8T细胞磁珠分选试剂盒中操作阴性分选对应的免疫细胞。
(5)分选出的免疫细胞用PBS重悬。200μL PBS重悬。每200μL PBS加3.6μL FITC荧光标记的CD33蛋白，室温孵育15分钟。加1-2ml PBS终止反应。随后离心收集细胞，用300微升PBS重悬。所得细胞通过流式细胞仪检测不同FITC绿色荧光阳性率，从而表征细胞的CAR表达效率。
以发病率高、恶性程度高的急性髓系淋巴细胞白血病为模型，以髓系淋巴白血病细胞普遍高表达的CD33为靶标，构建anti CD33-mRNA(图8a)，并用此mRNA构建CD8 Apt-LNP。首先，在体外通过CD8 Apt-LNP向T细胞系细胞Jurkat细胞内递送CD33 CAR mRNA，证明了CD8 Apt-LNP可使T细胞系细胞高表达CAR，表达效率高达91％(图8b)。随后，静脉注射包载CD33 CAR mRNA的CD8 Apt-LNP，6小时后对脾脏T细胞分析，结果显示CD8 Apt-LNP可使脾脏CD3+T的CAR表达效率14.9％，CD8+T的CAR表达效率26.5％，显示出了脾脏内细胞毒性T细胞(CD8T)特异性mRNA表达效果(图8c)。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种递送核酸药物的制剂，其特征在于，所述制剂的表面修饰有短链核酸，并装载有任选自siRNA、aiRNA、miRNA、dsRNA、aRNA、lncRNA、mRNA、DNA的核酸药物；
    优选地，核酸药物为mRNA；
    优选地，mRNA编码病原体抗原、肿瘤相关抗原、肿瘤特异性抗原、嵌合抗原受体CAR、细胞因子及其它蛋白或抗体、及其任意组合；
    优选地，mRNA编码嵌合抗原CAR，优选为编码anti-CD33 CAR；
    优选地，剂为纳米脂质体LNP、脂质体、聚合物纳米颗粒、无机纳米颗粒或上述制剂的组合。
  2. 根据权利要求1所述的制剂，其特征在于，所述制剂为纳米脂质体LNP；
    优选地，纳米脂质体LNP包括脂材和由脂材包载的核酸药物；
    优选地，脂材包括阳离子脂材、辅助脂材、结构脂质、PEG脂材；
    优选地，所述阳离子脂材选自SM-102、ALC-0315、ALC-0519、Dlin-MC3-DMA、DODMA、C12-200、DlinDMA；
    优选地，所述辅助脂材选自DSPC、DOTAP、DOPE、DOPC、DOPG或DOPS；
    优选地，所述结构脂质为胆固醇或胆固醇衍生物；
    优选地，PEG脂材选自DMG-PEG2000、DSPE-PEG2000、DTDA-PEG2000、TPGS；
    优选地，阳离子脂材、辅助脂材、结构脂质、PEG脂材的用量配比为50％∶10％∶38.5％∶1.5％；
    优选地，短链核酸为随机序列核酸或者核酸适体；优选地，核酸适体为RNA核酸适体或DNA核酸适体；更优选地，DNA核酸适体为ssDNA核酸适体。
  3. 根据权利要求1或2所述的制剂，其特征在于，所述制剂为靶向制剂；
    优选地，所述制剂可靶向脾脏及其内各类免疫细胞；
    优选地，核酸适体或制剂的靶标选自：免疫细胞、抗原呈递细胞、T细胞、驻留T细胞、B细胞、自然杀伤(NK)细胞、癌细胞、与疾病或病症相关的细胞、与疾病或病症相关的组织、脑组织、中枢神经***组织、肺组织、顶端表面组织、上皮细胞、内皮细胞、肝组织、肠组织、结肠组织、小肠组织、大肠组织、粪便、骨髓、巨噬细胞、脾脏组织、肌肉组织、关节组织、肿瘤细胞、病变组织、***组织、淋巴循环和它们的任意组合组成的组；
    优选地，核酸适体或制剂的靶标选自脾脏组织及其亚细胞；优选地，所述脾脏亚细胞为脾脏CD8+T细胞、CD4+T细胞、巨噬细胞、B细胞；
    优选地，ssDNA核酸适体的序列选自SEQ ID NO.1-7；优选地，ssDNA核酸适体序列选自SEQ ID NO.5-7；优选地，短链核酸为SEQ ID NO.5所示的CD4 Apt时，靶向脾脏CD4+T细胞；优选地，短链核酸为SEQ ID NO.6所示的CD8 Apt时，靶向脾脏CD8+T细胞；优选地，短链核酸为SEQ ID NO.7所示的DC-SIGN时，靶向脾脏巨噬细胞。
  4. 权利要求1-3任一项所述的制剂的制备方法，所述制备方法选自以下任一方法：
    方法1)：使用修饰有短链核酸的制剂成分制备制剂，得到所述修饰有短链核酸的制剂；
    方法2)：使用带有反应性基团的制剂成分制备制剂，得到表面具有反应性基团的制剂；然后，在合适条件下，使得带有另一反应性基团的短链核酸与表面具有反应性基团的制剂反应，得到修饰有短链核酸的制剂；
    方法3)：将短链核酸与制剂混合，得到修饰有短链核酸的制剂；优选地，短链核酸与制剂通过物理连接作用混合，得到修饰有短链核酸的制剂；物理连接作用例如静电吸附连接、疏水插膜连接；
    优选地，方法1)中，修饰有短链核酸的制剂成分为修饰有短链核酸的脂材，例如修饰有短链核酸的PEG脂材、修饰有短链核酸的阳离子脂材、修饰有短链核酸的辅助脂材、修饰有短链核酸的结构脂质；
    优选的，使用方法2)制备所述修饰有短链核酸的制剂；
    优选的，方法2)中，所述反应性基团与另一反应基团为发生点击化学反应的基团；
    优选地，方法2)中，所述反应性基团与另一反应基团发生的反应为化学连接反应，包括但不限于：无催化剂的叠氮-炔基偶合反应、有催化剂的叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等；
    优选地，方法2)中，反应性基团和/或另一反应基团选自：叠氮基团、炔基基团(例如二苯基环辛炔基团)、磷酸化基团、甲基化基团、氨基化基团、巯基化基团、生物素基团、荧光标记物基团、放射性同位素基团等；
    优选地，制剂是纳米脂质体LNP；优选地，带有反应性基团的制剂成分为带有反应性基团的脂材，例如带有反应性基团的PEG脂材、带有反应性基团的阳离子脂材、带有反应性基团的辅助脂材、带有反应性基团的结构脂质；优选地，上述制备方法包括以下步骤：PEG脂材、辅助脂材、结构脂质、阳离子脂材和核酸药物合成纳米脂质体，随后纳米脂质体与短链 核酸发生点击化学反应，形成短链核酸修饰的纳米脂质体；优选地，上述制备方法包括以下步骤：叠氮修饰的PEG脂材、辅助脂材、结构脂质、阳离子脂材和核酸药物合成纳米脂质体，随后纳米脂质体与氮杂环辛炔DBCO修饰的短链核酸发生点击化学反应，形成短链核酸修饰的纳米脂质体。
  5. 一种组合物，所述组合物包括权利要求1-3任一项所述的制剂；
    优选地，所述组合物是疫苗组合物或药物组合物；优选地，疫苗选自RNA疫苗、DNA疫苗；更优选地，疫苗是mRNA疫苗；
    优选地，上述组合物中进一步包括其他治疗或预防剂；优选地，其他治疗或预防剂选自核酸药物、小分子药物、蛋白质药物、药物活性分子中的任意至少一种；
    优选地，所述组合物可靶向脾脏及其内各类免疫细胞；
    优选地，所述组合物将核酸药物递送至靶标(例如靶细胞和/或靶器官)；
    优选地，所述靶标选自：免疫细胞、抗原呈递细胞、T细胞、驻留T细胞、B细胞、自然杀伤(NK)细胞、癌细胞、与疾病或病症相关的细胞、与疾病或病症相关的组织、脑组织、中枢神经***组织、肺组织、顶端表面组织、上皮细胞、内皮细胞、肝组织、肠组织、结肠组织、小肠组织、大肠组织、粪便、骨髓、巨噬细胞、脾脏组织、肌肉组织、关节组织、肿瘤细胞、病变组织、***组织、淋巴循环和它们的任意组合组成的组；
    优选地，所述靶标选自脾脏组织及其亚细胞；优选地，所述脾脏亚细胞为脾脏CD8+T细胞、CD4+T细胞、巨噬细胞、B细胞。
  6. 权利要求1-3任一项所述的制剂、权利要求5所述的组合物在制备预防和/或治疗疾病或病症的药物中的应用；
    优选地，所述疾病或病症选自肿瘤、自身免疫病、代谢类疾病、病毒感染、细菌感染、真菌感染、寄生虫感染、流感感染、癌症、关节炎、心脏病、心血管疾病、神经病症或疾病、遗传病、胎儿疾病、影响胎儿发育的遗传病或它们的任意组合中选择的至少一种。
  7. 一种向有需要的受试者递送核酸药物的方法，所述方法包含向所述受试者施用治疗有效量的权利要求1-3任一项所述的制剂、权利要求5所述的组合物。
  8. 一种在有需要的受试者中预防和/或治疗疾病或病症的方法，包括向受试者施用治疗有效量的权利要求1-3任一项所述的制剂、权利要求5所述的组合物；
    优选地，所述疾病或病症选自肿瘤、自身免疫病、代谢类疾病、病毒感染、细菌感染、真菌感染、寄生虫感染、流感感染、癌症、关节炎、心脏病、心血管疾病、神经病症或疾病、遗传病、胎儿疾病、影响胎儿发育的遗传病或它们的任意组合中选择的至少一种。
  9. 一种短链核酸修饰的PEG脂材，结构为短链核酸-A-PEG；
    其中，A为任意的化学连接基团或连接臂；优选地，A为点击化学生成的连接结构；
    优选地，A为叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等所生成的连接结构；
    优选地，PEG脂材选自DMG-PEG2000、DSPE-PEG2000、DTDA-PEG2000、TPGS；优选地，PEG脂材选自DMG-PEG2000、DSPE-PEG2000；
    优选地，短链核酸为随机序列核酸或者核酸适体；优选地，核酸适体为RNA核酸适体或DNA核酸适体；更优选地，DNA核酸适体为ssDNA核酸适体；
    优选地，ssDNA核酸适体的序列选自SEQ ID NO.1-7；优选地，ssDNA核酸适体序列选自SEQ ID NO.5-7。
  10. 权利要求9所述的短链核酸修饰的PEG脂材的制备方法，所述制备方法包括：
    带有反应性基团的PEG脂材，与带有另一反应性基团的短链核酸发生反应，得到短链核酸修饰的PEG脂材；
    优选地，发生的反应为化学连接反应，包括但不限于：无催化剂的叠氮-炔基偶合反应、有催化剂的叠氮-炔基偶合反应、四氮杂苯-反式环辛烯连接反应、马来酰亚胺-巯基偶联反应等；
    优选地，反应性基团和/或另一反应基团选自：叠氮基团、炔基基团(例如二苯基环辛炔基团)、磷酸化基团、甲基化基团、氨基化基团、巯基化基团、生物素基团、荧光标记物基团、放射性同位素基团等。
PCT/CN2023/132420 2022-12-19 2023-11-17 基于核酸适体的脾脏及其亚细胞的mRNA靶向递送*** WO2024131403A1 (zh)

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