Method for enhancing mRNA-LNP tissue targeting delivery

Abstract

The invention discloses a method for enhancing mRNA-LNP tissue targeting delivery. The invention provides a method for enhancing tissue targeting delivery of nucleic acid lipid nanoparticles, which comprises the following steps: modifying the surfaces of the nucleic acid lipid nanoparticles with tissue targeting with antibodies to obtain the nucleic acid lipid nanoparticles with modified antibodies and tissue targeting, the tissue targeting delivery property of the nucleic acid lipid nanoparticles is enhanced. On the basis of novel lung targeting LNP, an antibody modification strategy (double targeting) is combined, more efficient, specific and safer lung targeting mRNA delivery is successfully achieved, and the strategy is suitable for any type of targeting LNP (charge-driven DOTAP LNP and structure-driven STAR LNP in the project).

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for enhancing mRNA-LNP tissue targeted delivery. Background Art

[0002] With the successful development and rapid approval of the new coronavirus mRNA vaccine, mRNA drugs have shown strong application prospects. By designing the sequence of messenger ribonucleic acid (mRNA), any protein can be expressed in theory, such as antigens, therapeutic proteins, gene editing elements, etc., so it can be used in many fields such as preventive vaccine development, protein replacement therapy, gene editing, etc. In addition, due to the simple production process, low cost and short cycle of mRNA, it is also favored in clinical transformation. However, mRNA carries a negative charge and is easily degraded by enzymes. In practical applications, it relies on safe and efficient delivery carriers, among which lipid nanoparticles (LNP) are considered to be one of the best carrier options. More than 90% of clinical mRNA drug projects under development rely on LNP.

[0003] LNPs are usually composed of ionizable cationic lipids, auxiliary phospholipids, cholesterol and polyethylene glycol lipids. Among them, ionizable cationic lipids are the key to achieving efficient mRNA loading and lysosomal escape, and are also the focus of various scientific research units and R&D companies. Ionizable cationic lipid molecules can be protonated in a low pH environment (such as pH 4.0), and then bind to mRNA to complete its encapsulation, while in a physiological environment (pH 7.4), they are uncharged, making LNP electrically neutral. Currently, most mRNA-LNPs can be used for local intramuscular injection of vaccines, or intravenous administration for distribution and expression in the liver. However, how to achieve efficient and specific extrahepatic organ-targeted mRNA delivery is an important and critical issue in the field, which is expected to broaden the application prospects of mRNA.

[0004] In recent years, some progress has been made in the delivery of extrahepatic organ-targeted mRNA. The main ones are as follows: 1) Charge-mediated targeting strategy, by introducing positively charged cationic lipids (such as quaternary ammonium lipid DOTAP) into LNP, lung-targeted mRNA delivery can be achieved, and by introducing negatively charged anionic lipids (such as phosphate lipid 18:1PA), spleen-targeted mRNA delivery can be achieved, which is called SORTLNP technology (Cheng et al., Nat. Nanotechnol., 15: 313-320, 2020); 2) Developing new structures of ionizable cationic lipids to achieve mRNA targeted delivery (such as Qiu et al., Proc. Natl. Acad. Sci. USA, 119: e2116271119, 2022); 3) Through antibody-modified LNP strategy, such as modifying CD31 antibody on the surface of LNP, mRNA expression in the lungs was successfully achieved (Parhiz et al., J. Control Release, 291: 106-115, 2018).

[0005] The above methods all have certain advantages and disadvantages, such as the potential toxicity of positively charged cationic lipids, the low delivery efficiency of new structures, and the poor specificity of antibody-modified LNPs. The new mRNA-LNP drugs have very broad application prospects, but the targeted delivery of mRNA to extrahepatic organs and cells is currently difficult. Summary of the invention

[0006] The problem solved by the present invention is to provide a method for enhancing tissue-targeted delivery of mRNA-LNP.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a method for enhancing the tissue-targeted delivery of nucleic acid lipid nanoparticles, comprising the following steps: modifying the surface of nucleic acid lipid nanoparticles with tissue-targeting properties with antibodies to obtain antibody-modified nucleic acid lipid nanoparticles with tissue-targeting properties, thereby enhancing the tissue-targeted delivery of the nucleic acid lipid nanoparticles.

[0008] In the above method, the tissue is lung, brain or spleen.

[0009] Alternatively, the antibody is a lung-targeting antibody, a brain-targeting antibody, or a spleen-targeting antibody.

[0010] In the above method, the lung-targeting antibody is CD31 antibody, plasmalemma vesicle-associated protein (PV1) antibody or CD326 antibody.

[0011] Alternatively, the brain-targeting antibody is a transferrin receptor monoclonal antibody (specifically, it may be any one or a combination of the following: OX26, R17217 and 8D377).

[0012] Or, the spleen targeting antibody is F4 / 80 antibody, CD5 antibody, CD4 antibody, CD8 antibody, CD3 antibody or CD19 antibody.

[0013] In the method described above, the tissue-targeted nucleic acid lipid nanoparticles are charge-driven tissue-targeted nucleic acid lipid nanoparticles or non-charge-driven tissue-targeted nucleic acid lipid nanoparticles;

[0014] The lipids in the charge-driven tissue-targeted nucleic acid lipid nanoparticles include ionizable cationic lipids, maleimide-modified polyethylene glycol lipids and permanent cationic lipids;

[0015] The lipids in the non-charge-driven tissue-targeting nucleic acid lipid nanoparticles include ionizable cationic lipids and maleimide-modified polyethylene glycol lipids.

[0016] In the method described above, the lipids in the charge-driven tissue-targeted nucleic acid lipid nanoparticles further include auxiliary lipids;

[0017] The lipids in the non-charge-driven tissue-targeting nucleic acid lipid nanoparticles further include auxiliary lipids;

[0018] The helper lipids include any one, any two or three of steroids, phospholipids and other PEG lipids.

[0019] In the above method, the permanent cationic lipid is a lipid molecule having quaternary ammonium salt properties;

[0020] Alternatively, the phospholipid is a lipid having a pharmaceutically acceptable amphipathic lipid phospholipid structure.

[0021] In the above method, the lipid molecule having quaternary ammonium salt properties is DOTAP, DDAB, DOTMA or EPC;

[0022] Or, the lipid having a pharmaceutically acceptable amphiphilic lipid phospholipid structure is DOPE, DSPE, POPE, 4ME, DOPC or DSPC;

[0023] Or, the steroid is cholesterol or an analog thereof;

[0024] Or the other PEG lipid is DMG-PEG, DSG-PEG or DSPE-PEG.

[0025] In the method described above, the charge-driven tissue-targeted nucleic acid lipid nanoparticles are charge-driven lung-targeted nucleic acid lipid nanoparticles; wherein the molar ratios of the various lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 20%-80%, the molar ratio of the permanent cationic lipids to the total lipids is 10%-50%, the molar ratio of the maleimide-modified polyethylene glycol lipids to the total lipids is 0.5-5%, and the molar ratio of the auxiliary lipids to the total lipids is 0-40%.

[0026] Or, in the method described above, the non-charge-driven tissue-targeted nucleic acid lipid nanoparticles are non-charge-driven lung-targeted nucleic acid lipid nanoparticles, wherein the molar ratios of each lipid are as follows: the molar ratio of the ionizable cationic lipid to the total lipids is 20%-80%, the molar ratio of the maleimide-modified polyethylene glycol lipid to the total lipids is 0.5-5%, and the molar ratio of the auxiliary lipid to the total lipids is 0-45%.

[0027] In the above method, the molar ratio of the permanent cationic lipid to the total lipids in the charge-driven lung-targeted nucleic acid lipid nanoparticles is 30%;

[0028] Alternatively, the molar ratio of the ionizable cationic lipid to the total lipids in the non-charge-driven lung-targeted nucleic acid lipid nanoparticles is 50%.

[0029] The ionizable cationic lipid in the charge-driven lung-targeted nucleic acid lipid nanoparticles may be SM-102, ALC-0315, MC3, C12-200, CKK-E12, 4A3-SC8 or 5A2-SC8, but is not limited thereto;

[0030] The ionizable cationic lipid in the non-charge-driven lung-targeted nucleic acid lipid nanoparticles may be A3-N11F, 306-N16B, 113-N16B, 98N12-5 or 7C1, but is not limited thereto.

[0031] The polyethylene glycol lipid in the maleimide-modified polyethylene glycol lipid may be DSPE-PEG or DMG-PEG, but is not limited thereto.

[0032] The cholesterol analogue may be β-sitosterol, 7α-hydroxycholesterol or 25-hydroxycholesterol, but is not limited thereto.

[0033] The DMG-PEG may be DMG-PEG2000, but is not limited thereto.

[0034] The DSPE-PEG may be DSPE-PEG2000, but is not limited thereto.

[0035] The DSPE-PEG-Mal may be DSPE-PEG2000-Mal, but is not limited thereto.

[0036] In one embodiment of the present invention, the charge-driven nucleic acid lipid nanoparticles targeting the lung are composed of lipids in the following molar ratio: 27%-50% SM-102, 10-50% DOTAP, 1.5% DSPE-PEG-Mal and 20-40% Chol; the total molar ratio does not exceed 100% (specifically, Figure 1 c).

[0037] Furthermore, the charge-driven lung-targeted nucleic acid lipid nanoparticles are composed of lipids in the following molar ratio: 38.7% SM-102, 30% DOTAP, 1.5% DSPE-PEG-Mal and 29.8% cholesterol; the total molar ratio does not exceed 100% (specifically 4C-DOTAP LNP in the embodiment).

[0038] In another embodiment of the present invention, the non-charge-driven nucleic acid lipid nanoparticles targeting the lung are composed of lipids in the following molar ratio: 23-50% A3-N11F, 10-24% DOPE, 1.5-5% DSPE-PEG-Mal and 38-48% cholesterol; the total molar ratio does not exceed 100% (specifically, Figure 7 c).

[0039] Furthermore, the non-charge-driven lung-targeted nucleic acid lipid nanoparticles are composed of lipids in the following molar ratio: 50% A3-N11F, 10% DOPE, 1.5% DSPE-PEG-Mal and 38.5% cholesterol; the total molar ratio does not exceed 100% (A3-N11F LNP).

[0040] The nucleic acid mentioned above is a chemically modified or non-chemically modified nucleic acid molecule.

[0041] The above chemically modified or unmodified nucleic acid molecules, such as DNA molecules or RNA molecules, preferably have a mass ratio of total lipids to nucleic acid molecules in LNP of about 1-100:1.

[0042] The above-mentioned chemically modified or unmodified DNA molecules can be any type of DNA molecules (but not limited to this), including but not limited to linear or circular DNA, double-stranded or single-stranded or multi-stranded assembled DNA, coding or non-coding DNA, which are optionally selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA probes, DNA aptamers, DNA nanoframes, DNA primers, homology repair DNA templates, immunostimulatory DNA or a combination thereof.

[0043] The chemically modified or unmodified RNA molecules may be any type of RNA molecules (but not limited thereto), including but not limited to messenger RNA (mRNA), small interfering RNA (siRNA), circular RNA (circRNA or oRNA), guide RNA (sgRNA), nicking guide RNA (nicking sgRNA), small hairpin RNA (shRNA), viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immunostimulatory RNA (isRNA), micro RNA (miRNA), small nuclear RNA (snRNA), RNA aptamer, antisense RNA, RNA nanoframe, RNA ribonucleoprotein complex or a combination thereof.

[0044] Furthermore, the mRNA expresses a protein with therapeutic function, a fluorescent reporter protein or a tumor-killing protein.

[0045] The antibody is obtained by connecting the thiol group on its surface with the maleimide group on the surface of the nucleic acid lipid nanoparticle with tissue targeting.

[0046] The delivery effect of the above-mentioned antibody-modified and tissue-targeted nucleic acid lipid nanoparticles is higher than that of the nucleic acid lipid nanoparticles with tissue targeting.

[0047] In a second aspect, the present invention provides antibody-modified nucleic acid lipid nanoparticles with tissue targeting prepared by the method described in the first aspect.

[0048] Alternatively, the present invention provides the use of the antibody-modified nucleic acid lipid nanoparticles having tissue targeting in the preparation of targeted tissue products.

[0049] Or the present invention provides a method for preparing the antibody-modified nucleic acid lipid nanoparticles with tissue targeting, comprising the steps described in the first aspect.

[0050] The above method for preparing antibody-modified nucleic acid lipid nanoparticles with tissue targeting specifically comprises the following steps:

[0051] A1) dissolving each lipid in the tissue-targeted nucleic acid lipid nanoparticles in an organic solvent to obtain a lipid organic phase; and then mixing with the mRNA aqueous phase to obtain tissue-targeted nucleic acid lipid nanoparticles;

[0052] A2) modifying the antibody with the tissue-targeting nucleic acid lipid nanoparticles to obtain antibody-modified nucleic acid lipid nanoparticles with tissue-targeting.

[0053] Or the present invention provides a tissue-targeted product, the active ingredient of which is the modified antibody and the nucleic acid-lipid nanoparticles with tissue targeting.

[0054] The above products are targeted delivery platforms or drugs.

[0055] The above-mentioned tissue is lung, brain or spleen.

[0056] Based on the above background, the present invention proposes that: combining the above-mentioned multiple strategies is expected to make up for the shortcomings of the current targeted mRNA-LNP. Therefore, the following experiments were conducted: 1) Referring to the SORT LNP technology, targeted LNP was obtained by introducing charged lipids. Unlike the 5-component SORT LNP, the targeted LNP maintained 4 components, and the PEG-DMG lipids in the LNP were completely replaced with DSPE-PEG-Mal (the lipids can provide active groups for antibody modification); 2) CD31 antibodies were used to establish LNP surface modification technology, and the optimal ratio of antibodies to LNPs was obtained; 3) In the charge-driven lung-targeted LNP and the structure-driven lung-targeted LNP, the CD31 antibody modification significantly improved the delivery specificity, safety and effectiveness of mRNA-LNP. 4) Using the above-mentioned enhanced targeted LNP, the lung-targeted delivery of interleukin 15 superagonist mRNA was completed, and high-efficiency tumor immunotherapy was mediated in the mouse model of lung tumor metastasis. This novel, antibody-enhanced lung-targeted mRNA-LNP delivery strategy can bring new breakthroughs in the treatment of lung diseases and greatly broaden the depth and breadth of application of mRNA-LNP drugs.

[0057] In this study, we integrated the research and development strategies of targeted LNP in the field, hoping to improve the performance of mRNA-LNP delivery through "dual targeting". The main innovations and key points are as follows:

[0058] 1) Referring to the SORT LNP technology (lung-targeted delivery is achieved by adding cationic lipids to the 4-component LNP), the lung-targeted LNP formulation based on the positive charge of DOTAP is optimized and improved, and the auxiliary phospholipid DSPC in the LNP is completely replaced with the positively charged lipid DOTAP, and DMG-PEG is completely replaced with DSPE-PEG-Mal. Unlike SORT LNP, not only the 4 components are retained, but also the original DMG-PEG is completely replaced with DSPE-PEG-Mal, providing the active group of Mal for the subsequent antibody-modified LNP. Among them, the LNP formulation containing 30% DOTAP (named 4C-DOTAP LNP) has the best transfection effect and good specificity, and the effect is better than the previously reported 5-component SORT LNP (5C-DOTAP LNP).

[0059] 2) Establish the optimal ratio of CD31 antibody to LNP. By optimizing the dosage of CD31 antibody, efficient antibody-modified lung-targeted mRNA delivery (named Anti LNP) can be achieved.

[0060] 3) The efficiency of lung-targeted mRNA delivery was further significantly improved by modifying the CD31 antibody on 4C-DOTAP LNP (named Synergistic Targeted LNP, SynTar LNP). SynTar LNP can efficiently transfect and edit lung endothelial cells, epithelial cells and immune cells, and has good stability.

[0061] 4) Compared with other lung-targeted LNP strategies, SynTar LNP has superior specificity compared to Anti LNP, significantly reducing off-target expression in the liver. SynTar LNP has higher lung delivery efficiency than 4C-DOTAP LNP and Anti LNP, and better safety than 5-component SORT LNP.

[0062] 5) In the previously established structure-driven targeted LNP technology (referred to as STAR LNP, patent application number PCT / CN2023 / 137998), the ionizable cationic lipid A3-N11F LNP has the function of lung-targeted mRNA delivery. Using the above-mentioned SynTar strategy, the modification of CD31 antibody on A3-N11F LNP further enhanced the delivery efficiency, verifying the universality of this combination strategy.

[0063] 6) Finally, based on the previously developed interleukin-15 superagonist mRNA technology (patent application number PCT / CN2024 / 084406), the SynTar LNP system was used to mediate efficient IL-15 mRNA lung-targeted delivery, and achieved efficient inhibition of mouse lung tumor metastasis, demonstrating the effectiveness of the SynTar LNP platform and its ability to be applied in disease treatment.

[0064] 7) Based on 1-6, a broad-spectrum, universal, and mRNA-LNP lung-targeted delivery technology with better delivery efficiency and specificity was developed, which can efficiently and specifically transfect endothelial cells, epithelial cells, and immune cells in the lungs, providing a new treatment for lung diseases. And there are reasonable reasons to infer that the "dual targeting" strategy of combining antibodies with targeted LNPs is expected to be extended to mRNA delivery and treatment outside the lungs, such as the brain, liver, kidneys, heart, etc.

[0065] The present invention successfully achieves more efficient, specific and safe lung-targeted mRNA delivery by combining antibody modification strategy (dual targeting) on ​​the basis of novel lung-targeted LNP. And this strategy is applicable to any type of targeted LNP (there are two types of charge-driven DOTAP LNP and structure-driven STAR LNP in this project). Finally, the therapeutic IL-15 superagonist mRNA is delivered through this novel LNP platform to achieve effective inhibition of lung tumors. This achievement has important reference significance and value for the treatment of diseases such as hereditary lung diseases, lung injury, idiopathic pulmonary fibrosis, cystic fibrosis, lung cancer, etc. In addition, there is reason to believe that as long as suitable antibodies and targeted LNPs are selected, this strategy can be expanded to organ-targeted delivery and treatment outside the lungs, such as the brain, heart, kidneys, pancreas, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The simplified 4-component DOTAP LNP can achieve efficient lung-targeted mRNA delivery.

[0067] Figure 2 Validation and dose optimization of CD31 antibody modifications.

[0068] Figure 3 SynTar LNP has significantly enhanced gene editing efficiency and is able to target multiple cell types in the lung.

[0069] Figure 4 SynTar LNP has higher delivery efficiency and specificity than Anti LNP.

[0070] Figure 5 SynTar LNP has higher delivery efficiency, specificity and safety than 5C-DOTAP LNP.

[0071] Figure 6 Antibody modification for CD31 can also enhance lung-targeted mRNA delivery based on the novel ionizable lipid A3-N11F (structure-mediated).

[0072] Figure 7 SynTar LNP lung-specific delivery of a novel IL-15 superagonist mRNA is highly effective in treating LLC lung metastases in mice. DETAILED DESCRIPTION

[0073] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0074] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0075] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0077] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0078] The ionizable cationic lipids in the following examples are: SM-102, A3-N11F;

[0079] Auxiliary phospholipids in the following examples: DSPC, DOPE;

[0080] Permanent cationic lipids (lipid molecules with quaternary ammonium salt properties) in the following examples: DOTAP;

[0081] PEG lipids in the following examples: DMG-PEG 2000 (abbreviated as DMG-PEG), DSPE-PEG 2000 , DSPE-PEG 2000 -Maleimide (abbreviated as DSPE-PEG-Mal).

[0082] The structural formulas of the compounds in the following examples are shown in Table 1:

[0083] Table 1 shows the structural formula of the compounds

[0084]

[0085]

[0086] In the following examples, sodium citrate buffer (10 mM, pH 4.0) includes 5.962 mM citric acid, 4.037 mM trisodium citrate, and the balance is water.

[0087] In the following examples, 1×PBS solution: pH 7.4, containing 1.06 mM KH2PO4, 155 mM NaCl, 2.97 mM Na2HPO4-7H2O, and the balance being water, was purchased from Gibco.

[0088] The various mRNA-LNPs in the following examples are produced by rapidly mixing an ethanol phase containing lipid molecules and an aqueous phase containing mRNA molecules. The specific synthesis method is as follows:

[0089] 1) Aqueous phase containing mRNA molecules

[0090] The mRNA molecules were dissolved in sodium citrate buffer (pH 4.0, 10 mM) to obtain an mRNA solution with a concentration of 0.2 mg / mL; recorded as the aqueous phase containing the mRNA molecules;

[0091] 2) Ethanol phase containing lipid molecules

[0092] The LNP components corresponding to each example were dissolved in ethanol according to the corresponding specific molar ratio to obtain an ethanol phase containing lipid molecules, and the concentration of the lipid molecules in the ethanol phase was 30 mM.

[0093] 3) Preparation of LNPs encapsulating mRNA

[0094] The aqueous phase containing mRNA molecules obtained in step 1) and the ethanol phase containing lipid molecules obtained in step 2) were quickly mixed at a volume ratio of 3:1 (a small batch was produced by rapid pipetting with a pipette, and a large batch was produced by a microfluidic chip), and the mass ratio of total lipid to mRNA was fixed at 40 / 1 or 20 / 1. Incubate at room temperature for 10 minutes, then dialyze in 1×PBS solution for 3 hours to obtain an mRNA-LNP solution with a concentration of 0.4 mg / mL (the solvent was 1×PBS solution).

[0095] The encapsulation efficiency detection method in the following embodiment is as follows: the mRNA-LNP to be tested is diluted to about 100 ng / mL with 1×TE buffer (1×TE buffer: 20×TE buffer is diluted 20 times with sterile water, 20×TE buffer is purchased from invitrogen), and the samples to be tested are divided into two groups according to Table 2 below and added to a black 96-well plate, and the corresponding volumes of buffer, Triton-100 lysate and LNP sample are added respectively. Then shake at 200 rpm for 30 minutes at room temperature. Dilute RiboGreen 2000 times with 1×TE, add 100 μL RiboGreen reaction solution to each well, and read the results after shaking in the microplate reader for 3 minutes. The specific parameters are as follows: excitation wavelength: 480 nm, emission wavelength: 520 nm. The encapsulation efficiency is then calculated based on the readings:

[0096] Encapsulation Efficiency (%) = [(AB) / A] * 100%

[0097] (A represents the calculated value of samples treated with Triton-100, and B represents the calculated value of samples not treated with Triton-100)

[0098] Table 2 shows the components of each group

[0099]

[0100] Example 1, Preparation and Identification of Lung-Targeted DOTAP LNPs of 4 Components

[0101] The composition and formulation of LNP have an important impact on its in vivo delivery efficiency and organ / tissue targeting. First, the SM-102LNP of the new crown mRNA vaccine (Spikevax) approved by the US FDA was selected, and on this basis, the selective organ targeted delivery technology (SORT) was modified and optimized. For the purpose of achieving lung targeted delivery and simplifying the prescription, the cationic lipid DOTAP completely replaced the traditional auxiliary phospholipid DSPC. For the purpose of subsequent antibody coupling and simplified prescription, the traditional DMG-PEG lipid was completely replaced by the DSPE-PEG-Mal lipid with maleimide at the end.

[0102] In order to find LNPs with efficient and specific delivery capabilities to the lungs, the molar ratio of DOTAP in LNPs was adjusted (10%-50%), and a series of new LNPs (simplified four-component LNPs containing DOTAP and Mal groups) were synthesized by microfluidic technology. The above LNP formulations were screened in vivo ( Figure 1 a).

[0103] 1. Synthesis of lipid nanoparticles (LNPs) encapsulating mRNA

[0104] According to the above mRNA-LNP synthesis method, Luciferase mRNA (sequence 1) and Figure 1 c shows the individual LNP lipids (SM-102, Chol, DSPE-PEG-Mal and DOTAP) and the corresponding molar ratios ( Figure 1 c) to prepare mRNA-LNP solutions, and the following LNPs encapsulating Luciferase mRNA were obtained: F1, F2, F3 (also denoted as 4C-DOTAP in the figure), F4, and F5 (the mass ratio of total lipid to RNA was 40:1).

[0105] Figure 1 4C-DSPC in c is synthesized by the aforementioned mRNA-LNP method by combining Luciferase mRNA and Figure 1c The LNP lipids (SM-102, DSPC, Chol, and DSPE-PEG-Mal) and the corresponding molar ratios were used to prepare the mRNA-LNP solution, which was named 4C-DSPC LNP encapsulating Luciferase mRNA (total lipid to RNA mass ratio of 40:1).

[0106] Traditional 5-component 30% DOTAP SORT LNP (abbreviated as 5C-DOTAP): According to the aforementioned mRNA-LNP synthesis method, Luciferase mRNA and various LNP lipids (SM-102, DSPC, chol, DMG-PEG and positively charged lipid DOTAP) and their respective molar ratios (the molar ratios of SM-102, DSPC, chol, DMG-PEG and positively charged lipid DOTAP are 35:7:27.0:1:30, respectively) are prepared to obtain an mRNA-LNP solution, namely, 5C-DOTAP LNP encapsulating Luciferase mRNA (total lipid to RNA mass ratio is 40:1).

[0107] 2. Characterization of LNPs

[0108] 1. LNP prescription screening

[0109] The following experiments were conducted on the above LNPs encapsulating Luciferase mRNA: F1, F2, F3 (also referred to as 4C-DOTAP in the figure), F4, F5, 4C-DSPC and 5C-DOTAP:

[0110] Each of the above LNP solutions encapsulating Luciferase mRNA was diluted with 1×PBS to obtain a LNP injection solution encapsulating Luciferase mRNA with a concentration of 0.1 mg / mL.

[0111] C57BL / 6J mice were injected with 0.2 mg / kg of LNP injection solution encapsulating Luciferase mRNA through the tail vein, and bioluminescence imaging of major organs was performed 6 hours later. Specifically, 150 mg / kg of luciferin potassium salt (Meilun Bio) was injected intraperitoneally 6 hours after injection, and bioluminescence imaging was performed using the IVIS Lumina system (Perkin Elmer) 10 minutes later.

[0112] The image results are as follows Figure 1 As shown in b, the total fluorescence intensity of the mouse lungs ( Figure 1 d) The mean fluorescence intensity ratio of lung / liver ( Figure 1 e) and the percentage of total fluorescence of different organs ( Figure 1f), it can be seen that 4C-DOTAP achieved the strongest total fluorescence value in the lungs, and compared with the traditional 5C-DOTAP, it was enhanced by 2.07 times ( Figure 1 d). The average lung / liver intensity ratio of 4C-DOTAP was higher than 40, and the level was comparable to that of 5C-DOTAP ( Figure 1 e), proving its high lung delivery specificity. By analyzing the percentage of total fluorescence in different organs, it was found that the lung expression percentage of 4C-DOTAP was similar to that of 5C-DOTAP, both higher than 85%, which also proved that its lung targeting has high specificity ( Figure 1 f).

[0113] 2. DLS detection of particle size

[0114] The particle sizes of the above-mentioned LNPs encapsulating Luciferase mRNA: F1, F2, F3 (also referred to as 4C-DOTAP in the figure), F4, F5, 4C-DSPC and 5C-DOTAP were detected by DLS.

[0115] The results are as follows: Through DLS detection, it was found that the particle size of each LNP was approximately between 90nm and 130nm, and the PDI was less than 0.2, with good uniformity ( Figure 1 g).

[0116] The particle size of 4C-DOTAP LNP was detected by DLS after being stored at 4℃ for 7 days. This LNP has good stability and maintains normal particle size and PDI ( Figure 1 h, denoted as 4C-DOTAP LNP (F3)).

[0117] Therefore, through LNP formulation optimization and screening, 4C-DOTAP LNP with strong lung delivery efficiency, high specificity and good stability was obtained.

[0118] Example 2. Obtaining Anti LNP Modified with CD31 Antibody

[0119] Next, we modified LNPs with CD31 antibodies to achieve lung-targeted mRNA delivery. The antibody modification technology was verified and optimized. Figure 2 As shown in a. First, the antibody is modified using a SATA reagent to insert a thiol group into the antibody, and the antibody modification of LNP is achieved through the connection reaction between the thiol group on the antibody and the maleimide (Mal) group on the surface of LNP.

[0120] 1. Exploration of the ratio of modified antibodies

[0121] 1. Antibody thiol modification (-SH)

[0122] The commercial antibody CD31 Ab (Brand: Biolegend, Catalog No.: 102402) was thiol-modified (-SH) according to the instructions of SATA molecule (N-succinic acid, S-acetylthioglycol ester) (thermo):

[0123] A) Allow the antibody to react with SATA (N-Succinimidyl S-Acetylthioacetate): Dissolve SATA in DMSO to 0.1 mg / mL to obtain a SATA solution.

[0124] CD31 Ab solution with a concentration of 0.5 mg / mL (solvent is phosphate buffer at pH 7.2).

[0125] The SATA solution was added to the antibody solution at a fixed SATA / antibody molar ratio of 6:1 or 9:1, and incubated at room temperature for 30 minutes to obtain a reaction product;

[0126] B) The above reaction product was filtered through ultrafiltration (membrane pore size: 10 kd, rotation speed: 8500 rpm, replacement fluid was Reaction Buffer) to remove excess reactants and by-products to obtain a SATA modified antibody solution (solvent was Reaction Buffer, specific composition: 0.1 M phosphate, 0.15 M sodium chloride, dissolved in ultrapure water, pH 7.3).

[0127] C) Deprotection of SATA-modified antibodies to generate reactive sulfhydryl groups

[0128] To 250 μL of SATA modified antibody solution, 25 μL of deacetylation solution (0.5 M hydroxylamine hydrochloride, 25 mM EDTA, dissolved in Reation Buffer, pH 7.3) was added, mixed thoroughly and incubated at room temperature for 2 hours. The excess hydroxylamine in the deacetylation solution was then removed by ultrafiltration, and the solution was replaced with 1×PBS containing 10 mM EDTA to reduce the formation of disulfide bonds, to obtain a 2 mg / mL thiol-modified antibody solution (solvent was 1×PBS containing 10 mM EDTA).

[0129] 2. Antibody coupling to the surface of mRNA-LNP

[0130] Reacting LNPs containing DSPE-PEG-Mal with thiolated antibodies:

[0131] The LNP solution containing DSPE-PEG-Mal (concentration of 0.4 mg / mL) was mixed with the 2 mg / mL thiol-modified antibody solution obtained in 1 above according to the molar ratio of Mal to Ab in LNP of 40:1, 20:1 or 10:1, and stirred at room temperature for 2 hours to couple the antibody to the LNP surface to obtain the Anti LNP solution of each modified antibody.

[0132] 3. Purification of antibody-coupled LNP

[0133] The above-mentioned modified antibody Anti LNP solution was purified by size exclusion chromatography (using Sepharose CL-4B agarose gel), eluted with 1×PBS solution as the mobile phase, and separated to obtain the purified modified antibody Anti LNP solution.

[0134] The solute in the purified antibody-modified Anti LNP solution is the antibody-modified Anti LNP, the solvent is 1×PBS, and the concentration is 0.1 mg / mL.

[0135] 2. Characterization of LNPs modified with antibodies

[0136] 1. LNP prescription screening

[0137] Anti-LNP solutions of modified antibodies with different molar ratios of Mal to Ab were prepared using LNPs containing DSPE-PEG-Mal (specifically 4C-DSPC LNPs encapsulating Luciferase mRNA) according to the method described above: 40:1, 20:1, and 10:1;

[0138] Then, the above-mentioned Anti LNP solutions of different modified antibodies were dissolved in 1×PBS at a ratio of 40:1, 20:1, and 10:1, respectively, to obtain various Anti LNP injection solutions with a concentration of 0.1 mg / mL.

[0139] Bioluminescence imaging of major organs of C57BL / 6J mice was performed 6 hours after the tail vein injection of 0.1 mg / kg of various Anti LNP injection solutions (denoted as 40:1, 20:1 and 10:1 in the figure). The LNPs prepared in Example 1: 5C-DOTAP and 4C-DSPC were used as controls.

[0140] The image results are as follows Figure 2 As shown in b, quantitative analysis of bioluminescent signal intensity showed that there was no signal in the lungs of the unmodified antibody group, and most of the signals were in the liver and spleen, while the lung signal of the 40:1 group was improved, and the lung signal of the 20:1 group was significantly improved compared with the 40:1 group, but there was no further improvement when it was further increased to 10:1 ( Figure 2c). In addition, the lung-targeted delivery efficiency of the modification ratio of 20:1 is higher than that of 5C-DOTAP ( Figure 2 c) and has very high lung targeting specificity ( Figure 2 d).

[0141] 2. DLS detection of particle size

[0142] The method is the same as in Example 1.

[0143] The particle size of the 20:1 group Anti LNP prepared by 4C-DSPC was detected by DLS.

[0144] The results demonstrated the successful modification of the antibody and the good stability of the modified LNP, with a PDI of less than 0.2 ( Figure 2 e).

[0145] Example 3. Preparation and detection of SynTar LNP

[0146] 1. Preparation of SynTar LNPs encapsulating Cre mRNA

[0147] According to the method of Example 1, Cre mRNA (sequence 2) was encapsulated in liposomes of specific components and molar ratios to obtain 4C-DOTAP LNPs encapsulating Cre mRNA (denoted as DOTAP in the figure, with a total lipid to RNA mass ratio of 40:1), and then the 4C-DOTAP LNPs encapsulating Cre mRNA were modified with antibody CD31 Ab according to the method of Example 2, wherein the molar ratio of SATA / antibody was 9:1, and the molar ratio of Mal in the 4C-DOTAP LNP to CD31 Ab was 20:1, to obtain a SynTar LNP solution prepared by 4C-DOTAP LNPs encapsulating Cre mRNA, the solvent was 1×PBS, and the concentration was 0.1 mg / mL.

[0148] 2. In vivo testing

[0149] The Ai14 reporter mouse (CAG-loxP-stop-loxP-tdTomato) model was used (this animal model has been published in the following literature: Kauffman, KJ; Oberli, MA; Dorkin, JR; Hurtado, JE; Kaczmarek, JC; Bhadani, S.; Wyckoff, J.; Langer, R.; Jaklenec, A.; Anderson, DG Pilot, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse. Mol. Ther. Nucleic. Acids 2018, 10, 55–63. https: / / doi.org / 10.1016 / j.omtn.2017.11.005. The name in the article is Ai14). The mice were injected with Cre mRNA-encapsulated SynTar LNPs through the tail vein. Cre The mRNA is translated into Cre recombinase in the cell, which then cuts the loxP site to make the target cell express tdTomato fluorescent protein ( Figure 3 a). The details are as follows:

[0150] The Cre mRNA-encapsulated SynTar LNP prepared in 1 above was diluted with 1×PBS solution to obtain SynTar LNP injection solutions encapsulating Cre mRNA at a concentration of 0.03 mg / mL.

[0151] The 4C-DOTAP LNPs encapsulating Cre mRNA prepared in 1 above were diluted with 1×PBS solution to obtain DOTAP LNP injection solutions encapsulating Cre mRNA at a concentration of 0.03 mg / mL.

[0152] Ai14 mice were injected with 0.2 mg / kg SynTar LNP injection containing Cre mRNA (denoted as SynTar in the figure) through the tail vein. The same dose of 4C-DOTAP LNP injection containing Cre mRNA (denoted as DOTAP in the figure) and PBS were injected as controls.

[0153] 1) Bioluminescence imaging of major organs

[0154] Bioluminescence imaging of major organs 5 days after injection.

[0155] The results are as follows Figure 3As shown in b and 3c, the results of lung fluorescence quantification showed that the 4C-DOTAP LNP group was significantly improved compared with the PBS group, and the antibody-modified (SynTar LNP) was further significantly improved by 3 times compared with DOTAP LNP.

[0156] 2) Flow cytometry analysis

[0157] Flow cytometry was used to analyze the transfection and editing of different LNPs on lung cell types.

[0158] Five days after the injection, the lungs of mice in each group were collected and digested to obtain single-cell suspensions, which were stained and then subjected to flow cytometry analysis to identify the cell types and tdTomato expression.

[0159] The results are as follows: DOTAP LNP can effectively transfect and edit CD31+ endothelial cells, and SynTar LNP modified with CD31 antibody can further significantly increase the positive rate from 30.5% to 72% ( Figure 3 d). The tdTomato positive rate of all cells also increased significantly ( Figure 3 e), from 6.3% to 34%. Further analysis of other cell types showed that SynTar LNP also significantly improved the editing rate for epithelial cells and immune cells ( Figure 3 f), epithelial cells increased from 22% to 65%, and immune cells increased from 2% to 20%.

[0160] 3. Particle size and encapsulation rate detection

[0161] The particle size of the Cre mRNA-encapsulated SynTar LNP was detected by DLS after being stored at 4°C for 7 days, using the same method as in Example 1.

[0162] Results: SynTar LNP had good stability. After storage at 4℃ for 7 days, the particle size and PDI remained normal ( Figure 3 g).

[0163] The encapsulation efficiency of SynTar LNPs encapsulating Cre mRNA (denoted as SynTar (7 days) in the figure) stored at 4°C for 7 days was measured by Ribogreen. SynTar LNPs encapsulating Cre mRNA (denoted as SynTar (0 days) in the figure) and DOTAPLNPs (denoted as DOTAP in the figure) were used as controls.

[0164] The results are as follows: It was found that DOTAP LNP had a high mRNA encapsulation rate of more than 95%, and the modified antibody had no effect on the encapsulation rate (SynTar LNP could still reach more than 95%). The stability was also good, and the encapsulation rate was still higher than 90% after 7 days of storage ( Figure 3 h).

[0165] 4. Comparison of delivery efficiency and specificity of lung-targeted LNPs

[0166] The delivery efficiency and specificity of four LNPs, namely 4C-DSPC LNP encapsulating Cre mRNA, 4C-DOTAP LNP encapsulating Cre mRNA, Anti LNP encapsulating Cre mRNA, and SynTar LNP encapsulating Cre mRNA, were compared by IVIS organ fluorescence imaging. Figure 4 a).

[0167] The Anti LNP encapsulating Cre mRNA is prepared by antibody-modifying the 4C-DSPC LNP encapsulating Cre mRNA according to the method of Example 3, wherein the molar ratio of SATA / antibody is 9:1, and the molar ratio of Mal to CD31 Ab in the 4C-DSPC LNP is 20:1, respectively, to obtain the Anti LNP encapsulating Cre mRNA.

[0168] Ai14 mice were injected with 0.2 mg / kg of each of the above LNPs (concentration was 0.1 mg / mL) through the tail vein.

[0169] Five days after injection, the mice were killed to obtain liver and lung tissues, which were then embedded in OCT compound ( Sakura) and stored in a -80 degree refrigerator. After slicing with a cryostat, place the tissue slices on a slide and add ProLong TM Gold antifade mounting medium, cover with coverslip, store in -20 degree refrigerator, and observe the image using confocal microscope.

[0170] IVIS organ fluorescence imaging detected the tdTomato fluorescence of the main organs of each group. The test results are as follows: SynTar LNPs encapsulating CremRNA showed higher lung-targeted delivery efficiency than DOTAP LNPs encapsulating Cre mRNA and Anti LNPs encapsulating Cre mRNA (lung fluorescence quantification increased by more than 2 times, 2.83 and 2.09 times, respectively), and better lung-targeted specificity than Anti LNPs encapsulating Cre mRNA, and significantly reduced off-target expression in the liver (liver fluorescence quantification decreased by 5.47 times compared with Anti LNPs encapsulating Cre mRNA ( Figure 4 b,c)).

[0171] Confocal microscopy was used to detect liver and lung tissue sections of mice in different LNP treatment groups.

[0172] The results are as follows: The tdTomato fluorescence signal in the liver sections of the SynTar LNP group was significantly reduced compared with the Anti LNP group, and obvious fluorescence signals were observed in the lung sections ( Figure 4 d), Demonstrating the superior lung-targeted delivery efficiency and specificity of Cre mRNA-encapsulated SynTar LNPs.

[0173] 5. Testing of delivery efficiency, specificity and safety

[0174] SynTar LNPs were compared with the classic 5C-DOTAP LNPs in terms of delivery efficiency, specificity, and safety.

[0175] 5C-DOTAP LNPs encapsulating luciferase mRNA: 5C-DOTAP LNPs encapsulating luciferase mRNA were prepared according to the method of Example 1 (the mass ratio of total lipid to RNA was 40:1).

[0176] According to the method of Example 1, luciferase mRNA was encapsulated in liposomes of specific components and molar ratios according to the method of Example 1 to obtain 4C-DOTAP LNPs encapsulating luciferase mRNA (the mass ratio of total lipids to RNA was 40:1), and then the 4C-DOTAP LNPs encapsulating luciferase mRNA were modified with antibody CD31 Ab according to the method of Example 3, wherein the molar ratio of SATA / antibody was 9:1, and the molar ratio of Mal in 4C-DOTAP LNP to CD31Ab was 20:1, to obtain SynTar LNPs encapsulating luciferase mRNA, the solvent was 1×PBS, and the concentration was 0.1 mg / mL.

[0177] The method for preparing the injection is the same as before.

[0178] The mice were injected with 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg SynTar LNP injections encapsulating luciferase mRNA and 1 mg / kg 5C-DOTAP LNP injections encapsulating luciferase mRNA via tail vein.

[0179] The results are as follows: SynTar LNP at a dose of 0.5 mg / kg showed a lung-targeted delivery efficiency comparable to that of 1.0 mg / kg 5C-DOTAP LNP 6 hours after injection ( Figure 5a, b, c), demonstrating the superior lung-targeted mRNA delivery efficiency of SynTar LNP. In addition, the lung / liver bioluminescence intensity ratio, lung / spleen bioluminescence intensity ratio, and lung expression ratio of SynTar LNP were significantly improved compared with the 5C-DOTAP LNP group, demonstrating that SynTar LNP has better lung targeting specificity ( Figure 5 d, e).

[0180] C57BL / 6J mice were injected with 0.5 mg / kg SynTar LNP injection containing luciferase mRNA and 1 mg / kg 5C-DOTAP LNP injection containing luciferase mRNA through the tail vein, and untreated mice were used as controls (marked as untreated in the figure). After 6 and 24 hours of administration, serum and lung tissues of mice were collected for cytokine and liver function tests to evaluate safety ( Figure 5 f).

[0181] The results are as follows: Due to the possible toxicity caused by the positively charged lipid DOTAP, mice showed adverse reactions such as lethargy and decreased activity after administration of 1.0 mg / kg 5C-DOTAP LNP, while mice injected with 0.5 mg / kg SynTar LNP had no obvious adverse reactions. The levels of typical inflammatory cytokines and chemokines IL-1β, TNF-α, and CCL2 in the lungs and serum of the SynTar LNP-treated group were only slightly increased, but those in the 5C-DOTAP group were significantly higher than those in the SynTar group ( Figure 5 g, 5i). The liver function AST level in the SynTar LNP group was comparable to that in the untreated control group, while that in the 5C-DOTAP group was also significantly increased ( Figure 5 h). Body weight was measured for 15 consecutive days after administration (administered once every 5 days; LPS (lipopolysaccharide, intraperitoneal administration, 5 mg / kg), 1.0 mg / kg 5C-DOTAPLNP group caused a more rapid and significant weight loss ( Figure 5 j and 5k).

[0182] The above results indicate that SynTar LNP is superior to 5C-DOTAPLNP in terms of delivery efficiency, specificity and safety.

[0183] Example 4: Application of DOTAP-based SynTar LNP in the preparation of products loaded with mRNA for inhibiting lung metastasis

[0184] Using DOTAP-based SynTar LNP, IL-15 superagonist mRNA (IL-15mRNA) was efficiently delivered to the lungs for expression, achieving effective inhibition of lung metastasis ( Figure 6 a).

[0185] IL-15 superagonist mRNA (IL-15mRNA, sequence 3) was prepared according to the method of Example 3 to obtain SynTar LNP encapsulating IL-15mRNA.

[0186] SynTar LNPs encapsulating Cre mRNA were used as a control.

[0187] The injection solution was prepared as before.

[0188] Each mouse was injected intravenously with 1×10 6 Lewis Lung Cancer (LLC) (purchased from Yuchun Biological, catalog number: CM1999) cells were used to construct a lung metastasis tumor model. On the third day of modeling, IVIS in vivo imaging was performed, and the mice were randomly divided into PBS group, SynTar LNP group encapsulating Cre mRNA, and SynTar LNP group encapsulating IL-15 mRNA according to the bioluminescence intensity of the mouse lungs. The corresponding drug treatment (such as 0.5 mg / kg) was given on the 3rd, 6th, and 9th days. Figure 6 b).

[0189] The above drugs are SynTar LNP injection solution encapsulating IL-15mRNA (denoted as IL-15mRNA@SynTar LNP in the figure), SynTar LNP injection solution encapsulating Cre mRNA (denoted as Cre mRNA@SynTar LNP in the figure) and PBS.

[0190] IVIS in vivo imaging was performed on days 3, 6, 9, 11, and 13 after administration to observe the growth of lung tumors.

[0191] The results of bioluminescent in vivo imaging of mice in each group are shown in Figure 6 As shown in Figure c, the bioluminescent signal in the lungs of mice in the PBS group and the Cre mRNA@SynTarLNP group increased significantly and rapidly, indicating the rapid development of lung tumors. The bioluminescent signal in the lungs of mice in the IL-15mRNA@SynTar LNP group increased slowly.

[0192] The average lung bioluminescence quantification of each group of mice ( Figure 6 d) and individual tumor development ( Figure 6 e) It also showed that IL-15mRNA@SynTar LNP treatment significantly inhibited the development of lung tumors in mice, demonstrating superior therapeutic effects.

[0193] On the 15th day of administration, some mice were sacrificed for ex vivo bioluminescence imaging and quantification of the lungs. The results further demonstrated that the treatment with IL-15mRNA@SynTar LNP significantly reduced the tumor burden in the lungs ( Figure 6 f).

[0194] Weighing the lungs of mice, the weight change also proves that IL-15mRNA@SynTar LNP treatment effectively inhibits lung tumor progression ( Figure 6 g). Statistical survival rate Figure 6 As shown in h, it can be seen that IL-15mRNA@SynTar LNP improves the survival rate of mice.

[0195] The lungs of mice were taken and sliced ​​for H&E staining. It was found that there were obvious and numerous tumor nodules in the lungs of mice in the PBS group and the Cre mRNA@SynTar LNP group, but no obvious tumor nodules were found in the IL-15mRNA@SynTar LNP group ( Figure 6 i). In addition, immunohistochemical staining showed that the lungs of the IL-15mRNA@SynTar LNP group had more CD8+T lymphocytes than the control group, proving that the IL-15 superagonist effectively recruited and activated T cells for tumor immunotherapy ( Figure 6 j).

[0196] In summary, the SynTar LNP platform can efficiently deliver IL-15 mRNA to the lungs and efficiently express the target protein, thereby achieving effective inhibition of lung tumor growth.

[0197] Example 5: The strategy of CD31 antibody modification to enhance lung targeting is also applicable to structure-mediated lung-targeted LNPs

[0198] We validated that the strategy of CD31 antibody modification to enhance lung targeting is also applicable to structure-mediated lung-targeted LNPs.

[0199] 1. Preparation of LNPs with different formulations

[0200] The ionizable lipid A3-N11F (molecular structure as shown in Figure 2) in the previous STAR LNP was selected. Figure 7 a), and based on this, a series of LNPs with different formulations were prepared, including SM-102, 50%, 35%, 23.8%, and 50% (Mal) for screening (the specific lipid composition ratio is shown in Figure 7 c).

[0201] According to the above mRNA-LNP synthesis method, Luciferase mRNA and Figure 7mRNA-LNPs were prepared by using the respective LNP lipids (A3-N11F, DOPE, Chol, and DMG-PEG) and the corresponding molar ratios shown in c, and the following various LNPs were obtained: named as 50%, 35%, and 23.8% (the mass ratio of total lipid to RNA was 40:1);

[0202] According to the above mRNA-LNP synthesis method, Luciferase mRNA and Figure 7 mRNA-LNPs were prepared with the respective LNP lipids (SM-102, DSPC, Chol, and DMG-PEG) and the corresponding molar ratios shown in c, and the following LNPs were obtained: SM-102 (total lipid to RNA mass ratio of 40:1);

[0203] According to the above mRNA-LNP synthesis method, Luciferase mRNA and Figure 7 mRNA-LNPs were prepared with the respective LNP lipids (A3-N11F, DOPE, Chol, and DSPE-PEG-Mal) and the corresponding molar ratios shown in c, and the following LNPs were obtained: 50% (Mal) (total lipid to RNA mass ratio of 40:1);

[0204] A3-N11F SynTar LNP (denoted as SynTar in the figure) is a liposome obtained by modifying CD31 antibody with 50% (Mal) according to the method of Example 3; wherein the molar ratio of SATA / antibody is 9:1, and the molar ratio of Mal and CD31Ab in LNP is 20:1, respectively.

[0205] 2. Targeted detection

[0206] Mice were injected with 0.1 mg / kg of LNPs encapsulating luciferase obtained in 1 above through the tail vein, and bioluminescence imaging of major organs was performed 6 hours later.

[0207] The results are as follows Figure 7 As shown in Figure 2, when the molar percentage of ionizable lipid A3-N11F is 50%, effective lung-targeted mRNA delivery can be achieved. When the DMG-PEG component is completely replaced with DSPE-PEG-Mal, LNP can still achieve a considerable level of lung-targeted mRNA delivery. Figure 7 As shown in d and e, CD31 antibody modification (SynTar) further improved the lung-targeted delivery efficiency (lung fluorescence quantification increased by approximately 4 times) and specificity (lung expression percentage was higher than 95%).

[0208] 3. DLS detection of particle size

[0209] The particle sizes of the LNPs encapsulating Luciferase mRNA in each group obtained in 1 above were tested.

[0210] The results are as follows: the particle sizes are between 90nm and 150nm, and the PDI is less than 0.2, with good uniformity ( Figure 7 f) Representative particle size distribution results of A3-N11F LNP before and after antibody modification are shown in Figure 2. Figure 7 As shown in g.

[0211] 4. Encapsulation rate detection

[0212] The encapsulation efficiency of 50% (Mal) LNP and SynTar LNP was detected by Ribogreen.

[0213] The results are as follows: CD31 antibody modification did not affect the mRNA encapsulation efficiency of LNP, which remained at a high level ( Figure 7 h).

[0214] Therefore, the antibody modification enhancement strategy is also applicable to the structure-mediated lung-targeted LNP system, demonstrating the universality of this strategy.

[0215] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for enhancing tissue-targeted delivery of nucleic acid lipid nanoparticles, comprising the following steps: modifying the surface of nucleic acid lipid nanoparticles with tissue-targeted properties with antibodies to obtain antibody-modified nucleic acid lipid nanoparticles with tissue-targeted properties, thereby enhancing the tissue-targeted delivery of the nucleic acid lipid nanoparticles.

2. The method according to claim 1, characterized in that: The tissue is lung, brain or spleen; Alternatively, the antibody is a lung-targeting antibody, a brain-targeting antibody, or a spleen-targeting antibody.

3. The method according to claim 2, characterized in that: The lung-targeting antibody is a CD31 antibody, a plasmalemma vesicle-associated protein antibody or a CD326 antibody; Or, the brain-targeting antibody is a transferrin receptor monoclonal antibody; Or, the spleen targeting antibody is F4 / 80 antibody, CD5 antibody, CD4 antibody, CD8 antibody, CD3 antibody or CD19 antibody.

4. The method according to any one of claims 1 to 3, characterized in that: The tissue-targeting nucleic acid lipid nanoparticles are charge-driven tissue-targeting nucleic acid lipid nanoparticles or non-charge-driven tissue-targeting nucleic acid lipid nanoparticles; The lipids in the charge-driven tissue-targeted nucleic acid lipid nanoparticles include ionizable cationic lipids, maleimide-modified polyethylene glycol lipids and permanent cationic lipids; The lipids in the non-charge driven tissue-targeting nucleic acid lipid nanoparticles include ionizable cationic lipids and maleimide-modified polyethylene glycol lipids.

5. The method according to claim 4, characterized in that: The lipids in the charge-driven tissue-targeted nucleic acid lipid nanoparticles further include auxiliary lipids; The lipids in the non-charge-driven tissue-targeting nucleic acid lipid nanoparticles further include auxiliary lipids; The helper lipids include any one, any two or three of steroids, phospholipids and other PEG lipids.

6. The method according to claim 4 or 5, characterized in that: The permanent cationic lipid is a lipid molecule having quaternary ammonium salt properties; Alternatively, the phospholipid is a lipid having a pharmaceutically acceptable amphipathic lipid phospholipid structure.

7. The method according to claim 6, characterized in that: The lipid molecule with quaternary ammonium salt characteristics is DOTAP, DDAB, DOTMA or EPC; Or, the lipid having a pharmaceutically acceptable amphiphilic lipid phospholipid structure is DOPE, DSPE, POPE, 4ME, DOPC or DSPC; Or, the steroid is cholesterol or an analog thereof; Or the other PEG lipid is DMG-PEG, DSG-PEG or DSPE-PEG.

8. The method according to any one of claims 5 to 7, characterized in that: The charge-driven tissue-targeted nucleic acid lipid nanoparticles are charge-driven lung-targeted nucleic acid lipid nanoparticles; wherein the molar ratios of the lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 20%-80%, the molar ratio of the permanent cationic lipids to the total lipids is 10%-50%, the molar ratio of the maleimide-modified polyethylene glycol lipids to the total lipids is 0.5-5%, and the molar ratio of the auxiliary lipids to the total lipids is 0-40%; Alternatively, the non-charge-driven tissue-targeted nucleic acid lipid nanoparticles are non-charge-driven lung-targeted nucleic acid lipid nanoparticles, wherein the molar ratios of the various lipids are as follows: the molar ratio of the ionizable cationic lipids to the total lipids is 20%-80%, the molar ratio of the maleimide-modified polyethylene glycol lipids to the total lipids is 0.5-5%, and the molar ratio of the auxiliary lipids to the total lipids is 0-45%.

9. The method according to claim 8, characterized in that: The molar ratio of the permanent cationic lipid to the total lipids in the charge-driven lung-targeted nucleic acid lipid nanoparticles is 30%; Alternatively, the molar ratio of the ionizable cationic lipid to the total lipids in the non-charge-driven lung-targeted nucleic acid lipid nanoparticles is 50%.

10. Antibody-modified nucleic acid lipid nanoparticles with tissue targeting prepared by the method of any one of claims 1 to 9; Or, use of the antibody-modified and tissue-targeted nucleic acid lipid nanoparticles in the preparation of tissue-targeted products; Or a method for preparing the antibody-modified nucleic acid lipid nanoparticles with tissue targeting, comprising the steps in any one of claims 1 to 9; Or a tissue-targeted product, whose active ingredients are the modified antibodies and nucleic acid lipid nanoparticles with tissue targeting.

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