A method of enhancing tissue-targeted delivery of mRNA-LNP

Abstract

The application discloses a method for enhancing tissue-targeted delivery of mRNA-LNP. The application provides a method for enhancing tissue-targeted delivery of nucleic acid lipid nanoparticles, comprising the following steps: modifying antibodies on the surface of nucleic acid lipid nanoparticles with tissue targeting to obtain nucleic acid lipid nanoparticles modified with antibodies and with tissue targeting, and realizing enhanced tissue-targeted delivery of the nucleic acid lipid nanoparticles. The application successfully realizes more efficient, specific and safe lung-targeted mRNA delivery by combining the antibody modification strategy (double targeting) on the basis of the novel lung-targeted LNP, and the strategy is applicable to any kind of targeted LNP (two kinds of charge-driven DOTAP LNP and structure-driven STAR LNP in the project).

Description

Technical Field

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

[0002] With the successful development and rapid approval of COVID-19 mRNA vaccines, mRNA drugs have demonstrated their strong application prospects. Theoretically, by designing messenger ribonucleic acid (mRNA) sequences, any protein can be expressed, such as antigens, therapeutic proteins, gene editing elements, etc., thus it can be used in many fields such as preventative vaccine development, protein replacement therapy, and gene editing. Furthermore, due to the simplicity, low cost, and short cycle of mRNA production processes, it is highly favored in clinical translation. However, mRNA carries a negative charge and is easily degraded by enzymes, so practical applications rely on safe and efficient delivery vectors. Lipid nanoparticles (LNPs) are considered one of the best vector choices, and over 90% of mRNA drug projects in clinical development rely on LNPs.

[0003] LNPs typically consist of ionizable cationic lipids, helper phospholipids, cholesterol, and polyethylene glycol lipids. Among these, ionizable cationic lipids are crucial for achieving efficient mRNA loading and lysosomal escape, and are a key focus for research institutions and R&D companies. Ionizable cationic lipid molecules can be protonated in low-pH environments (e.g., pH 4.0), thus binding and encapsulating mRNA, while in physiological environments (pH 7.4), they are uncharged, making LNPs electroneutrally neutral. Currently, most mRNA-LNPs are used for local intramuscular injection in vaccines or for intravenous administration to distribute and express mRNA in the liver. However, achieving efficient and specific extrahepatic organ-targeted mRNA delivery remains an important and critical challenge in the field, potentially broadening the application prospects of mRNA.

[0004] In recent years, some progress has been made in extrahepatic organ-targeted mRNA delivery. The main approaches are as follows: 1) Charge-mediated targeting strategies: Lung-targeted mRNA delivery can be achieved by introducing electropositive cationic lipids (e.g., quaternary ammonium lipid DOTAP) into LNPs, and spleen-targeted mRNA delivery can be achieved by introducing electronegative anionic lipids (e.g., phospholipid 18:1PA), a technique known as SORTLNP technology (Cheng et al., Nat. Nanotechnol., 15:313-320, 2020); 2) Developing novel ionizable cationic lipid structures to achieve mRNA-targeted delivery (e.g., Qiu et al., Proc. Natl. Acad. Sci. USA, 119:e2116271119, 2022); 3) Antibody-modified LNP strategies, such as modifying the LNP surface with CD31 antibodies, have successfully achieved mRNA expression in the lungs (Parhiz et al., J. Control Release, 291:106-115, 2018).

[0005] The methods described above each have certain advantages and disadvantages, such as the potential toxicity of positively charged cationic lipids, the possible low delivery efficiency of novel structures, and the poor specificity of antibody-modified LNPs. Novel mRNA-LNP drugs have very broad application prospects, but targeted delivery of mRNA to extrahepatic organs and cells remains a challenge. Summary of the Invention

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

[0007] To address the aforementioned technical problems, the first aspect of this invention provides a method for enhancing the tissue-targeted delivery of nucleic acid lipid nanoparticles, comprising the following steps: modifying the surface of tissue-targeting nucleic acid lipid nanoparticles with antibodies to obtain modified antibody-modified nucleic acid lipid nanoparticles with tissue targeting, thereby enhancing the tissue-targeted delivery of the nucleic acid lipid nanoparticles.

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

[0009] Alternatively, the antibody may be an antibody targeting the lungs, an antibody targeting the brain, or an antibody targeting the spleen.

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

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

[0012] Alternatively, the antibody targeting the spleen may be an F4 / 80 antibody, a CD5 antibody, a CD4 antibody, a CD8 antibody, a CD3 antibody, or a CD19 antibody.

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

[0014] The lipids in the charge-driven tissue-targeting 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-targeting nucleic acid lipid nanoparticles also include auxiliary lipids;

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

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

[0019] In the method described above, the permanently cationic lipid is a lipid molecule with quaternary ammonium salt properties;

[0020] Alternatively, the phospholipid may be a lipid having a pharmaceutically acceptable amphiphilic lipid phospholipid structure.

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

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

[0023] Alternatively, the steroid may be cholesterol or an analogue thereof;

[0024] Or the other PEG lipids mentioned are DMG-PEG, DSG-PEG or DSPE-PEG.

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

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

[0027] In the above method, the permanent cationic lipids in the charge-driven lung-targeting nucleic acid lipid nanoparticles account for 30% of the total lipids in a molar ratio.

[0028] Alternatively, the ionizable cationic lipids in the non-charge-driven lung-targeting nucleic acid lipid nanoparticles account for 50% of the total lipids.

[0029] The ionizable cationic lipids in the above-mentioned charge-driven lung-targeting nucleic acid lipid nanoparticles may be SM-102, ALC-0315, MC3, C12-200, CKK-E12, 4A3-SC8 or 5A2-SC8, but are not limited to these.

[0030] The ionizable cationic lipids in the aforementioned non-charge-driven lung-targeting nucleic acid lipid nanoparticles may be A3-N11F, 306-N16B, 113-N16B, 98N12-5, or 7C1, but are not limited to these.

[0031] The polyethylene glycol lipid in the maleimide-modified polyethylene glycol lipids mentioned above can be DSPE-PEG or DMG-PEG, but is not limited to these.

[0032] The cholesterol analogues mentioned above may be β-sitosterol, 7α-hydroxycholesterol or 25-hydroxycholesterol, but are not limited to these.

[0033] The aforementioned DMG-PEG can be DMG-PEG2000, but is not limited to this.

[0034] The aforementioned DSPE-PEG can be DSPE-PEG2000, but is not limited to this.

[0035] The aforementioned DSPE-PEG-Mal can be DSPE-PEG2000-Mal, but is not limited to this.

[0036] In one embodiment of the present invention, the above-mentioned charge-driven lung-targeting nucleic acid lipid nanoparticles 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 as follows). Figure 1 c).

[0037] Furthermore, the charge-driven lung-targeting 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 examples).

[0038] In another embodiment of the present invention, the non-charge-driven lung-targeting nucleic acid lipid nanoparticles 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 as follows). Figure 7 c).

[0039] Furthermore, the non-charge-driven lung-targeting 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 aforementioned nucleic acids are either chemically modified or unmodified nucleic acid molecules.

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

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

[0043] The aforementioned chemically modified or unmodified RNA molecules can be any type of RNA molecule (but are not limited to this), 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), microRNA (miRNA), small nuclear RNA (snRNA), RNA aptamers, antisense RNA, RNA nanoframeworks, RNA-ribonucleoprotein complexes, or combinations thereof.

[0044] Furthermore, the mRNA expresses proteins with therapeutic functions, fluorescent reporter proteins, or tumor-killing proteins.

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

[0046] The above-mentioned modified antibody- and tissue-targeting nucleic acid lipid nanoparticles have a higher delivery efficiency than the tissue-targeting nucleic acid lipid nanoparticles.

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

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

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

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

[0051] A1) Dissolve each lipid in the tissue-targeting nucleic acid lipid nanoparticles in an organic solvent to obtain a lipid organic phase; then mix it with the mRNA aqueous phase to obtain tissue-targeting nucleic acid lipid nanoparticles;

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

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

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

[0055] The aforementioned tissues are the lungs, brain, or spleen.

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

[0057] This study integrates existing research strategies targeting LNPs to improve the performance of mRNA-LNP delivery through a "dual-targeting" approach. The main innovations and key points are as follows:

[0058] 1) Referring to SORT LNP technology (achieving lung-targeted delivery by adding cationic lipids to a 4-component LNP), we optimized and improved a lung-targeted LNP formulation based on positively charged DOTAP. We completely replaced the helper phospholipid DSPC in the LNP with the positively charged lipid DOTAP, and completely replaced DMG-PEG with DSPE-PEG-Mal. Unlike SORT LNP, this formulation not only retains all four components but also completely replaces the original DMG-PEG with DSPE-PEG-Mal, providing the active group of Mal for subsequent antibody-modified LNPs. The LNP formulation containing 30% DOTAP (named 4C-DOTAP LNP) exhibited the best transfection efficiency and better specificity, outperforming 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-targeting mRNA delivery (named Anti LNP) is achieved.

[0060] 3) A synergistic targeting strategy using CD31 antibodies modified on 4C-DOTAP LNPs (named SynergisticTargeted LNP, SynTar LNP) significantly improves the efficiency of lung-targeting mRNA delivery. SynTar LNPs can efficiently transfect and edit lung endothelial cells, epithelial cells, and immune cells, and exhibit good stability.

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

[0062] 5) In the previously established structure-driven targeted LNP technology (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 SynTar strategy, modifying A3-N11F LNP with CD31 antibody further enhances the delivery efficiency, verifying the universality of this binding strategy.

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

[0064] 7) Based on 1-6, a broad-spectrum, universal mRNA-LNP lung-targeted delivery technology with improved delivery efficiency and specificity has been developed. This technology can efficiently and specifically transfect lung endothelial cells, epithelial cells, and immune cells, providing a novel treatment approach for lung diseases. Furthermore, there is reasonable reason to infer that the "dual-targeting" strategy of combining antibodies with LNP targeting has the potential to be extended to mRNA delivery and treatment outside the lungs, such as in the brain, liver, kidneys, and heart.

[0065] This invention achieves more efficient, specific, and safer lung-targeted mRNA delivery by combining a novel lung-targeting LNP with an antibody modification strategy (dual targeting). This strategy is applicable to any type of targeted LNP (this project includes both charge-driven DOTAP LNPs and structure-driven STAR LNPs). Finally, this novel LNP platform delivers therapeutic IL-15 superagonist mRNA, achieving effective inhibition of lung tumors. This achievement has significant reference value for the treatment of hereditary lung diseases, lung injury, idiopathic pulmonary fibrosis, cystic fibrosis, and lung cancer. Furthermore, it is reasonable to believe that, with the selection of appropriate antibodies and targeted LNPs, this strategy can be extended to targeted delivery and treatment of organs beyond the lungs, such as the brain, heart, kidneys, and pancreas. Attached Figure Description

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

[0067] Figure 2 To validate CD31 antibody modification and optimize dosage.

[0068] Figure 3 SynTar LNP exhibits significantly enhanced gene editing efficiency and can target multiple cell types in the lungs.

[0069] Figure 4 SynTar LNPs offer higher delivery efficiency and specificity compared to Anti LNPs.

[0070] Figure 5 SynTar LNP offers higher delivery efficiency, specificity, and safety compared to 5C-DOTAP LNP.

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

[0072] Figure 7 SynTar LNPs deliver novel IL-15 superagonist mRNA to the lungs for highly effective treatment of LLC lung metastases in mice. Detailed Implementation

[0073] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0075] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

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

[0077] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

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

[0079] The following examples use auxiliary phospholipids: DSPC, DOPE;

[0080] The following examples include a permanent cationic lipid (a lipid molecule with quaternary ammonium salt properties): 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 below:

[0083] Table 1 shows the structural formulas of the compounds.

[0084]

[0085]

[0086] The sodium citrate buffer (10 mM, pH 4.0) in the following examples consists of 5.962 mM citric acid, 4.037 mM trisodium citrate, and the balance being water.

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

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

[0089] 1) Aqueous phase containing mRNA molecules

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

[0091] 2) Ethanol phase containing lipid molecules

[0092] The LNP components corresponding to each embodiment were dissolved in ethanol according to the corresponding specific molar ratio to obtain an ethanol phase containing lipid molecules, with the concentration of lipid molecules in the ethanol phase being 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) are rapidly mixed at a volume ratio of 3:1 (for small-batch production, rapid pipetting is used; for large-batch production, microfluidic chip production is used). The total lipid to mRNA mass ratio is fixed at 40 / 1 or 20 / 1. The mixture is incubated at room temperature for 10 minutes, and then dialyzed in 1×PBS solution for 3 hours to obtain a 0.4 mg / mL mRNA-LNP solution (solvent: 1×PBS solution).

[0095] The encapsulation efficiency was detected as follows in the following examples: The mRNA-LNP to be tested was diluted to approximately 100 ng / mL with 1×TE buffer (1×TE buffer: obtained by diluting 20×TE buffer 20 times with sterile water; 20×TE buffer was purchased from Invitrogen). The samples were divided into two groups according to Table 2 below and added to black 96-well plates. The corresponding volumes of buffer, Triton-100 lysis buffer, and LNP sample were added to each well. The plates were then shaken at 200 rpm for 30 minutes at room temperature. RiboGreen was diluted 2000 times with 1×TE, and 100 μL of RiboGreen reaction solution was added to each well. After shaking in a microplate reader for 3 minutes, the readings were taken. The specific parameters were as follows: excitation wavelength: 480 nm, emission wavelength: 520 nm. The encapsulation efficiency was then calculated based on the readings.

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

[0097] (A represents the calculated value of the sample treated with Triton-100, and B represents the calculated value of the sample without Triton-100 treatment)

[0098] Table 2 shows the components.

[0099]

[0100] Example 1: Preparation and identification of lung-targeting DOTAP LNPs of four components

[0101] The composition and formulation of organ-targeted delivery systems (LNPs) have a significant impact on their in vivo delivery efficiency and organ / tissue targeting. We first selected SM-102LNP, a COVID-19 mRNA vaccine (Spikevax) approved by the US FDA, and then modified and optimized the Selective Organ Targeted Delivery (SORT) technology based on it. To achieve lung-targeted delivery and simplify the formulation, the traditional helper phospholipid DSPC was completely replaced with the cationic lipid DOTAP. For subsequent antibody conjugation and further formulation simplification, the traditional DMG-PEG lipid was completely replaced with the DSPE-PEG-Mal lipid terminated with maleimide.

[0102] To identify lung-specific and efficient lung-deliverable LNPs, the molar ratio of DOTAP in LNPs was adjusted (10%-50%). A series of novel LNPs (simplified four-component LNPs containing DOTAP and Mal groups) were synthesized using microfluidic technology. These LNP formulations were then screened in vivo. Figure 1 a).

[0103] I. Synthesis of Lipid Nanoparticles (LNPs) Encapsulating mRNA

[0104] Following the aforementioned mRNA-LNP synthesis method, Luciferase mRNA (sequence 1) and Figure 1 c shows the various LNP lipids (SM-102, Chol, DSPE-PEG-Mal, and DOTAP) and their corresponding molar ratios. Figure 1 The mRNA-LNP solution was prepared by (the value in c) and the following LNPs encapsulating Luciferase mRNA were obtained: F1, F2, F3 (also referred to as 4C-DOTAP in the figure), F4, F5 (total lipid to RNA mass ratio of 40:1).

[0105] Figure 1 In c, 4C-DSPC is synthesized according to the aforementioned mRNA-LNP method, combining Luciferase mRNA and... Figure 1The LNP lipids (SM-102, DSPC, Chol, and DSPE-PEG-Mal) and their corresponding molar ratios shown in c were used to prepare an 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): Following the aforementioned mRNA-LNP synthesis method, Luciferase mRNA and various LNP lipids (SM-102, DSPC, chole, DMG-PEG, and positively charged lipid DOTAP) and their respective molar ratios (SM-102, DSPC, chole, DMG-PEG, and positively charged lipid DOTAP are 35:7:27.0:1:30, respectively) are used to prepare an mRNA-LNP solution, which is the 5C-DOTAP LNP encapsulating Luciferase mRNA (total lipid to RNA mass ratio is 40:1).

[0107] II. Characterization of LNP

[0108] 1. LNP prescription screening

[0109] The LNPs that encapsulate Luciferase mRNA—F1, F2, F3 (also denoted as 4C-DOTAP in the figure), F4, F5, 4C-DSPC, and 5C-DOTAP—were subjected to the following experiments:

[0110] The LNP solutions containing Luciferase mRNA were diluted with 1×PBS to obtain LNP injection solutions containing Luciferase mRNA with a concentration of 0.1 mg / mL.

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

[0112] Image results as follows Figure 1 As shown in b, the total fluorescence intensity in the mouse lungs was statistically analyzed. Figure 1 d) The mean fluorescence intensity ratio of the lungs to the liver ( Figure 1 e) and the percentage of total fluorescence in different organs ( Figure 1f) shows that 4C-DOTAP achieved the strongest total fluorescence value in the lungs, and showed a 2.07-fold enhancement compared to the traditional 5C-DOTAP. Figure 1 d). The mean lung / liver intensity ratio of 4C-DOTAP was higher than 40, and the level was comparable to that of 5C-DOTAP. Figure 1 e) demonstrates its high lung delivery specificity. Analysis of the percentage of total fluorescence in different organs revealed that the lung expression percentage of 4C-DOTAP was similar to that of 5C-DOTAP, both exceeding 85%, further demonstrating its high lung targeting specificity. Figure 1 f).

[0113] 2. DLS particle size detection

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

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

[0116] 4C-DOTAP LNPs were stored at 4℃ for 7 days, and the particle size was determined by DLS. This type of LNP exhibits good stability, maintaining normal particle size and PDI even after 7 days of storage at 4℃. Figure 1 h, denoted as 4C-DOTAP LNP(F3)).

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

[0118] Example 2: Obtaining CD31 antibody-modified Anti LNP

[0119] Next, lung-targeted mRNA delivery was achieved by modifying LNPs with CD31 antibody. The antibody modification technology was validated and optimized. The antibody modification and purification process for LNPs is as follows: Figure 2 As shown in Figure a, the antibody was first modified using SATA reagents to insert thiol groups into the antibody. The modification of the LNP was achieved through the linkage reaction between the thiol groups on the antibody and the maleimide (Mal) groups on the surface of the LNP.

[0120] I. Exploration of Modified Antibody Ratios

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

[0122] The commercially available antibody CD31 Ab (brand: Biolegend, catalog number: 102402) was modified by thiolation (-SH) according to the instructions for use of the SATA molecule (N-succinic acid, S-acetyl mercaptoethylene glycol ester) (thermo):

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

[0124] A 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 the reaction product.

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

[0127] C) Deprotection of SATA-modified antibodies to generate active thiol groups.

[0128] Add 25 μL of deacetylation solution (0.5 M hydroxylamine hydrochloride, 25 mM EDTA dissolved in Reation Buffer, pH 7.3) to 250 μL of SATA-modified antibody solution, mix thoroughly, and incubate at room temperature for 2 hours. Then, remove excess hydroxylamine from the deacetylation solution by ultrafiltration, and replace the solution with 1×PBS containing 10 mM EDTA to reduce disulfide bond formation, obtaining a 2 mg / mL thiol-modified antibody solution (solvent: 1×PBS containing 10 mM EDTA).

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

[0130] The LNP containing DSPE-PEG-Mal was reacted with a thiolized antibody:

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

[0132] 3. Purification of antibody-conjugated LNP

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

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

[0135] II. Characterization of antibody-modified LNPs

[0136] 1. LNP prescription screening

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

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

[0139] C57BL / 6J mice were injected via the tail vein with 0.1 mg / kg of various anti-LNP solutions (referred to in the figure as 40:1, 20:1, and 10:1) for 6 hours. Major organ bioluminescence imaging was performed. LNPs prepared in Example 1, 5C-DOTAP and 4C-DSPC, were used as controls.

[0140] Image results as follows Figure 2 As shown in b, quantitative analysis of bioluminescent signal intensity indicated that there was no signal in the lungs of the unmodified antibody group, with most signals in the liver and spleen. The 40:1 group showed increased lung signal intensity, and the 20:1 group showed a significant increase in lung signal intensity compared to the 40:1 group. Further increases to 10:1 did not result in any further increase in lung signal intensity. Figure 2c). Furthermore, a modification ratio of 20:1 already resulted in higher lung-targeted delivery efficiency than 5C-DOTAP ( Figure 2 c), and has very high lung-targeting specificity ( Figure 2 d).

[0141] 2. DLS particle size detection

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

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

[0144] The results demonstrated the successful modification of the antibody, and the modified LNP exhibited good stability 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] Following the method in Example 1, Cre mRNA (sequence 2) was encapsulated in liposomes with specific components and molar ratios to obtain Cre mRNA-encapsulated 4C-DOTAP LNPs (denoted as DOTAP in the figure, with a total lipid to RNA mass ratio of 40:1). Then, following the method in Example 2, the Cre mRNA-encapsulated 4C-DOTAP LNPs were modified with antibody CD31 Ab, wherein the molar ratio of SATA / antibody was 9:1, and the molar ratio of Mal to CD31 Ab in the 4C-DOTAP LNPs was 20:1, resulting in a SynTar LNP solution prepared from Cre mRNA-encapsulated 4C-DOTAP LNPs, with 1×PBS as the solvent and a concentration of 0.1 mg / mL.

[0148] 2. In vivo testing

[0149] The Ai14 reporter mouse (CAG-loxP-stop-loxP-tdTomato) model (published literature on this animal model: Kauffman, KJ; Oberli, MA; Dorkin, JR; Hurtado, JE; Kaczmarek, JC; Bhadani, S.; Wyckoff, J.; Langer, R.; Jaklenec, A.; Anderson, DGRapid, 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) was used. Mice were injected intravenously with SynTar LNP encapsulated with CremRNA. mRNA is translated into Cre recombinase within the cell, which then cleaves the loxP site to induce the expression of tdTomato fluorescent protein in target cells. Figure 3 a). Specifically as follows:

[0150] The SynTar LNPs encapsulating Cre mRNA prepared in step 1 were diluted with 1×PBS solution to obtain SynTar LNP injection solutions with a concentration of 0.03 mg / mL encapsulating Cre mRNA.

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

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

[0153] 1) Bioluminescence imaging detection 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 quantitative fluorescence results of the lungs showed that the 4C-DOTAP LNP group was significantly higher than that of the PBS group, and the modified antibody (SynTar LNP) was even more significantly higher than that of DOTAP LNP by 3 times.

[0156] 2) Flow cytometry analysis

[0157] The effects of different LNPs on the transfection and editing of lung cell types were analyzed by flow cytometry.

[0158] Five days after injection, the lungs of mice in each group were harvested and digested to obtain single-cell suspensions. After staining, flow cytometry analysis was performed to identify cell types and tdTomato expression.

[0159] The results are as follows: DOTAP LNP can effectively transfect CD31+ edited endothelial cells, and SynTar LNP modified with CD31 antibody significantly increased the positive rate from 30.5% to 72%. Figure 3 d). The tdTomato positivity rate in all cells also increased significantly. Figure 3 e), increasing 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 efficiency testing

[0161] SynTar LNPs encapsulating Cre mRNA were stored at 4°C for 7 days, and their particle size was detected by DLS using the same method as in Example 1.

[0162] Results: SynTar LNP showed good stability, with particle size and PDI remaining normal after 7 days of storage at 4℃. Figure 3 g).

[0163] SynTar LNPs encapsulating Cre mRNA (denoted as SynTar(7 days) in the figure) stored at 4℃ for 7 days were analyzed for encapsulation efficiency using 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: DOTAP LNP showed a high encapsulation efficiency of over 95% for mRNA, and the modification antibody had no effect on the encapsulation efficiency (SynTar LNP still achieved over 95%). The stability was also good, with the encapsulation efficiency remaining above 90% even 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 types of LNPs—4C-DSPC LNP, 4C-DOTAP LNP, Anti LNP, and SynTar LNP—encapsulating Cre mRNA were compared using IVIS organ fluorescence imaging. Figure 4 a).

[0167] The anti-Cre mRNA-encapsulated LNP was prepared by modifying the 4C-DSPC LNP encapsulating Cre mRNA with an antibody according to the method in Example 3, wherein the molar ratio of SATA / antibody was 9:1, and the molar ratio of Mal to CD31 Ab in the 4C-DSPC LNP was 20:1, thus obtaining the anti-Cre mRNA-encapsulated LNP.

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

[0169] Five days after injection, mice were sacrificed to obtain liver and lung tissues, which were then embedded in an OCT compound. Sakura) and store in a -80°C freezer. After sectioning using a cryostat, mount the tissue sections onto a glass slide and add ProLong. TM Gold anti-quenching mounting medium was used, and a coverslip was placed on top. The sample was stored at -20 degrees Celsius and the images were subsequently observed using a confocal microscope.

[0170] IVIS organ fluorescence imaging was used to detect tdTomato fluorescence in major organs of each group. The results are as follows: SynTar LNPs encapsulating CremRNA showed higher lung-targeting delivery efficiency (more than 2-fold increase in lung fluorescence quantification, 2.83-fold and 2.09-fold increases, respectively) and better lung-targeting specificity than Anti LNPs encapsulating CremRNA, significantly reducing off-target expression in the liver (liver fluorescence quantification was 5.47-fold lower than that of Anti LNPs encapsulating CremRNA). Figure 4 b,c)).

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

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

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

[0174] The SynTar LNP was compared with the classic 5C-DOTAP LNP 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 in Example 1 (total lipid to RNA mass ratio of 40:1).

[0176] Following the method of Example 1, luciferase mRNA was encapsulated in liposomes with specific components and molar ratios to obtain 4C-DOTAP LNPs encapsulating luciferase mRNA (total lipid to RNA mass ratio of 40:1). Then, following the method of Example 3, the 4C-DOTAP LNPs encapsulating luciferase mRNA were modified with antibody CD31 Ab, wherein the molar ratio of SATA / antibody was 9:1, and the molar ratio of Mal to CD31Ab in the 4C-DOTAP LNPs 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 preparation method for the injection solution is the same as before.

[0178] Mice were administered 0.1 mg / kg, 0.2 mg / kg, and 0.5 mg / kg of SynTarLNP injection containing luciferase mRNA, and 1 mg / kg of 5C-DOTAP LNP injection containing luciferase mRNA via tail vein injection.

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

[0180] C57BL / 6J mice were administered 0.5 mg / kg of SynTar LNP injection containing luciferase mRNA and 1 mg / kg of 5C-DOTAP LNP injection containing luciferase mRNA via tail vein injection. Untreated mice served as controls (marked as untreated in the figure). Serum and lung tissue were collected from the mice 6 and 24 hours after administration for cytokine and liver function tests to evaluate safety. Figure 5 f).

[0181] The results are as follows: Due to the potential toxicity of positively charged lipid DOTAP, mice treated with 1.0 mg / kg 5C-DOTAP LNP experienced adverse reactions such as lethargy and decreased activity, while mice injected with 0.5 mg / kg SynTar LNP showed no significant 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 the levels 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 the 5C-DOTAP group also showed a significant increase (g, 5i). Figure 5 h). Weight was monitored for 15 consecutive days after administration (administered every 5 days; LPS (lipopolysaccharide, intraperitoneal administration, 5 mg / kg), 1.0 mg / kg 5C-DOTAPLNP group caused faster and more significant weight loss). Figure 5 j and 5k).

[0182] The results above 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 that inhibits lung metastases.

[0184] The use of DOTAP-based SynTar LNP to efficiently deliver IL-15 superagonist mRNA (IL-15 mRNA) to the lungs for expression achieved effective inhibition of lung metastases. Figure 6 a).

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

[0186] SynTar LNP, which encapsulates Cre mRNA, was used as a control.

[0187] The preparation of the injection solution is the same as before.

[0188] Administer 1×10 via tail vein injection to each mouse 6 Lewis Lung Cancer (LLC) cells (purchased from Yuchun Bio, catalog number: CM1999) were used to construct a lung metastasis model. On day 3 of modeling, IVIS in vivo imaging was performed. Based on the bioluminescence intensity of the mouse lungs, mice were randomly divided into three groups: a PBS group, a SynTar LNP group encapsulating Cre mRNA, and a SynTar LNP group encapsulating IL-15 mRNA. On days 3, 6, and 9, mice were treated with the corresponding drugs at a dose of 0.5 mg / kg (e.g., PBS, SynTar LNP, and IL-15, SynTar LNP). Figure 6 b).

[0189] The aforementioned drugs are SynTar LNP injection encapsulating IL-15 mRNA (denoted as IL-15mRNA@SynTar LNP in the figure), SynTar LNP injection 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 drug administration to observe the growth of lung tumors.

[0191] Bioluminescent in vivo imaging results of mice in each group are as follows: 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 showed a very significant and rapid increase, indicating the rapid development of lung tumors. The bioluminescent signal in the lungs of mice in the IL-15 mRNA@SynTar LNP group, however, showed a slow increase.

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

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

[0194] Weighing the mouse lungs and observing the changes in weight further demonstrates that IL-15 mRNA@SynTar LNP treatment effectively inhibits lung tumor progression. Figure 6 g). Statistical survival rate, such as Figure 6 As shown in h, it can be seen that IL-15mRNA@SynTar LNP improves mouse survival rate.

[0195] Lung tissues from mice were sectioned and stained with H&E. Numerous and obvious tumor nodules were observed in the lungs of mice in the PBS group and the Cre mRNA@SynTar LNP group, while no obvious tumor nodules were observed in the IL-15 mRNA@SynTar LNP group. Figure 6 i). Furthermore, immunohistochemical staining revealed a greater enrichment of CD8+ T lymphocytes in the lungs of the IL-15 mRNA@SynTar LNP group compared to the control group, demonstrating 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, thus 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-targeting LNP.

[0198] The strategy of enhancing lung targeting through CD31 antibody modification has been validated and is also applicable to structure-mediated lung-targeting LNPs.

[0199] 1. Preparation of LNPs with different formulations

[0200] The previously selected ionizable lipid A3-N11F from STAR LNP (molecular structure as follows) was chosen. Figure 7 As shown in a), a series of LNPs with different formulations were prepared based on this, including SM-102, 50%, 35%, 23.8%, and 50% (Mal) for screening (see figure for specific lipid composition ratios). Figure 7 c).

[0201] Following the aforementioned mRNA-LNP synthesis method, Luciferase mRNA and Figure 7mRNA-LNPs were prepared using the various LNP lipids (A3-N11F, DOPE, Chol, and DMG-PEG) and their corresponding molar ratios shown in c, resulting in the following LNPs: named 50%, 35%, and 23.8% (total lipid to RNA mass ratio of 40:1).

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

[0203] Following the aforementioned mRNA-LNP synthesis method, Luciferase mRNA and Figure 7 mRNA-LNPs were prepared using the various LNP lipids (A3-N11F, DOPE, Chol, and DSPE-PEG-Mal) and their corresponding molar ratios shown in c, resulting in the following LNPs: 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 50% (Mal) of CD31 antibody according to the method of Example 3; wherein, the molar ratio of SATA / antibody is 9:1, and the molar ratio of Mal to CD31Ab in LNP is 20:1.

[0205] 2. Targeted detection

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

[0207] The results are as follows Figure 7 As shown in b, effective lung-targeted mRNA delivery can be achieved when the molar percentage of ionizable lipid A3-N11F is 50%. Even when the DMG-PEG component is completely replaced with DSPE-PEG-Mal, LNP still achieves a comparable level of lung-targeted mRNA delivery. Figure 7 As shown in d and e, the CD31 antibody modification (SynTar) further improved the lung-targeted delivery efficiency (approximately 4-fold increase in lung fluorescence quantification) and specificity (lung expression percentage exceeding 95%).

[0208] 3. DLS particle size detection

[0209] The particle size of LNPs encapsulating Luciferase mRNA obtained in step 1 above was detected.

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

[0211] 4. Encapsulation efficiency test

[0212] Encapsulation efficiency of 50% (Mal) LNP and SynTar LNP was determined using Ribogreen.

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

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

[0215] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for enhancing the lung-targeted delivery of nucleic acid lipid nanoparticles, comprising the following steps: modifying the surface of lung-targeting nucleic acid lipid nanoparticles with antibodies to obtain lung-targeting nucleic acid lipid nanoparticles modified with antibodies, thereby enhancing the lung-targeted delivery of the nucleic acid lipid nanoparticles; The lung-targeting nucleic acid lipid nanoparticles are either charge-driven lung-targeting nucleic acid lipid nanoparticles or non-charge-driven lung-targeting nucleic acid lipid nanoparticles. The charge-driven, lung-targeting nucleic acid lipid nanoparticles are composed of lipids in the following molar ratio: 27%-44.35% SM-102, 20-50% DOTAP, 1.5% DSPE-PEG-Mal, and 20-34.15% Chol; total molar ratio is 100%. The non-charge-driven, lung-targeting 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 is 100%. The antibody is a lung-targeting antibody; the lung-targeting antibody is a CD31 antibody; The antibody is obtained by linking its surface thiol groups to the maleimide groups on the surface of the lung-targeting nucleic acid lipid nanoparticles in a molar ratio of 1:

20.

2. Modified antibody and tissue-targeting nucleic acid lipid nanoparticles prepared by the method of claim 1.

3. The application of the modified antibody and lung-targeting nucleic acid lipid nanoparticles of claim 2 in the preparation of lung-targeting products.

4. A method for preparing modified antibody- and lung-targeting nucleic acid lipid nanoparticles, comprising the following steps: modifying the surface of lung-targeting nucleic acid lipid nanoparticles with antibodies to obtain modified antibody- and lung-targeting nucleic acid lipid nanoparticles; The lung-targeting nucleic acid lipid nanoparticles are either charge-driven lung-targeting nucleic acid lipid nanoparticles or non-charge-driven lung-targeting nucleic acid lipid nanoparticles. The charge-driven, lung-targeting nucleic acid lipid nanoparticles are composed of lipids in the following molar ratio: 27%-44.35% SM-102, 20-50% DOTAP, 1.5% DSPE-PEG-Mal, and 20-34.15% Chol; total molar ratio is 100%. The non-charge-driven, lung-targeting 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 is 100%. The antibody is a lung-targeting antibody; the lung-targeting antibody is a CD31 antibody; The antibody is obtained by linking its surface thiol groups to the maleimide groups on the surface of the lung-targeting nucleic acid lipid nanoparticles in a molar ratio of 1:

20.

5. A lung-targeting product, wherein the active ingredient is the modified antibody of claim 2 and the lung-targeting nucleic acid lipid nanoparticles.

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