Protonatable cationic lipid, nucleic acid delivery system and use thereof

Abstract

The present application relates to the technical fields of biological medicine and gene therapy, and particularly relates to a protonatable cationic lipid, a nucleic acid delivery system and application thereof. The protonatable cationic lipid has a structure as shown in formula (I), and contains a protonatable group such as a hydroxyl group or an amino group at the end. The protonatable cationic lipid can form a lipid nanoparticle for loading various nucleic acid substances. The lipid nanoparticle nucleic acid delivery system has high cell transfection efficiency in vitro, and can be targeted to organs such as liver in vivo to achieve efficient gene editing. The lipid nanoparticle constructed by the protonatable cationic lipid provided by the present application has high encapsulation efficiency, good stability and excellent delivery efficiency, and has a wide application prospect in the fields of gene therapy and cell engineering.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and gene therapy, and in particular to a protonable cationic lipid and nucleic acid delivery system and its applications. Background Technology

[0002] Gene therapy is a hot research topic in modern biomedicine. It utilizes nucleic acid drugs to modify, transcribe, and translate genes, enabling the treatment and control of genetic diseases and cancer, as well as the prevention of infectious diseases and cancer. However, because nucleic acids are negatively charged, have large molecular weights, and are easily degraded by nucleases in the body, they are difficult to spontaneously cross the cell membrane and enter the cell to exert their effects. Therefore, developing safe and efficient delivery vectors to protect and deliver nucleic acid substances into cells has become a prerequisite for the various practical applications of gene therapy, especially highly efficient targeted delivery.

[0003] Lipid nanoparticles (LNPs) are currently a research hotspot in the field of non-viral gene vectors, comprising cationic lipids, sterol lipids, helper phospholipids, and polymer-coupled lipids. Among them, cationic lipids are the core of LNP function. They typically protonate under acidic conditions to carry a positive charge, thereby efficiently encapsulating negatively charged nucleic acids through electrostatic interactions to form complexes. After entering cells in vivo, they can be protonated again in the acidic environment of endosomes, promoting endosome escape and releasing nucleic acids into the cytoplasm.

[0004] Although LNP technology has been successfully applied, its performance is largely limited by the cationic lipids used. Existing cationic lipids still face many challenges: (1) insufficient protonation efficiency and weak endosome escape ability result in low nucleic acid delivery efficiency to difficult-to-transfect cells such as primary cells, stem cells, and immune cells, limiting the potential for widespread application of LNPs in cutting-edge fields such as cell therapy and gene editing; (2) efficient in vivo delivery often requires targeted modification of LNPs. However, many cationic lipids have poor compatibility with target ligands and other lipid components, leading to decreased self-assembly stability and nucleic acid encapsulation efficiency of targeted modified LNPs, which restricts the ability of LNPs to achieve efficient and tissue-specific delivery. These factors make it difficult for existing LNPs to meet the application requirements of cell therapy, targeted gene editing, and other fields.

[0005] Therefore, developing cationic lipid compounds with novel structures, simple synthesis, high efficiency in delivery, and good compatibility is of great significance for expanding the application scope of gene therapy and developing more precise delivery tools. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the present invention provides novel protonable cationic lipids, lipid nanoparticles comprising the protonable cationic lipids, and nucleic acid delivery systems constructed therefrom. The lipid nanoparticle nucleic acid delivery system can efficiently transfect nucleic acid drugs into cells or deliver them into organs, improving the delivery efficiency of nucleic acid drugs and thus enhancing their therapeutic effects.

[0007] To achieve the above and other related objectives, the first aspect of the present invention provides a protonable cationic lipid, which is a compound having the structure shown in formula (I) below, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof:

[0008] (I)

[0009] R1 is selected from C1-C8 alkyl groups;

[0010] R2 and R3 are each independently selected from C2-C10 alkyl groups;

[0011] X1 and X2 are each independently selected from -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -S-, -SS-, -C(=S)-, -NH-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NH-;

[0012] Y1 and Y2 are each independently selected from C6-C20 straight-chain or branched alkyl groups, amino acid derivatives, retinol and its derivatives, tocopherol and its derivatives, pantothenic acid and its derivatives, and panthenol and its derivatives.

[0013] G is selected from -OH, -SH, -O-(CH2)2-OH, , , , , , .

[0014] A second aspect of the present invention provides a lipid nanoparticle comprising one or more of the above-described protonable cationic lipids or pharmaceutically acceptable salts thereof.

[0015] In some embodiments of the present invention, the lipid nanoparticles further comprise any one or more combinations of sterol lipids, cofactor phospholipids, and polymer lipids.

[0016] A third aspect of the present invention provides a nucleic acid delivery system comprising the above-mentioned lipid nanoparticles and nucleic acid-like substances.

[0017] A fourth aspect of the present invention provides a pharmaceutical composition comprising the above-described nucleic acid delivery system and pharmaceutically acceptable excipients.

[0018] In some embodiments of the invention, the pharmaceutical composition is used to deliver nucleic acids encoding gene-editing tools in vivo.

[0019] In some embodiments of the present invention, the gene editing tool is selected from the CRISPR / Cas system, an adenine base editor, or a cytosine base editor.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The protonable cationic lipids provided by this invention, through the design of terminal protonation, cleavable linkages, and functionalized tails, simultaneously improve the stability, encapsulation efficiency, and transfection efficiency of the resulting lipid nanoparticles. The ends contain protonable groups such as hydroxyl and amino groups, which can be efficiently protonated in a weakly acidic environment, forming an amphiphilic structure with a positively charged hydrophilic head and a hydrophobic tail. This not only allows them to bind tightly to negatively charged nucleic acids through electrostatic interactions, significantly improving the encapsulation efficiency, but more importantly, in the acidic endosome environment within cells, the positive charge generated by protonation effectively promotes the instability and fusion of the endosome membrane, driving the efficient escape of nucleic acid drugs from the endosome into the cytoplasm. This comprehensively improves the nucleic acid delivery efficiency from both the encapsulation and release stages. Experiments show that the delivery system based on the cationic lipids of this invention maintains high delivery efficiency over a wide range of nitrogen-to-phosphorus ratios (N / P) (e.g., 4~10:1), demonstrating good formulation tolerance and process adaptability.

[0022] 2. Experiments have demonstrated that the LNP composed of the cationic lipids of this invention exhibits significantly higher transfection efficiency than the commercially available control ALC-0315 when delivering reporter genes (such as EGFP mRNA and Luciferase mRNA) to cell lines such as 293T in vitro. In in vitro cell transfection, the delivery of compound 3 increased the average expression intensity (fluorescence intensity) of the reporter gene by more than an order of magnitude compared to the commercially available control ALC-0315, demonstrating excellent delivery performance.

[0023] 3. The LNP provided by this invention exhibits good liver targeting and functional release capabilities in in vivo experiments. It can be easily coupled with target ligands. After modification, it can further improve liver targeting and gene editing efficiency in vivo, demonstrating good modifiability and application flexibility.

[0024] 4. The cationic lipid and LNP system provided by this invention is compatible with various types of nucleic acid loads and is suitable for various gene operations, including gene editing (such as CRISPR / Cas9, base editing) and gene overexpression. Its efficient delivery performance is insensitive to changes in the charge ratio (nitrogen-phosphorus ratio) in the formulation, providing a wider parameter window and higher controllability for large-scale production processes, and has broad application prospects in the fields of genetic disease treatment and tumor immunotherapy. Attached Figure Description

[0025] Figure 1 This is the proton spectrum of protonable cationic lipid compound 1 provided by the present invention.

[0026] Figure 2 This is the proton spectrum of protonable cationic lipid compound 2 provided by the present invention.

[0027] Figure 3 This is the proton spectrum of protonable cationic lipid compound 3 provided by the present invention.

[0028] Figure 4 This is the proton spectrum of protonable cationic lipid compound 4 provided by the present invention.

[0029] Figure 5 This is the proton spectrum of protonable cationic lipid compound 5 provided by the present invention.

[0030] Figure 6 This is the proton spectrum of protonable cationic lipid compound 6 provided by the present invention.

[0031] Figure 7 This is the in vivo fluorescence imaging result of the delivery of luciferase nucleic acid to the liver by the nucleic acid lipid nanoparticles (LNP-RNA) of the present invention.

[0032] Figure 8 This is a bar chart showing the gene editing efficiency of the nucleic acid lipid nanoparticle conjugate complex provided by this invention for delivering nucleic acid to the liver for gene editing.

[0033] Figure 9 This is a bar chart showing the gene editing efficiency of the nucleic acid lipid nanoparticle conjugate complex provided by this invention for delivering nucleic acid to the liver of APOC3 model mice for gene editing. Detailed Implementation

[0034] To address the shortcomings of existing technologies, the present invention aims to provide a novel, highly efficient protonable cationic lipid for nucleic acid delivery, a nucleic acid delivery system, and its application in gene therapy. The protonable cationic lipid readily protonates under weakly acidic conditions, enabling it not only to efficiently encapsulate nucleic acids but also to promote nucleic acid release in the acidic environment of endosomes, thereby significantly improving in vitro and in vivo delivery efficiency. Lipid nanoparticles constructed based on the protonable cationic lipid have demonstrated excellent transfection performance and gene editing capabilities in various cell lines, primary cells, and animal models, making them particularly suitable for liver-targeted delivery and systemic gene therapy.

[0035] In this application, unless otherwise stated, the following terms have the following meanings:

[0036] "Protonable cationic lipids" refer to compounds having the structure shown in formula (I) of this invention, whose molecular ends contain hydroxyl, amino, or other groups that can accept protons and carry a positive charge in a weakly acidic environment (such as pH 4-6), thereby enabling them to bind and deliver nucleic acids through electrostatic interactions.

[0037] "Pharmaceutically acceptable salt" refers to a salt formed by the protonable cationic lipid of the present invention and a pharmaceutically acceptable inorganic acid, organic acid or base through ionic bonding.

[0038] "Stereoisomers" are isomers with different atomic spatial arrangements resulting from the presence of one or more chiral centers in a molecule, including enantiomers, diastereomers, and mixtures thereof (such as racemates). The compounds of this invention cover all such stereoisomers.

[0039] "Solvate" refers to a complex formed by the non-covalent combination of the compound of the present invention or its pharmaceutically acceptable salt with one or more solvent molecules (such as water, ethanol, acetone).

[0040] "Non-covalent complex" refers to a stable complex formed by the compound of this invention combining with other molecules through non-covalent intermolecular interactions such as hydrogen bonds, ionic bonds, and van der Waals forces.

[0041] "Independently" is used to describe the same symbols that appear multiple times in a general formula (such as R2 and R3, X1 and X2), indicating that these groups can independently choose the same or different options within the defined range each time they appear.

[0042] "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group consisting only of carbon and hydrogen atoms and containing no unsaturated bonds. When a range of carbon atoms is specified, such as "C1-C8 alkyl", it means that the alkyl group contains 1 to 8 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, etc.

[0043] "Lipid nanoparticles (LNPs)" refer to nanoparticles with a particle size typically in the range of 50-200 nm, formed by the self-assembly of protonable cationic lipids as described in this invention, optionally with sterol lipids (such as cholesterol), cofactor phospholipids (such as DSPC, DOPE) and polymer lipids (such as DMG-PEG2000), for encapsulating and protecting nucleic acids.

[0044] "Nucleic acid delivery system" refers to a composition comprising the aforementioned lipid nanoparticles and nucleic acid-like substances encapsulated thereon. Optionally, the system may also include a small targeting ligand molecule (such as GalNAc) coupled to the surface of the LNP.

[0045] "Nitrogen-to-phosphorus ratio (N / P ratio)" refers to the ratio of the total number of moles of protonable nitrogen atoms (N) in the protonable cationic lipid used to prepare LNP to the total number of moles of phosphate groups (P) in the nucleic acid it carries.

[0046] The polydispersity index (PDI) is a parameter used in dynamic light scattering technology to characterize the width of particle size distribution. The closer its value is to 0, the more uniform the particle size distribution.

[0047] "Encapsulation rate" refers to the percentage of nucleic acids successfully encapsulated inside LNPs out of the total nucleic acids fed in. It is usually quantitatively determined by destroying free nucleic acids and is one of the key indicators for evaluating the quality of LNP preparation processes.

[0048] To achieve the above objectives, the present invention adopts the following technical solution:

[0049] The first aspect of this invention provides protonable cationic lipids, which are compounds having the structure shown in formula (I), or pharmaceutically acceptable salts, stereoisomers, or solvates thereof:

[0050]

[0051] (I)

[0052] R1 is selected from C1-C8 alkyl groups;

[0053] R2 and R3 are each independently selected from C2-C10 alkyl groups;

[0054] X1 and X2 are each independently selected from -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -S-, -SS-, -C(=S)-, -NH-, -OC(=O)NH-, -NHC(=O)NH-, -C(=O)NH-;

[0055] Y1 and Y2 are each independently selected from C6-C20 straight-chain or branched alkyl groups, amino acid derivatives, retinol and its derivatives, tocopherol and its derivatives, pantothenic acid and its derivatives, and panthenol and its derivatives.

[0056] G is selected from -OH, -SH, -O-(CH2)2-OH, , , , , , .

[0057] Preferably, R1 is selected from C1 alkyl, C2 alkyl or C4 alkyl.

[0058] Preferably, R2 is selected from C3 alkyl, C4 alkyl or C6 alkyl.

[0059] Preferably, R3 is selected from C4 alkyl or C6 alkyl.

[0060] Preferably, Y1 is selected from , , or ;

[0061] Preferably, Y2 is selected from C9 alkyl groups. , or .

[0062] In some embodiments of the present invention, the compound is selected from any of the structures shown in Table 1 below:

[0063] Table 1. Chemical structures of protonable cationic lipids

[0064] .

[0065] A second aspect of the present invention provides a lipid nanoparticle comprising one or more combinations of the above-mentioned protonable cationic lipids or pharmaceutically acceptable salts thereof.

[0066] Preferably, the lipid nanoparticles further comprise any one or more combinations of sterol lipids, cofactor phospholipids, and polymer lipids.

[0067] Preferably, the sterol lipids are selected from one or more combinations of cholesterol, β-sitosterol, stigmasterol, fucosterol, lanosterol, campesterol, taraxasterol, brassosterol, 24,25-dihydrolanosterol, and dehydroergosterol.

[0068] Preferably, the auxiliary phospholipid is selected from 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanol (DOPE), 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (... The combination of one or more of the following: POPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), N-octadecanoyl-D-erythrosphoylphosphatidylcholine (SM18:0), N-hexadecanoyl-D-erythrosphoylphosphatidylcholine (SM16:0), and 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE).

[0069] In this invention, the polymer lipid is selected from polyethylene glycol (PEG) modified lipids. The average molecular weight of the PEG is 1000-5000 Daltons. Preferably, the polymer lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, N-(carbonyl-methoxy polyethylene glycol)-1,2-distearyl-sn-glycerol-3-phosphoethanolamine, N-(carbonyl-methoxy polyethylene glycol)-1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, N-(carbonyl-biotinyl polyethylene glycol)-1,2-distearyl-sn-glycerol-3-phosphoethanolamine, and pharmaceutically acceptable salts thereof. Specifically, the polymer lipid is selected from 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearyl-SN-glycerol-3-phosphoethanolamine sodium salt (DSPE-mPEG2000), and N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine. Sodium salt (DMPE-mPEG2000), N-(carbonyl-methoxy polyethylene glycol 3400)-1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine, sodium salt (DMPE-mPEG3400), N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine, sodium salt (DPPE-mPEG2000), N-(carbonyl-methoxy polyethylene glycol 3400)-1 2-Dipalmitoyl-sn-glycerol-3-phosphoethanolamine, sodium salt (DPPE-mPEG3400), N-(carbonyl-biotinyl polyethylene glycol 2000)-1,2-distearyl-SN-glycerol-3-phosphoethanolamine, sodium salt (DSPE-PEG2000-Biotin), N-(carbonyl-biotinyl polyethylene glycol 3400)-1,2-distearyl-SN-glycerol-3-phosphoethanolamine, sodium salt (DSPE-PE) One or more combinations of G3400-Biotin, N-(carbonyl-biotinyl polyethylene glycol 2000)-1,2-dimyristoyl-SN-glycerol-3-phosphorylethanolamine sodium salt (DMPE-PEG2000-Biotin), and N-(carbonyl-biotinyl polyethylene glycol 3400)-1,2-dimyristoyl-SN-glycerol-3-phosphorylethanolamine sodium salt (DMPE-PEG3400-Biotin).In some embodiments of the present invention, the polymer lipid may be a PEG-modified lipid with a reactive functional group at the end, such as N-(carbonyl-azidopolyethylene glycol 2000)-1,2-distearyl-sn-glycerol-3-phosphorylethanolamine (DSPE-PEG2000-N3) or N-(carbonyl-dibenzocyclooctylenepolyethylene glycol 2000)-1,2-distearyl-sn-glycerol-3-phosphorylethanolamine (DSPE-PEG2000-DBCO). These polymer lipids with reactive functional groups (such as azides and DBCO) can be used to efficiently couple targeting ligands (such as GalNAc) to the surface of lipid nanoparticles via click chemistry, thereby achieving the active targeting function of the delivery system.

[0070] Preferably, in the lipid nanoparticles, the molar percentage of sterol lipids: cofactor phospholipids: protonable cationic lipids: polymer lipids is 20~55:0~30:10~60:0.5~3. More preferably, the molar percentage of sterol lipids: cofactor phospholipids: protonable cationic lipids: polymer lipids is 35~52:2~12:45~60:1.0~2.0.

[0071] In some specific embodiments of the present invention, the following preferred molar percentage formulations all yield LNPs with high encapsulation efficiency, uniform particle size, and excellent delivery performance:

[0072] Formula A: The molar percentage of the sterol lipids: co-phospholipids: protonable cationic lipids: polymer lipids is 37~42:8~12:48~52:1.2~1.8, for example 38.5:10:50:1.5.

[0073] Formula B: The molar percentage of the sterol lipids: co-phospholipids: protonable cationic lipids: polymer lipids is 40~43:2~4:53~56:1.2~1.8, for example 41.5:3:54:1.5.

[0074] Formula C: The molar percentage of the sterol lipids: co-phospholipids: protonable cationic lipids: polymer lipids is 49~52:2~4:43~46:1.2~1.8, for example 50.5:3:45:1.5.

[0075] A third aspect of the present invention provides a nucleic acid delivery system comprising the above-mentioned lipid nanoparticles and nucleic acid-like substances.

[0076] Preferably, the lipid nanoparticles are further coupled with a targeting ligand.

[0077] In this invention, a "targeting ligand" refers to a molecule or molecular fragment that can be stably linked to the surface of lipid nanoparticles through covalent bonds or high-affinity interactions, thereby endowing the particles with the ability to recognize and bind to specific cells, tissues, or organs. The targeting ligand includes, but is not limited to: small molecules (such as GalNAc, folic acid), peptides, antibodies or fragments thereof (such as single-chain antibodies, Fab), aptamers, and carbohydrates. It achieves targeted delivery through binding to cell surface specific receptors (such as desialyl glycoprotein receptors).

[0078] Preferably, the targeting ligand is selected from one or more of N-acetylgalactosamine, folic acid, mannose, biotin, and phenylboronic acid. In a preferred embodiment of the invention, N-acetylgalactosamine is coupled to the LNP surface via a click chemistry reaction to achieve efficient liver targeting. Other ligands, such as folic acid and mannose, can be selected according to the targeting requirements, and the coupling method can refer to the coupling method of N-acetylgalactosamine or conventional techniques in the art.

[0079] In this invention, "nucleic acid-like substances" refer to a class of biological macromolecules that can be encapsulated and delivered into cells by the lipid nanoparticles to exert their biological functions. These are deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and their types include, but are not limited to:

[0080] (1) RNA

[0081] Messenger RNA (mRNA): mRNA used to express target proteins within cells, such as reporter proteins (e.g., luciferase, green fluorescent protein), therapeutic proteins, or gene editing tools (e.g., CRISPR / Cas system, base editor).

[0082] Regulatory RNAs include small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), and small activating RNA (saRNA), which are used to specifically regulate the expression of target genes through RNA interference or activation pathways.

[0083] RNA related to gene editing systems: mainly refers to guide RNA (gRNA), including single-stranded guide RNA (sgRNA) used in the classic CRISPR / Cas9 system, and pegRNA used in emerging technologies such as Prime editing.

[0084] Other structural RNAs, such as circular RNAs (circRNAs).

[0085] Antisense oligonucleotides: can specifically bind to target RNA to regulate its function.

[0086] (2) DNA

[0087] Plasmid DNA: Circular double-stranded DNA, commonly used for gene cloning and long-term expression.

[0088] Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA): can be used as homologous repair templates or direct expression elements for gene editing.

[0089] Circular DNA (circDNA): DNA molecules with a special circular structure.

[0090] In some specific embodiments of the present invention, mRNAs encoding luciferase or adenine base editors (ABEs) and sgRNAs targeting specific genes are used by way of example. Those skilled in the art will understand that the above examples are merely illustrations, and any other nucleic acid molecules with similar function or structure are within the scope of this invention.

[0091] Preferably, the molar ratio (i.e. nitrogen-phosphorus ratio) of the protonable nitrogen atom in the protonable cationic lipid to the phosphate group in the nucleic acid substance is 3~12:1, which can be 3~4:1, 4~5:1, 5~6:1, 6~7:1, 7~8:1, 8~9:1, 9~10:1, 10~11:1, or 11~12:1, more preferably 4~10:1, such as 4:1, 6:1, 8:1, 10:1.

[0092] A fourth aspect of the present invention provides a pharmaceutical composition comprising the above-described nucleic acid delivery system and pharmaceutically acceptable excipients.

[0093] The "pharmaceuticalally acceptable excipients" described in this invention should be compatible with the active ingredient, meaning they can be mixed with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable excipients include alcohols such as ethanol, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protectants; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffer solutions, etc., and combinations thereof. These substances are used, as needed, to improve the stability of the formulation or to contribute to enhancing its activity or bioavailability.

[0094] Preferably, the pharmaceutical composition is used to perform gene manipulation in a subject. The "subject" is preferably a mammal, more preferably a human. The gene manipulation includes gene editing, base editing, gene overexpression, or gene expression inhibition.

[0095] In some embodiments of the invention, the pharmaceutical composition is used to deliver nucleic acids encoding a gene-editing tool within a subject to edit the subject's cells. In other embodiments of the invention, the pharmaceutical composition can also be used for gene manipulation of cells cultured in vitro or ex vivo. For example, the pharmaceutical composition can be used to edit immune cells (such as T cells), stem cells, or progenitor cells isolated from the subject. Preferably, the gene-editing tool is selected from the CRISPR / Cas system, an adenine base editor, or a cytosine base editor.

[0096] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0097] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention.

[0098] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0099] Example 1: Synthesis of protonable cationic lipids

[0100] 1. Synthesized compound 1: (2-(2-1,3-dioxolane)ethyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0101] The synthesis route is as follows:

[0102]

[0103] (1) Synthesis of compounds 1-2

[0104] Compound 1-1, 2-hexyldecanoic acid (1.00 g, 3.9 mmol, 1.0 eq) and 6-bromo-1-hexanol (1.06 g, 5.85 mmol, 1.5 eq) were added to 20 mL of dichloromethane and stirred at room temperature until dissolved. Then, EDCl (897.1 mg, 4.68 mmol, 1.2 eq), DMAP (95.3 mg, 779.94 μmol, 0.2 eq), and DIEA (2 mL, 11.7 mmol, 3 eq) were added sequentially. After reacting for 2 hours, the mixture was extracted with dichloromethane, washed with 1N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 1-2 (1.45 g). MS: m / z (ESI) = 419.1 [M+H] + .

[0105] (2) Synthesis of Compound 1

[0106] Compound 2-(2-aminoethyl)-1,3-dioxolane (100.0 mg, 853.62 μmol, 1 eq), compounds 1-2 (895.2 mg, 2.13 mmol, 2.5 eq), and sodium carbonate (452.4 mg, 4.27 mmol, 5 eq) were added to 10 mL of THF. The reaction mixture was heated to 50 °C for 8 hours, then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 1 (536.2 mg). MS: m / z (ESI) = 794.42 [M+H] + .

[0107] 1 1H NMR (300 MHz, CDCl3) δ: 0.875 (s, 12H), 1.254–1.550 (m, 57H), 1.577–1.645 (m, 8H), 1.781–1.813 (m, 3H), 2.290–2.308 (m, 2H), 2.367–2.416 (m, 4H), 2.568 (t, J = 7.5 Hz, 2H), 3.837–3.860 (m, 2H), 3.941–3.964 (m, 2H), 4.058 (t, J = 6.6 Hz, 4H), 4.890 (s, 1H). The 1H NMR spectrum of compound 1 is shown below. Figure 1 .

[0108] 2. Synthesize compound 2: ((3-ethoxy-2-hydroxypropyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0109] The synthesis route is as follows:

[0110]

[0111] (1) Synthesis of compound 2-2

[0112] Compound 2-1 (1 g, 5.46 mmol, 1 eq) and ammonia (3 ml) were added to 10 ml of THF and reacted at 50°C for 8 hours. The mixture was then concentrated, lyophilized, and purified by column chromatography to obtain compound 2-2 (867.5 mg). MS: m / z (ESI) = 120.2 [M+H] + .

[0113] (2) Synthesis of compound 2

[0114] Compound 2-2 (200 mg, 1 mmol, 1 eq), compounds 1-2 (1.05 g, 2.5 mmol, 2.5 eq), and sodium carbonate (529.7 mg, 5 mmol, 5 eq) were added to 10 mL of THF and reacted at 50 °C for 8 hours. The mixture was then extracted with dichloromethane, washed with 1 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 2 (685.2 mg). MS: m / z (ESI) = 796.90 [M+H] + .

[0115] 1 1H NMR (300 MHz, CDCl3) δ: 0.853–0.897 (m, 11H), 1.187–1.456 (m, 55H), 1.552–1.646 (m, 8H), 2.279–2.338 (m, 2H), 2.406–2.519 (m, 5H), 3.413–3.432 (m, 2H), 3.500–3.570 (m, 3H), 4.057 (t, J = 6.6 Hz, 4H). The 1H NMR spectrum of compound 2 is shown below. Figure 2 .

[0116] 3. Synthesized compound 3: ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(N-octanoyl-S-hexylcysteine ​​ester)

[0117] The synthesis route is as follows:

[0118]

[0119] (1) Synthesis of compound 3-1

[0120] Cysteine ​​(1 g, 8.25 mmol, 1 eq), 1-bromohexane (2.04 g, 12.38 mmol, 1.5 eq), and sodium hydroxide (990.4 mg, 24.76 mmol, 3 eq) were added to 10 mL of anhydrous ethanol. The mixture was reacted at room temperature for 6 hours, then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 3-1 (1.22 g). MS: m / z (ESI) = 205.92 [M+H] + .

[0121] (2) Synthesis of compound 3-2

[0122] Compound 3-1 (1 g, 4.87 mmol, 1 eq) and DIEA (1.89 g, 14.61 mmol, 3 eq) were added to 10 mL of DCM and placed on an ice bath. Octanoyl chloride (950.7 mg, 5.84 mmol, 1.2 eq) was dissolved in 5 mL of DCM and slowly added dropwise to compound 3-1 over 20 min. The mixture was then heated to room temperature and reacted for 2 h. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 3-2 (1.43 g). MS: m / z (ESI) = 330.31 [MH] - .

[0123] (3) Synthesis of compound 3-3

[0124] Compound 3-2 (1 g, 3.02 mmol, 1 eq) and 6-bromo-1-hexanol (819.3 mg, 4.52 mmol, 1.5 eq) were added to 20 mL of dichloromethane and stirred at room temperature until dissolved. Then, EDCl (693.9 mg, 3.62 mmol, 1.2 eq), DMAP (73.7 mg, 603.3 μmol, 0.2 eq), and DIEA (1.17 g, 9.05 mmol, 3 eq) were added sequentially. After reacting for 2 hours, the mixture was extracted with dichloromethane, washed with 1 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 3-3 (1.18 g). MS: m / z (ESI) = 516.35 [M + Na] + .

[0125] (4) Synthesis of compound 3

[0126] Compound 4-amino-1-butanol (49.92 mg, 0.56 mmol, 1 eq), compound 3-3 (693.6 mg, 1.4 mmol, 2.5 eq), and sodium carbonate (297.3 mg, 2.8 mmol, 5 eq) were added to 10 mL of THF and reacted at 50°C for 8 hours. The mixture was then extracted with dichloromethane, washed with 1N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 3 (441.2 mg). MS: m / z (ESI) = 916.72 [M+H] + .

[0127] 1 ¹H NMR (400 MHz, CDCl₃) δ: 0.85–0.90 (m, 12H), 1.24–1.38 (m, 36H), 1.45–1.59 (m, 8H), 1.60–1.72 (m, 12H), 2.20–2.28 (m, 4H), 2.37–2.57 (m, 10H), 2.87–3.08 (m, 4H), 3.54 (d, J = 5.2 Hz, 2H), 4.06–4.23 (m, 4H), 4.71–4.87 (m, 2H), 6.32 (d, J = 7.5 Hz, 2H). The ¹H NMR spectrum of compound 3 is shown below. Figure 3 .

[0128] 4. Synthesized compound 4: ((2-O-lacticoyl-hydroxyethyl)azadialkyl)bis(hexane-6,1-diyl)-(2-hexyldecanoate-decanoate)

[0129]

[0130] (1) Synthesis of compound 4-1

[0131] Compounds 1-2 (1.0 g, 2.38 mmol, 1 eq), aminoethanol (364.0 mg, 5.96 mmol, 2.5 eq), and sodium carbonate (1.26 mg, 11.92 mmol, 5 eq) were added to 20 mL of DCM and reacted overnight at room temperature. The mixture was then extracted with dichloromethane, washed with 1 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 4-1 (723.8 mg). MS: m / z (ESI) = 400.11 [M+H] + .

[0132] (2) Synthesis of compound 4-2

[0133] Decanoic acid (1.00 g, 5.80 mmol, 1.0 eq) and 6-bromo-1-hexanol (1.58 g, 8.71 mmol, 1.5 eq) were added to 20 mL of dichloromethane and stirred at room temperature until dissolved. Then, EDCl (1.34 g, 6.97 mmol, 1.2 eq), DMAP (141.8 mg, 1.16 mmol, 0.2 eq), and DIEA (2.25 g, 17.41 mmol, 3 eq) were added sequentially. After reacting for 2 hours, the mixture was extracted with dichloromethane, washed with 1 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 4-2 (1.63 g). MS: m / z (ESI) = 357.24 [M+Na] + .

[0134] (3) Synthesis of compound 4-3

[0135] Compound 4-1 (600.0 mg, 1.5 mmol, 1 eq), compound 4-2 (755.1 mg, 2.25 mmol, 1.5 eq), and sodium carbonate (477.4 mg, 4.5 mmol, 3 eq) were added to 10 mL of THF and reacted overnight at room temperature. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 4-3 (755.8 mg). MS: m / z (ESI) = 654.81 [M+H] + .

[0136] (4) Synthesis of compound 4-4

[0137] Compound 4-3 (500.0 mg, 764.44 μmol, 1.0 eq) and O-benzyl-L-lactic acid (165.3 mg, 917.33 μmol, 1.2 eq) were added to 10 mL of dichloromethane and stirred at room temperature until dissolved. Then, EDCl (219.8 mg, 1.15 mmol, 1.5 eq), DMAP (18.7 mg, 152.89 μmol, 0.2 eq), and DIEA (296.4 mg, 2.29 mmol, 3 eq) were added sequentially. After reacting for 2 hours, the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 4-4 (488.5 mg). MS: m / z (ESI) = 816.47 [M+H] + .

[0138] (5) Synthesis of compound 4

[0139] Compound 4-4 (400.0 mg, 490.04 μmol, 1.0 eq) and palladium on carbon (52.15 mg, 49 μmol, 0.1 eq) were added to 10 mL of dichloromethane, and H2 was bubbled through. The reaction was carried out overnight at room temperature. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 4 (328.4 mg). MS: m / z (ESI) = 726.78 [M+H] + .

[0140] 1 1H NMR (400 MHz, CDCl3) δ: 4.33–4.15 (m, 3H), 4.06 (td, J=6.7, 3.3 Hz, 4H), 2.90 (s, 1H), 2.70 (t, J=6.2 Hz, 2H), 2.50–2.39 (m, 4H), 2.36–2.25 (m, 3H), 1.59 (dd, J=18.7, 7.6 Hz, 8H), 1.48–1.19 (m, 49H), 0.88 (dd, J=6.9, 6.1 Hz, 9H). The 1H NMR spectrum of compound 4 is shown below. Figure 4 .

[0141] 5. Synthesized compound 5: ((4-hydroxybutyl)azadialkyl)bis(butane-4,1-diyl)bis(O,O'-dioctanoyl-pantothenate)

[0142]

[0143] (1) Synthesis of compound 5-1

[0144] Compound calcium pantothenate (1.0 g, 2.1 mmol, 1.0 eq), TBAI (1.55 g, 4.2 mmol, 2 eq), and benzyl bromide (897.3 mg, 5.25 mmol, 2.5 eq) were added to 20 mL of DMF. The mixture was heated to 80°C and reacted overnight. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 5-1 (1.18 g). MS: m / z (ESI) = 332.25 [M + Na] + .

[0145] (2) Synthesis of compound 5-2

[0146] Compound 5-1 (1.00 g, 3.23 mmol, 1 eq) and pyridine (1.02 g, 12.93 mmol, 4 eq) were added to 20 mL of DCM and placed on an ice bath. Octanoyl chloride (1.31 g, 8.08 mmol, 2.5 eq) was dissolved in 5 mL of DCM and slowly added dropwise to compound 5-1. After 20 min of addition, the mixture was allowed to react at room temperature for 2 h. The mixture was then extracted with dichloromethane, washed with saturated sodium carbonate aqueous solution, 1 N hydrochloric acid, and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 5-2 (1.63 g). MS: m / z (ESI) = 584.20 [M + Na] + .

[0147] (3) Synthesis of compound 5-3

[0148] Compound 5-2 (1.00 g, 1.78 mmol, 1.0 eq) and palladium on carbon (189.4 mg, 178.01 μmol, 0.1 eq) were added to 20 mL of THF, and H2 was bubbled through. The reaction was allowed to proceed overnight at room temperature. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 5-3 (749.8 mg). MS: m / z (ESI) = 470.48 [MH] - .

[0149] (4) Synthesis of compound 5-4

[0150] Compound 5-3 (600.0 mg, 1.27 mmol, 1.0 eq) and 4-bromo-1-butanol (292.0 mg, 1.91 mmol, 1.5 eq) were added to 10 mL of dichloromethane and stirred at room temperature until dissolved. Then, EDCl (292.6 mg, 1.53 mmol, 1.2 eq), DMAP (31.0 mg, 254.43 μmol, 0.2 eq), and DIEA (493.2 mg, 3.82 mmol, 3 eq) were added sequentially. After reacting for 2 hours, the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 5-4 (585.8 mg). MS: m / z (ESI) = 628.51 [M + Na] + .

[0151] (5) Synthesis of compound 5

[0152] Compound 4-amino-1-butanol (20.0 mg, 224.37 μmol, 1 eq), compound 5-3 (340.2 mg, 560.93 μmol, 2.5 eq), and sodium carbonate (118.9 mg, 1.12 mmol, 5 eq) were added to 10 mL of DCM and reacted overnight at room temperature. The mixture was then extracted with dichloromethane, washed with 1 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 5 (178.9 mg). MS: m / z (ESI) = 1168.64 [M+H] + .

[0153] 1 ¹H NMR (400 MHz, CDCl₃) δ: 6.67 (t, J = 6.0 Hz, 2H), 4.97 (s, 2H), 4.09 (t, J = 5.6 Hz, 4H), 4.01 (d, J = 11.2 Hz, 2H), 3.85 (d, J = 11.2 Hz, 2H), 3.63–3.41 (m, 6H), 2.67–2.49 (m, 9H), 2.40 (t, J = 7.6 Hz, 4H), 2.32 (t, J = 7.6 Hz, 4H), 1.73–1.56 (m, 20H), 1.37–1.22 (m, 34H), 1.06 (s, 6H), 1.01 (s, 6H), 0.91–0.85 (m, 12H). The ¹H NMR spectrum of compound 5 is shown below. Figure 5 .

[0154] 6. Synthesized compound 6: N-(2-hydroxyethoxy-2-ethyl)-N-6-(2-hexyldecanoate)hexyl-γ-aminobutyric acid-α-tocopherol ester

[0155]

[0156] (1) Synthesis of compound 6-1

[0157] Compound 1-2 (2 g, 4.77 mmol, 1 eq) and 2-(2-aminoethoxy)ethanol (1.25 g, 11.92 mmol, 2.5 eq) were added to 20 mL of anhydrous ethanol. The mixture was heated to 90°C and reacted overnight. The solution was then concentrated and purified by column chromatography to give compound 6-1 (1.25 g). MS: m / z (ESI) = 444.60 [M+H] + .

[0158] (2) Synthesis of compound 6-2

[0159] Compound 6-1 (1.00 g, 2.25 mmol, 1 eq), compound 4-bromobutyrate benzyl ester (869.2 mg, 3.38 mmol, 1.5 eq), and DIEA (873.86 mg, 6.76 mmol, 3 eq) were added to 20 mL of acetonitrile and reacted overnight at room temperature. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 6-2 (1.16 g). MS: m / z (ESI) = 620.56 [M+H] + .

[0160] (3) Synthesis of compound 6-3

[0161] Compound 6-2 (1.00 g, 1.61 mmol, 1 eq), compound DHP (271.4 mg, 3.23 mmol, 2 eq), and PPTS (81.0 mg, 322.62 μmol, 0.2 eq) were added to 10 mL of THF and reacted overnight at room temperature. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 6-3 (1.01 g). MS: m / z (ESI) = 704.44 [M+H] + .

[0162] (4) Synthesis of compound 6-4

[0163] Compound 6-3 (1.00 g, 1.42 mmol, 1.0 eq) and palladium on carbon (151.1 mg, 142.04 μmol, 0.1 eq) were added to 10 mL of THF, and H2 was bubbled through. The reaction was allowed to proceed overnight at room temperature. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to give compound 6-4 (806.2 mg). MS: m / z (ESI) = 614.44 [M+H] + .

[0164] (5) Synthesis of compound 6-5

[0165] Compound 6-4 (500.0 mg, 814.44 μmol, 1.0 eq) and α-tocopherol (420.9 mg, 977.32 μmol, 1.1 eq) were added to 20 mL of dichloromethane and stirred at room temperature until dissolved. Then, EDCl (234.2 mg, 1.22 mmol, 1.5 eq), DMAP (19.9 mg, 162.8 μmol, 0.2 eq), and DIEA (421.0 mg, 3.26 mmol, 4 eq) were added sequentially. After reacting for 2 hours, the mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 6-5 (632.7 mg). MS: m / z (ESI) = 1026.68 [M+H] + .

[0166] (6) Synthesis of compound 6

[0167] Compound 6-5 (500.0 mg, 487.0 μmol, 1.0 eq) and PPTS (12.24 mg, 48.70 μmol, 0.1 eq) were added to 10 mL of anhydrous ethanol and heated to 50°C for 1 hour. The mixture was then extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give compound 6 (398.6 mg). MS: m / z (ESI) = 943.01 [M+H] + .

[0168] 1 1H NMR (400 MHz, CDCl3) δ: 4.06 (t, J = 6.7 Hz, 2H), 3.75–3.67 (m, 2H), 3.66–3.55 (m, 4H), 2.73–2.55 (m, 8H), 2.55–2.48 (m, 2H), 2.35–2.21 (m, 3H), 2.08 (s, 3H), 2.01 (s, 3H), 1.99–1.89 (m, 5H), 1.85–1.72 (m, 2H), 1.65–1.47 (m, 9H), 1.43–1.21 (m, 40H), 1.16–1.02 (m, 6H), 0.92–0.81 (m, 18H). The 1H NMR spectrum of compound 6 is shown below. Figure 6 .

[0169] The nitrogen-to-phosphorus ratio (N / P ratio) is one of the key parameters for preparing nucleic acid-loaded LNPs. It refers to the molar ratio of protonable nitrogen atoms (N) in the cationic lipid to phosphate groups (P) in the nucleic acid. In the following examples, unless otherwise specified, an N / P ratio of 6 was used for preparation.

[0170] In the following examples, unless otherwise specified, all mice used were 6-8 week old female C57BL / 6 mice weighing 18-22g. All animal experiments were conducted in accordance with relevant animal ethics guidelines.

[0171] Example 2: Preparation of nucleic acid lipid nanoparticles (LNP-RNA)

[0172] (1) Prepare materials: reporter gene mRNA encoding luciferase (obtained by in vitro transcription and modified with capping and pseudouridine), cholesterol, DSPC, DMG-PEG, compounds 1-6 and ALC-0315, anhydrous ethanol, sodium citrate buffer (pH 4, 50 mM), PBS, 1 ml syringe, and ultrafiltration tube (100 kDa).

[0173] (2) Prepare working solutions of cholesterol, DSPC, cationic lipids (compounds 1-6 and ALC-0315) and DMG-PEG2000 in advance with anhydrous ethanol. The concentration of each working solution is 8 mg / ml. Prepare an organic phase according to the nitrogen-phosphorus ratio of 6 and the molar percentage of each component as cholesterol:DSPC:cationic lipids:DMG-PEG2000=38.5:10:50:1.5. Then dilute 100 μg Luciferase mRNA in sodium citrate buffer.

[0174] (3) Organic and aqueous phases were respectively drawn up with a 1 ml syringe and loaded into an LNP microfluidic device at an organic phase:water phase flow rate of 1:3 and a total flow rate of 9 ml / min. After preparation, PBS was added for dilution. Then, the mixture was concentrated and washed using an ultrafiltration tube. The average particle size and PDI (polydispersity index) of LNP-RNA (lipid nanoparticles encapsulating RNA) were characterized using a Malvern Zetasizer Nano ZS, and the encapsulation efficiency of RNA was determined using a Ribogreen RNA quantification kit. The results are shown in Table 2.

[0175] Table 2 Characterization data of LNP-RNA in each group

[0176]

[0177] Note: The structural formula of ALC-0315 is as follows:

[0178] .

[0179] The results showed that using compounds 1-6 provided in Example 1 of this invention as protonable cationic lipids, lipid LNPs encapsulating mRNA could be successfully prepared. The average particle size distribution of the obtained LNP-RNAs was in the range of 75-120 nm, the PDI was generally lower than 0.15, and the RNA encapsulation efficiency was higher than 88%. This indicates that these compounds can form a nanodelivery system with uniform particle size, stable structure, and high encapsulation efficiency. In the comparative experiment with the commercially available cationic lipid ALC-0315, under the same formulation and process conditions, the LNP-RNA prepared based on ALC-0315 had a larger average particle size (108.4 nm) and a higher PDI (0.18). In addition, combining different cationic lipids of this invention (such as 1+3, 3+4, etc.) also resulted in composite lipid LNP-RNAs that exhibited good particle size, distribution, and encapsulation efficiency, indicating that the lipids of this invention have good applicability in multi-component lipid formulations.

[0180] Example 3: Delivery of nucleic acid to 293T cells using nucleic acid lipid nanoparticles (LNP-RNA).

[0181] 293T cells were seeded and cultured in 24-well plates containing DMEM high-glucose medium (HyClone, SH30022.01B) supplemented with 10% FBS, with 1×10⁶ cells per well. 5 Cells containing penicillin (100 U / ml) and streptomycin (100 µg / ml) were cultured. Two hours before transfection, the medium was replaced with antibiotic-free medium. Then, 50 μl of the LNP-RNA complex prepared in Example 2 (equivalent to 2 μg EGFP mRNA) was added to each well, and the cells were cultured for 24–48 hours. The transfection efficiency of the LNP-RNA complex was determined by flow cytometry and analyzed for fluorescence, as shown in Table 3.

[0182] Table 3. Transfection efficiency and expression intensity of different LNP-RNAs in 293T cells.

[0183]

[0184] The results showed that, as shown in Table 3, the LNPs prepared from compounds 1-6 of this invention achieved transfection efficiencies (82.1%-99.0%) and average fluorescence intensities (2.6 × 10⁻⁶) of EGFP mRNA delivery in 293T cells. 5 ~1.6×10 6 Both were significantly higher than the control group LNP prepared from commercially available cationic lipid ALC-0315 (transfection efficiency 67.6%, average fluorescence intensity 7.6 × 10⁻⁶). 4 This indicates that the protonable cationic lipids provided by the present invention have significant advantages in in vitro cell transfection.

[0185] Example 4: Delivery of luciferase nucleic acid to the liver using nucleic acid lipid nanoparticles (LNP-RNA).

[0186] LNPs of compounds 1-6 or the control ALC-0315 were prepared using a microfluidic device, with the component ratio being cholesterol:DSPC: cationic lipid:PEG lipid = 41.5:3:54:1.5 (mol / mol), and Luciferase mRNA was encapsulated in each component.

[0187] The LNP-RNA was administered to mice via tail vein injection at a dose of 0.5 mg / kg (based on mRNA). In vivo imaging was performed 4 hours post-injection to analyze the fluorescence intensity in the liver region. Results are as follows: Figure 7 As shown.

[0188] like Figure 7 As shown, the LNPs prepared by compounds 1-6 of this invention exhibited strong fluorescence signals in the liver region of mice 4 hours after tail vein injection. The signal intensity was generally higher than that of the LNPs prepared by the positive control ALC-0315, proving that the LNP system constructed by the protonable cationic lipids provided by this invention can efficiently accumulate in the liver in vivo and mediate the rapid expression of reporter genes.

[0189] Example 5: Delivery of gene-editing nucleic acids to the liver via nucleic acid lipid nanoparticles (LNP-RNA) for gene editing.

[0190] LNPs of compounds 1–6 were prepared using a microfluidic device with a nitrogen-to-phosphorus ratio of 4–10:1 and a component ratio of cholesterol:DSPC:cationic lipid:PEG lipid = 50.5:3:45:1.5 (mol / mol). ABE mRNA and PCSK9s gRNA (mixed at a 1:1 mass ratio) were then encapsulated separately. LNPs prepared using the same formulation and procedure with cationic lipid ALC-0315 served as a control group.

[0191] The LNP-RNA was administered to mice via tail vein injection at a dose of 0.5 mg / kg (based on mRNA). Three days after injection, the mice were sacrificed, and their livers were dissected and collected for DNA extraction and gene editing identification. The gene editing efficiency was obtained through first-generation sequencing, as shown in Table 4 below.

[0192] Table 4. Editing efficiency of nucleic acid delivery by different compounds

[0193]

[0194] The results, as shown in Table 4, indicate that in in vivo gene editing experiments, the LNPs prepared from compounds 1-6 of this invention delivered ABE mRNA and sgRNA with gene editing efficiencies (44%-68%) in mouse livers that were generally comparable to the positive control ALC-0315 (45%-62%), and exhibited comparable or better editing effects under various nitrogen-phosphorus ratios. At nitrogen-phosphorus ratios of 8 and 10, compounds 4 and 6 both achieved editing efficiencies of 68%, significantly higher than the editing efficiency of ALC-0315 (62%) under the same conditions. This demonstrates that the cationic lipids provided by this invention can maintain efficient delivery and gene editing capabilities even at higher nitrogen-phosphorus ratios, exhibiting superior dose adaptability and in vivo stability. Furthermore, each compound showed a stable editing efficiency trend under different nitrogen-phosphorus ratios, proving that its structural design has good protonation controllability and endosomal escape capability, making it suitable for in vivo systemic delivery of gene editing tools and achieving efficient and stable gene editing effects.

[0195] Example 6: Delivery of nucleic acid to the liver via nucleic acid-lipid nanoparticle conjugate complex for gene editing

[0196] 1. Preparation of nucleic acid-lipid nanoparticle coupling complexes

[0197] (1) Prepare materials: reporter gene mRNA encoding luciferase (obtained by in vitro transcription and modified with capping and pseudouridine), cholesterol, DSPC, DMG-PEG, compound, anhydrous ethanol, sodium citrate buffer (pH4, 50mM), PBS, GalNAc-DBCO, 1ml syringe, ultrafiltration tube (100kDa).

[0198] (2) Prepare working solutions of cholesterol, DSPC, cationic lipids and DMG-PEG2000 in advance with anhydrous ethanol. The concentration of each working solution is 8 mg / ml. Prepare organic phases according to the nitrogen-phosphorus ratio of 6 and the molar percentage of cholesterol:DSPC:cationic lipids:DMG-PEG2000:DSPE-PEG2000-N3 of 41.5:3:54:1.0:0.5. Then dilute 100 μg of Luciferase mRNA in sodium citrate buffer.

[0199] (3) The organic phase and aqueous phase were respectively drawn up with a 1ml syringe and loaded into the LNP microfluidic device at an organic phase: aqueous phase flow rate of 1:3 and a total flow rate of 9ml / min. After preparation, PBS was added for dilution. Then, the solution was concentrated and washed using an ultrafiltration tube.

[0200] (4) GalNAc-DBCO was then added to LNP and placed at room temperature for 2 hours to achieve coupling via a click chemical reaction. The mixture was then concentrated and washed using an ultrafiltration tube to obtain the GalNAc-coupled LNP-RNA complex.

[0201] Using the same method as in Example 2, encapsulation efficiency, particle size, and PDI were tested, and the test results are shown in Table 5 below.

[0202] Table 5 Characterization data of nucleic acid lipid nanoparticle coupled complex samples in each group

[0203]

[0204] 2. Nucleic acid-lipid nanoparticle conjugates deliver nucleic acids to the liver for gene editing.

[0205] LNPs containing compounds 1-6 or the control ALC-0315 were prepared using a microfluidic device. The components were prepared with a nitrogen-to-phosphorus ratio of 8 and a component ratio of cholesterol:DSPC: cationic lipid: PEG lipid:DSPE-PEG-N3 = 41.5:3:54:1.0:0.5 (mol / mol). ABE mRNA and PCSK9 sgRNA were then encapsulated in these components. Subsequently, an N-acetylgalactosamine (GalNAc) targeting ligand was coupled to the DSPE-PEG on the surface of the LNPs via click chemistry.

[0206] Mice were administered the GalNAc-targeted LNP at a dose of 0.5 mg / kg via tail vein injection. Three days after injection, the mice were dissected and liver samples were collected for DNA extraction and gene editing identification. First-generation sequencing results yielded... Figure 8 The gene editing efficiency is shown.

[0207] The results show that: Figure 8 As shown, compared with the corresponding LNPs without GalNAc conjugation, the LNPs prepared based on compounds 1-6 of this invention showed significantly improved gene editing efficiency in mouse livers after surface conjugation with GalNAc targeting ligands. Furthermore, the editing efficiency of these conjugated LNPs was generally higher than that of the ALC-0315 control group LNPs prepared under the same conditions. This indicates that the LNPs prepared by the cationic lipids of this invention are easily modified for targeted surfaces, and that modification can further enhance gene editing efficiency by improving liver targeting.

[0208] 3. Delivery of nucleic acid to APOC3 model mice via nucleic acid-lipid nanoparticle conjugate complex.

[0209] LNPs containing compounds 1-6 were prepared using a microfluidic device. The component ratio was cholesterol:DSPC: cationic lipid: PEG lipid:DSPE-PEG-N3 = 41.5:3:54:1.0:0.5 (mol / mol), and ABE mRNA and PCSK9sgRNA were encapsulated separately. B6 / JGpt-Tg(hAPOC3)22 / Gpt model mice (with the human APOC3 gene transferred into them) were used. LNPs were administered to mice via tail vein injection at a dose of 0.5 mg / kg. Three days after injection, dissection and liver sampling were performed. DNA extraction and gene editing identification were conducted on the liver. First-generation sequencing results yielded the following... Figure 9 The gene editing efficiency is shown.

[0210] The results show that: Figure 9 As shown, in APOC3 gene-editing model mice, LNPs prepared from the compounds of this invention and conjugated with GalNAc exhibited highly efficient gene-editing capabilities, directly demonstrating that the nucleic acid delivery system provided by this invention can not only perform gene editing under normal physiological conditions (such as...) Figure 8 As shown, it can perform efficient gene editing and effectively target and edit specific pathogenic genes in disease models, demonstrating clear potential for therapeutic applications.

[0211] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A protonable cationic lipid, characterized in that, The compound having the structure shown in formula (Ⅰ), or a pharmaceutically acceptable salt or stereoisomer thereof: （Ⅰ） R1 is selected from C1 alkyl, C2 alkyl or C4 alkyl; R2 and R3 are selected from C6 alkyl groups; X1 and X2 are selected from -O-; Y1 and Y2 are selected from G is selected from -OH.

2. The protonable cationic lipid according to claim 1, characterized in that, The compound is selected from the structures shown below: 。 3. A lipid nanoparticle, characterized in that, Comprising one or more combinations of the protonable cationic lipids of claim 1 or 2 or their pharmaceutically acceptable salts.

4. The lipid nanoparticles according to claim 3, characterized in that, It also includes any one or more combinations of sterol lipids, cofactor phospholipids, and polymer lipids.

5. The lipid nanoparticles according to claim 4, characterized in that, Includes one or more of the following characteristics: (a) The sterol lipids are selected from one or more combinations of cholesterol, β-sitosterol, stigmasterol, fucosterol, lanosterol, campesterol, taraxasterol, brassosterol, 24,25-dihydrolanosterol and dehydroergosterol; (b) The auxiliary phospholipid is selected from one or more combinations of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanol, 1,2-distearyl-sn-glycerol-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, dipalmitoylphosphatidylglycerol, oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, distearylphosphatidylethanolamine, N-octadecanoyl-D-erythrosphoylphosphatidylcholine, N-hexadecanoyl-D-erythrosphoylphosphatidylcholine and 1-stearoyl-2-oleoylphosphatidylethanolamine; (c) The polymer lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, N-(carbonyl-methoxy polyethylene glycol)-1,2-distearyl-sn-glycerol-3-phosphoethanolamine, N-(carbonyl-methoxy polyethylene glycol)-1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, N-(carbonyl-biotinyl polyethylene glycol)-1,2-distearyl-sn-glycerol-3-phosphoethanolamine, and pharmaceutically acceptable salts thereof.

6. The lipid nanoparticles according to claim 4, characterized in that, The molar percentage of the sterol lipids: cofactor phospholipids: protonable cationic lipids: polymer lipids is 20~55:0~30:10~60:0.5~3.

7. A nucleic acid delivery system, characterized in that, This includes lipid nanoparticles as described in any one of claims 3 to 6 and nucleic acid-like substances.

8. The nucleic acid delivery system according to claim 7, characterized in that, Includes one or more of the following characteristics: (a) The lipid nanoparticles are also coupled with a targeting ligand on their surface; (b) The nucleic acid material is selected from one or more of the following: messenger RNA, small interfering RNA, microRNA, short hairpin RNA, circular RNA, antisense oligonucleotide, guide RNA, plasmid DNA, single-stranded or double-stranded DNA; (c) The molar ratio of the protonable cationic lipid to the phosphate group in the nucleic acid is 3~12:

1.

9. The nucleic acid delivery system according to claim 8, characterized in that, Includes one or more of the following characteristics: (a1) The targeting ligand is selected from one or more of N-acetylgalactosamine, folic acid, mannose, biotin, and phenylboronic acid; (b1) The nucleic acid substances mentioned are mRNA and sgRNA encoding reporter proteins or gene editing tools; (c1) The molar ratio of protonable nitrogen atoms in the protonable cationic lipid to phosphate groups in the nucleic acid is 4~10:

1.

10. A pharmaceutical composition, characterized in that, It comprises a nucleic acid delivery system as described in any one of claims 7 to 9 and pharmaceutically acceptable excipients.

11. The pharmaceutical composition of claim 10, characterized in that, The pharmaceutical composition is used to deliver nucleic acids encoding gene-editing tools in the body of a subject.

12. The pharmaceutical composition according to claim 11, characterized in that, The gene editing tool is selected from the CRISPR / Cas system, an adenine base editor, or a cytosine base editor.

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