Nucleic acid delivery carrier composition and use thereof

Abstract

A nucleic acid delivery carrier composition and use thereof. The composition comprises a cationic lipid, a helper phospholipid, cholesterol, and a PEG-conjugated lipid having a molar ratio of (30-50):(4-16):(31.5-63.5):(0.5-2.5); the helper phospholipid is one or a combination of two of DOPE and DSPC, and the cationic lipid is one or a combination of more structures selected from the following structures. The composition can wrap mRNA used for protein expression to treat related deficiency diseases, is efficient, and does not have toxic side effects.

Description

A nucleic acid delivery vector composition and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nucleic acid delivery vector composition and application thereof. Background Art

[0002] In recent years, LNP (lipid nanoparticles) have shown great potential as an effective means of delivering mRNA in the fields of viral infectious diseases, malignant solid tumors, protein replacement therapy, and adoptive cell therapy. In the development of drugs in the field of protein replacement or protein supplementation, protein peptides or DNA plasmids are usually used as functional components for research and development. Compared with the above-mentioned treatment methods, mRNA therapy has higher safety and efficiency. This is reflected in the fact that mRNA can directly translate proteins in the cytoplasm without entering the cell nucleus, and there is no risk of chromosomal insertion. At the same time, mRNA uses its own living cells to translate and express the target protein, and after post-translational modification, it is closer to the natural conformation and properties of the target protein. In addition, for some diseases, effective protein drugs and broad-spectrum multimeric proteins cannot be formed in vitro. Specific sequences and multiple subunits can be expressed through mRNA to form effective functional components in vivo.

[0003] Numerous basic research and clinical trials have demonstrated that intracellular delivery of mRNA can complete the synthesis and processing of target proteins, generating corresponding functions. In vitro-transcribed mRNA has become a hot topic in the past few years. Moderna's mRNA-3927, encoding the α or β subunits (PCCA and PCCB) of propionyl-CoA carboxylase (PCC) for the treatment of propionic acidemia (PA), has completed interim data from a Phase I / II clinical trial. The mRNA-3927, encoding PCCA and PCCB subunits, is encapsulated in LNPs to restore functional PCC enzyme activity in the liver. LNPs are constructed by combining ionizable cationic lipids with auxiliary phospholipids, PEG lipids, and cholesterol to form nanoparticles. mRNA is encapsulated by binding to negatively charged mRNA under acidic conditions of pH 4.0. After dialysis, the LNP structure remains neutral under near-physiological conditions. Upon internalization into lysosomes, the cationic lipids transform to positive charge under acidic conditions, rapidly releasing the mRNA into the cytoplasm through the proton sponge effect, where it is translated and produced into proteins.

[0004] The ionizable lipids currently used in clinical practice include DLin-MC3-DMA ( Common auxiliary phospholipids include saturated lipid DSPC and unsaturated lipid DOPE. Cholesterol mainly reduces the rigidity of nanoparticles by filling the gaps between lipids, maintaining fluidity and thus increasing stability. Cholesterol extracted from lanolin is commonly used. Currently, relevant research has also been done on sterols to replace cholesterol, such as β-sitosterol. PEG-conjugated lipids are mainly used to maintain the hydrophilicity of nanoparticles, mask their surface positive charge, reduce adsorption in the body, increase circulation time, and extend the in vivo half-life of nanoparticles. The auxiliary lipid currently selected in LNPs of marketed products is DSPC, which makes it difficult to ensure the delivery of long-chain mRNA to the target site required for protein replacement therapy. The current LNP delivery efficiency needs to be improved. Summary of the Invention

[0005] One object of the present invention is to provide a nucleic acid delivery vector composition.

[0006] Another object of the present invention is to provide an application of the nucleic acid delivery vector composition.

[0007] To achieve the above-mentioned object, in one aspect, the present invention provides a nucleic acid delivery vector composition, wherein the composition comprises a cationic lipid, an auxiliary phospholipid, cholesterol, and a PEG-conjugated lipid in a molar ratio of (30-50): (4-16): (31.5-63.5): (0.5-2.5); the cationic lipid is selected from a combination of one or more of the following structures:

[0008] According to some specific embodiments of the present invention, the cationic lipid is selected from the following structures:

[0009] According to some specific embodiments of the present invention, the molar ratio of cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is (40-50): (10-16): (32-50): (0.5-2).

[0010] According to some specific embodiments of the present invention, the molar ratio of cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is (45-50):(10-16):(33.5-44.5):(0.5-1.5).

[0011] According to some specific embodiments of the present invention, the molar ratio of cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is (48-50):(10-16):(33.5-41.5):(0.5-1.5).

[0012] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid, the auxiliary phospholipid, the cholesterol and the PEG-conjugated lipid is 50:16:33.5:0.5.

[0013] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid, the auxiliary phospholipid, the cholesterol and the PEG-conjugated lipid is 48:10:40.5:1.5.

[0014] According to some specific embodiments of the present invention, the auxiliary phospholipid is one or a combination of two of DOPE and DSPC.

[0015] According to some specific embodiments of the present invention, the auxiliary phospholipid is DOPE.

[0016] According to some specific embodiments of the present invention, the PEG-conjugated lipid is PEG 2k -DMG.

[0017] According to some specific embodiments of the present invention, based on the total mass of the composition as 100%, the total mass ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is 94.6% to 97.0%.

[0018] According to some specific embodiments of the present invention, the nucleic acid is selected from one or more combinations of mRNA, siRNA, miRNA, shRNA and plasmid.

[0019] According to some specific embodiments of the present invention, the N / P ratio of the cationic lipid to the nucleic acid is 9.1 to 16.4.

[0020] According to some specific embodiments of the present invention, the N / P ratio of the cationic lipid to the nucleic acid is 13.5-16.4.

[0021] According to some specific embodiments of the present invention, the N / P ratio of the cationic lipid and the nucleic acid is 13.8.

[0022] According to some specific embodiments of the present invention, the N / P ratio of the cationic lipid and the nucleic acid is 16.4.

[0023] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is 50:16:33.5:0.5; and the N / P ratio of the cationic lipid and nucleic acid is 16.4.

[0024] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is 48:10:40.5:1.5; and the N / P ratio of the cationic lipid and nucleic acid is 16.4.

[0025] On the other hand, the present invention also provides use of the nucleic acid delivery vector composition of the present invention in the preparation of protein replacement therapeutic drugs.

[0026] In summary, the present invention provides a nucleic acid delivery vector composition and its application. The composition of the present invention has the following advantages:

[0027] The composition of the present invention is capable of encapsulating mRNA for protein expression to treat related defective diseases, and is highly effective and has no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a hydrogen spectrum of compound L1;

[0029] Figure 2 is a hydrogen spectrum of compound L1-1;

[0030] Figure 3 is a hydrogen spectrum of compound L5;

[0031] Figure 4 is a schematic diagram of fluorescence levels after tail vein injection of different combinations, wherein (a) is a schematic diagram of in vivo fluorescence levels 24 hours later, and (b) is a schematic diagram of liver fluorescence levels 24 hours later;

[0032] Figure 5 is a schematic diagram of fluorescence levels after intramuscular injection of different combinations, wherein (a) is a schematic diagram of in vivo fluorescence levels 24 hours later, (b) is a schematic diagram of liver fluorescence levels 24 hours later, and (c) is a schematic diagram of lymph node fluorescence levels 24 hours later;

[0033] Figure 6 is a schematic diagram of particle size and EE% of LNPs with different N / P ratios;

[0034] FIG7 is a schematic diagram of particle size and EE% of LNP with different molar ratios;

[0035] FIG8 is a schematic diagram of 6hEPO expression levels for combinations numbered 1 to 13;

[0036] FIG9 is a schematic diagram of 6hEPO expression levels in combinations numbered 14 to 18 and 8 and 13;

[0037] Figure 10 is a schematic diagram of the preliminary safety evaluation, wherein (a) is a schematic diagram of ALT levels; (b) is a schematic diagram of AST levels. DETAILED DESCRIPTION

[0038] Synthesis Example 1

[0039] The preparation of cationic lipid compound L1 is as follows:

[0040] Step 1:

[0041] Compound 1 (1.5 g) was dissolved in DCM (50 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.70 g), 4-dimethylaminopyridine (DMAP, 79 mg), triethylamine (1.95 g), and pentadecane-8-ol (2.94 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spotted with a standard sample (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml / min) was performed using a plate monitor. The pure product fraction was evaporated to obtain a colorless oily liquid compound 2 (2.5 g, 59.4% yield).

[0042] Step 2:

[0043] Trifluoroacetic acid (10 ml) was added to a solution of compound 2 (2.5 g) in dichloromethane (20 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (50 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (50 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the crude product after concentration was added with an appropriate amount of silica gel and DCM, mixed, and purified (25 g normal phase column, PE / EA, 0-0% 5 min, 0-50% 20 min, 50-50% 5 min, flow rate 30 ml / min) to obtain a colorless oily liquid compound 3 (1.8 g, 85% yield).

[0044] Step 3:

[0045] Compound 3 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (58 mg) and triphosgene (43 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was spotted with a standard control plate (PE / EA = 1 / 1, phosphomolybdic acid). New spots with decreasing polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and added dropwise to a mixed solution of 1-(3-hydroxypropyl)-4-methylpiperazine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% over 5 min, 0-70% over 20 min, 70-70% over 10 min, flow rate 20 ml / min) to afford Compound L1 (130 mg, 51% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound L1 is shown in Figure 1.

[0046] 1 H NMR (400MHz, Chloroform-d) δ4.95-4.86 (m, 2H), 4.14 (t, J = 8.0Hz 2H),4.11-4.03(m,4H),2.72-2.31(m,10H),2.30(s,3H),1.84-1.77(m,2 H), 1.56-1.48 (m, 8H), 1.30-1.21 (m, 40H), 0.90-0.85 (t, J = 6.8Hz, 12H).

[0047] Synthesis Example 2

[0048] The preparation of cationic lipid compound L1-1 is as follows:

[0049] Step 1:

[0050] Compound 3 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (58 mg) and triphosgene (43 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was spotted with a standard control plate (PE / EA = 1 / 1, phosphomolybdic acid). New spots with decreasing polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and added dropwise to a mixed solution of compound 1-(2-hydroxyethyl)-4-methylpiperazine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to obtain compound L1-1 (130 mg, 50% yield) as a pale yellow oily liquid. The hydrogen spectrum of compound L1-1 is shown in Figure 2.

[0051] 1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.08(s,2H),3.87(s,4H),2.79(s,2H),2.54(s,4H),2.49(s,2H),2.44(s,2H),2.27(s,3 H), 1.68 (d, J = 12.4Hz, 4H), 1.56 (d, J = 12.4Hz, 4H), 1.37 (d, J = 0.6Hz, 8H), 1.34 (d, J = 1.2Hz, 8H), 1.31-1.29 (m, 24H), 0.90 (s, 12H).

[0052] Synthesis Example 3

[0053] The preparation of cationic lipid compound L5 is as follows:

[0054] Step 1:

[0055] Compound 1-1 (5.0 g) was dissolved in DCM (200 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g), and 6-undecanol (7.39 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a standard sample of 1-1 (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml / min) was performed using a plate monitor. The pure product fractions were evaporated to give a colorless oily liquid compound 1-2 (8.0 g, 68.9% yield).

[0056] Step 2:

[0057] Trifluoroacetic acid (15 ml) was added to a solution of compound 1-2 (8.0 g) in dichloromethane (50 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 1-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (100 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 ml * 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then added with an appropriate amount of silica gel and DCM to mix the sample and purify (80 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% min, flow rate 50 ml / min) to obtain a colorless oily liquid compound 1-3 (5.8 g, 89% yield).

[0058] Step 3:

[0059] Compound 1-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (115 mg) and triphosgene (86 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard control sample of 1-3 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of compound N-(2-hydroxyethyl)hexamethylenediamine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% over 5 min, 0-70% over 20 min, 70-70% over 10 min, flow rate 20 ml / min) to afford compound L5 (170 mg, 41% yield) as a pale yellow oily liquid. The hydrogen spectrum of compound L5 is shown in Figure 3.

[0060] 1 H NMR(400MHz,Chloroform-d)δ4.95-4.86(m,2H),4.27-4.22(m,2H),4.09(d,J=12.0Hz,4H),2.78 -2.70(m,2H),2.65-2.50(m,4H),1.80-1.30(m,16),1.28-1.10(m,24H),0.87(t,J=4.0Hz,12H).

[0061] Composition Example 1: Preparation of LNP carriers combining cationic lipids and two auxiliary phospholipids

[0062] Prepare the aqueous phase: dilute the mRNA (Luc-mRNA, the nucleotide sequence corresponding to Luc-mRNA is shown in SEQ ID NO: 1 of patent application CN202210286081.0) in citric acid-sodium citrate buffer to a final concentration of 0.144 mg / mL.

[0063] Prepare the organic phase: cationic lipid L1 / L1-1 / L5:DSPC / DOPE:cholesterol:PEG 2k -DMG = 50:10:38.5:1.5 was dissolved in ethanol at a total concentration of 10 mg / mL.

[0064] Add 3ml of aqueous buffer and 1ml of lipid organic phase to a 15ml centrifuge tube, connect them to the A and B ends of the microfluidic control respectively, install the chip into the microfluidic device, set a certain flow rate ratio, and conduct a preliminary experiment with pure water and pure ethanol. When the pressure and flow rate are stable, add the feed liquid. The feed liquid flows through the chip while observing the color of the sample at the chip outlet. Discard the first and last 3 to 5 drops of milky white droplets (about 100μL), collect the middle end sample into the EP tube, and then quickly transfer the sample to the dialysis bag. Dialyze in 20mM Tris-HCl buffer for 12-24h. After dialysis, transfer to a 4℃ refrigerator for storage.

[0065] The Ribogreen kit was used to determine the encapsulation efficiency of the sample according to the operating instructions. Sample fluorescence was measured using a microplate reader at 485 nm excitation and 535 nm emission, and the sample encapsulation efficiency was calculated from the sample fluorescence value. A Zetasizer nano instrument from Malvern was used, and standard detection methods were used for particle size and PDI detection and Zeta potential analysis. The results of the particle size, PDI, and encapsulation efficiency of the mRNA-loaded LNPs prepared in this example are shown in Table 1.

[0066] Table 1. Physicochemical characterization of each combination

[0067] Luc-mRNA-lipid nanoparticles were injected into 6-8-week-old female Balb / c mice via the tail vein at a dose of 0.5 mg / kg. Five mice were used for each formulation. The L5+DSPC combination was abandoned due to low encapsulation efficiency. After 24 hours, a fluorescent substrate was injected via the tail vein. The mice were then anesthetized for in vivo imaging and dissected for ex vivo organ imaging. The results are shown in Figure 4. The results show that the L1-1+DOPE combination exhibited the highest fluorescence intensity in both whole-body and ex vivo livers, showing significant differences from the commercial SM102+DSPC combination.

[0068] Luc-mRNA-lipid nanoparticles were injected intramuscularly into 6-8 week old female Balb / c mice at a dose of 0.5 mg / kg. Five mice were used for each formulation. The L5+DSPC combination was abandoned due to low encapsulation efficiency. Fluorescent substrate was injected via the tail vein 24 hours later, and the mice were anesthetized for in vivo imaging and dissection for ex vivo organ imaging. The results are shown in Figure 5. The results showed that the L1-1+DOPE combination exhibited the highest fluorescence intensity in both in vivo imaging and in ex vivo liver imaging, showing significant differences compared to the commercial SM102+DSPC combination. In lymph nodes, this combination was equivalent and non-inferior to the SM102+DSPC combination.

[0069] Composition Example 2: Preparation of LNP carriers combining L1-1 and DOPE

[0070] (1) Examples of L1-1-LNP implementation effects with different N / P ratios

[0071] L1-1, DOPE, cholesterol, and PEG-DMG were prepared at a predetermined N / P ratio, following the LNP synthesis steps and ratios described in the Example for the Preparation of LNP Carriers Combining Cationic Lipids and Co-phospholipids. The LNPs were then tested for physical and chemical parameters, including particle size, potential, and encapsulation efficiency. The results are shown in Table 2 and Figure 6. The results show a favorable lipid / mRNA ratio between 9.1 and 16.4.

[0072] (2) Examples of L1-1-LNP implementation effects with different L1-1 mole percentages

[0073] LNPs were formed using L1-1, DOPE, cholesterol, and PEG-DMG, with L1-1 molar ratios ranging from 30% to 70%. The LNP synthesis steps were followed as described in the Example for the Preparation of LNP Carriers Combining Cationic Lipids and Co-phospholipids. The LNPs were then tested for physical and chemical parameters, including particle size, potential, and encapsulation efficiency. The results are shown in Table 3 and Figure 7. The results show a favorable trend when the molar percentage of L1-1 within the four lipid components ranges from 30% to 50%.

[0074] (3) Example of the effect of L1-1-LNP component ratio implementation

[0075] The specific composition formula and physicochemical characterization are shown in Table 4. Mice were injected with EPO-mRNA-lipid nanoparticles through the tail vein (the nucleotide sequence corresponding to EPO-mRNA is shown in SEQ ID NO: 2 of patent application CN202210286081.0). Five parallel mice were used for each formulation, and blood was collected from the mice at specific time points (6 hours and 12 hours). The resulting blood was centrifuged at 5000g for 10 minutes at 4°C to separate the serum. ELISA analysis was performed using commercially available kits. The test results are detailed in Figure 8.

[0076] Based on the data shown in Figure 8, we used the designed formula to encapsulate EPO mRNA for experiments and performed the same operations as the above-mentioned mouse experiments. The specific physicochemical properties and EPO expression levels are shown in Table 5 and Figure 9. The results showed that when the LNP ratio of L1-1: cholesterol: DOPE: PEG-DMG was 50:16:33.5:0.5 and the N / P ratio was 16.4, the EPO expression level was high; when the L1-1: cholesterol: DOPE: PEG-DMG was 48:10:40.5:1.5 and the N / P ratio was 16.4, the EPO expression level was also high. Both formulas showed significant differences in EPO expression compared with the control group SM102.

[0077] Table 2. Physicochemical parameters of LNPs with different N / P ratios

[0078] Table 3. Physicochemical parameters of LNPs with different L1-1 mole percentages

[0079] Table 4. Composition and physicochemical parameters of different L1-1-LNP combinations

[0080] Table 5. Composition and physicochemical parameters of different L1-1-LNP combinations

[0081] Preliminary safety of test example L1-1-LNP

[0082] LNPs were synthesized according to the formulations listed in Table 5. Mice were injected via the tail vein, with five mice per formulation used in parallel. Blood was collected from each mouse 24 hours later. The resulting blood was centrifuged at 5000 g for 10 minutes at 4°C to separate serum. AST and ALT levels were analyzed by ELISA using commercially available kits. The results are shown in Figure 10.

Claims

1. A nucleic acid delivery vector composition, wherein: The composition comprises a cationic lipid, an auxiliary phospholipid, cholesterol and a PEG-conjugated lipid in a molar ratio of (30-50): (4-16): (31.5-63.5): (0.5-2.5); the cationic lipid is selected from a combination of one or more of the following structures:

2. The nucleic acid delivery vector composition according to claim 1, wherein The molar ratio of cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is (40-50): (10-16): (32-50): (0.5-2).

3. The nucleic acid delivery vector composition according to claim 1, wherein The molar ratio of cationic lipid, auxiliary phospholipid, cholesterol and PEG conjugated lipid is (45-50):(10-16):(33.5-44.5):(0.5-1.5).

4. The nucleic acid delivery vector composition according to claim 1, wherein Taking the total mass of the composition as 100%, the total mass proportion of the cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is 94.6% to 97.0%.

5. The nucleic acid delivery carrier composition according to any one of claims 1 to 4, wherein The nucleic acid is selected from the group consisting of one or more combinations of mRNA, siRNA, miRNA, shRNA and plasmid.

6. The nucleic acid delivery carrier composition according to any one of claims 1 to 4, wherein The N / P ratio of the cationic lipid and nucleic acid is 9.1-16.

4.

7. The nucleic acid delivery carrier composition according to any one of claims 1 to 4, wherein The auxiliary phospholipid is one or a combination of DOPE and DSPC.

8. The nucleic acid delivery vector composition according to claim 7, wherein The auxiliary phospholipid is DOPE.

9. The nucleic acid delivery carrier composition according to any one of claims 1 to 4, wherein The PEG conjugated lipid is PEG 2k -DMG.

10. The nucleic acid delivery carrier composition according to any one of claims 1 to 4, wherein The molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is 50:16:33.5:0.5; and the N / P ratio of the cationic lipid and nucleic acid is 16.4; or The molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEG-conjugated lipid is 48:10:40.5:1.5; and the N / P ratio of the cationic lipid and nucleic acid is 16.

4.

11. Use of the nucleic acid delivery vector composition according to any one of claims 1 to 9 in the preparation of a protein replacement therapy drug.

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