Nucleic acid-lipid nanoparticles suitable for intramuscular administration, formulations thereof, and uses thereof

Abstract

The present invention discloses a nucleic acid-lipid nanoparticle, a preparation thereof and an application suitable for intramuscular injection, belonging to the field of biomedicine. The nucleic acid-lipid nanoparticle of the present invention is composed of the following components: (a) at least one nucleic acid; (b) at least one ionizable lipid, accounting for 20 mol% to 35 mol% of the total lipids; (c) at least one non-ionizable cationic lipid, accounting for 15 mol% to 30 mol% of the total lipids; (d) a lipid mixture of neutral phospholipids or their derivatives, accounting for 0 mol% to 10 mol% of the total lipids; (e) a mixture of cholesterol or its derivatives, accounting for 40 mol% to 56 mol% of the total lipids; (f) a mixture of PEG or its derivatives, accounting for 1.5 mol% to 3 mol% of the total lipids. The nucleic acid-lipid nanoparticle of the present invention can encapsulate mRNA or plasmid DNA, is particularly suitable for intramuscular injection, can be expressed for a long time at the injection site, produce high-titer specific neutralizing antibodies and reduce toxic and side effects, and can also significantly improve the thermal stability of the preparation, thus facilitating the transportation and distribution of nucleic acid vaccines and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a nucleic acid-lipid nanoparticle, a preparation thereof, and an application thereof that are suitable for intramuscular administration. Background Art

[0002] mRNA vaccines based on lipid nanoparticle (LNP) technology can simultaneously stimulate humoral immunity and cellular immunity, and are more protective, cost-effective, and capable of larger-scale production than other types of vaccines. After mRNA vaccines are administered, side effects such as fever, allergy, cytokine storm, and hepatotoxicity occur, and their incidence rate is higher than that of traditional vaccines. Although these are all mild and transient side reactions, this experience brings uncertainty to the popularization of nucleic acid vaccine technology in more application scenarios.

[0003] Currently, the composition and active ingredients of mRNA vaccines are as follows:

[0004] The lipid formulations of the first batch of mRNA vaccines to obtain market approval are all composed of an ionizable cationic lipid, a neutral phospholipid (DSPC), cholesterol, and PEG or its derivative lipid. The molar ratio of the four components follows the proportion range covered by US Patent US8058069, that is: the cationic lipid, neutral phospholipid, cholesterol or its derivative, and PEG or its derivative lipid account for 50-65 mol%, 4-10 mol%, 30-40 mol%, and 0.5-2 mol% of the total lipid, respectively. Another example is the patent 202210336875.3 recently applied for by CanSino Biologics in China, "A Novel Coronavirus mRNA Vaccine and Its Preparation Method and Use", the disclosed lipid components are: cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol-lipid molar ratio is 30-60:5-20:20-50:0.1-10.

[0005] mRNA vaccines simultaneously activate humoral immunity and cellular immunity:

[0006] As mentioned above, compared with traditional vaccines, the COVID-19 mRNA vaccine has protective efficacy, mainly because it can simultaneously activate humoral immunity and cellular immunity. Cellular immunity generates longer-lasting memory cells and T-cell immunity, which is not only beneficial for preventive vaccines to play a role, but also the main mechanism relied on by tumor vaccines.

[0007] Individuals vaccinated with a COVID-19 mRNA vaccine that is negative for antibodies but positive for neutralizing antibodies (e.g., the mRNA-1273 vaccine) are also protected by the vaccine. According to the estimates of Gilbert, P.B. et al., neutralizing antibodies contribute approximately two-thirds of the efficacy of mRNA vaccines. The corresponding values of vaccine protection for neutralizing antibody titers NT50 of 10, 100, and 1000 58 days after vaccination are 78%, 91%, and 96%. The neutralizing antibody titer of the clinical phase II vaccine has been used as a basis for vaccine approval as a means of "immune bridging" and has been adopted by many countries and regions.

[0008] Systemic expression of the LNP vector is one of the sources of acute side effects of nucleic acid vaccines:

[0009] According to reports by Patone, M. et al., Hassett, K.J. et al., and Pardi, N. et al., after intravenous injection of the mRNA-LNP formulation, nucleic acid lipid nanoparticles mainly attack liver tissue, and also have transfection effects on major organs and tissues such as the lungs, brain tissue, heart, and blood vessel endings, leading to risks of fever, chills, and other adverse reactions. Acetaminophen needs to be taken in advance before using Onpattro (MC3-LNP, Alnylam) to cope with potential systemic inflammation and neurotoxic side effects.

[0010] According to another report by Ndeupen, S. et al., nasal inhalation of LNP also leads to a significant increase in toxicity. Nasal inhalation of the same dose of LNP causes similar inflammatory reactions in the lungs and results in a high mortality rate.

[0011] Intramuscular injection allows for a relatively large volume at the injection site, so it may cause fewer adverse injection site reactions and is one of the best routes of administration for vaccines. Through intramuscular injection and subcutaneous injection, more persistent protein expression is achieved in tissues compared to intravenous administration. Nevertheless, studies have confirmed that after intramuscular injection, a significant number of nucleic acid lipid nanoparticles are still systemically delivered throughout the body via the circulatory system, targeting liver tissue, lung tissue, brain tissue, and myocardial tissue, and stimulating the generation of a large amount of foreign proteins in a short period. This systemically delivered lipid nanoparticles and a large amount of off-target expressed foreign proteins stimulate tissue damage, neurotoxicity, and the activation of cytokines and complement, resulting in transient side effects.

[0012] Ionizable lipids are essentially a potent immune adjuvant – causing systemic side effects:

[0013] Lipid nanoparticles themselves have adjuvant activity. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, researchers have demonstrated that empty lipid nanoparticle formulations (without bioactive substances such as nucleic acids) inherently have adjuvant activity, which can promote the induction of strong follicular helper T cells, germinal center B cells, long-lived plasma cells, and memory B cell responses in mice, and are associated with the generation of persistent protective antibodies. Lipid nanoparticles stimulate extremely strong humoral immunity, producing an excessive amount of antibodies in a short time, increasing the burden on the body's immune system.

[0014] Cheng et al. added a fifth charged lipid to the LNP system to regulate the internal charge of lipid nanoparticles. Without disrupting the original ratio of the four components (5A2-SC8:DOPE:Chol:PEG = 15:15:30:3), the ratio of DOTAP was adjusted from 0 to 100%, and a series of LNPs with different DOTAP contents were prepared. By intravenous injection of liposomes, the delivered genes showed organ selectivity. Maintaining the dose of ionizable lipid in LNP is the guarantee to provide sufficient adjuvant effect, especially under intramuscular injection, in order to produce enough vaccines. The above report by Cheng et al. used Dotap to change the surface charge of LNP, thereby changing the delivery targeting. Because it was administered by intravenous injection, in order to reduce the inflammatory response and systemic toxicity caused by ionizable lipids, the dosage had to be reduced, thus reducing the adjuvant effect of LNP.

[0015] Ionizable lipids are the key factors for the adjuvant activity of LNP and have an obvious dose effect. Non-ionizable cationic lipids have no adjuvant effect and cannot induce sufficient antibody titers. It is worth noting that all the above-mentioned charge-mediated lipid nanoparticle targeting selection methods use intravenous injection. To avoid the toxic reactions caused by intravenous administration, the concentration of ionizable lipids is forced to be reduced, so the immunoadjuvant efficacy of LNP is also reduced accordingly and is no longer suitable for nucleic acid vaccines.

[0016] In addition, the existing LNP preparations also have the problem of poor stability. mRNA-LNP finished products need to be stored at low temperatures of -20°C to -40°C, which brings inconvenience to vaccine distribution. To solve this problem, there are currently in-research mRNA-LNP freeze-dried powders, but this method increases the difficulty and cost of vaccine production. In addition, there are also patent reports on using synthetic thermostable imidazole-modified ionizable cationic LNPs to solve the ultra-low temperature storage and cold chain transportation problems of existing mRNA vaccines. However, this method also increases the production difficulty and cost. Summary of the Invention

[0017] One of the objectives of the present invention is to provide a nucleic acid-lipid nanoparticle suitable for intramuscular injection to solve the above problems.

[0018] To achieve the above object, the technical solution adopted by the present invention is as follows: A nucleic acid-lipid nanoparticle suitable for intramuscular administration, which is composed of the following components: (a) at least one nucleic acid; (b) at least one ionizable lipid, accounting for 20 mol% to 35 mol% of the total lipids; (c) at least one non-ionizable cationic lipid, accounting for 15 mol% to 30 mol% of the total lipids; (d) a lipid mixture of neutral phospholipids or their derivatives, accounting for 0 mol% to 10 mol% of the total lipids; (e) a mixture of cholesterol or its derivatives, accounting for 40 mol% to 56 mol% of the total lipids; (f) a mixture of PEG or its derivatives, accounting for 1.5 mol% to 3 mol% of the total lipids; the nucleic acid molecule of (a) is encapsulated inside the lipid nanoparticle composed of (b), (c), (d), (e) and (f). The lipid layer of the present invention protects the nucleic acid from enzymatic degradation in vivo.

[0019] The inventors of the present application have confirmed through experiments that: in traditional LNP, adding an appropriate amount of non-ionizable cationic lipid, high concentrations of non-ionizable cationic lipid can change the delivery targeting of lipid nanoparticles, reduce the systemic delivery ability of nucleic acid-lipid nanoparticles, and increase their stability. Its characteristics are that when administered intramuscularly, it can be expressed at the injection site for a long time, and the target gene is mainly expressed at the injection site, producing specific antibodies and neutralizing antibodies, and reducing transfection and gene expression in the liver, lung, brain and spleen.

[0020] As mentioned above, although it has been reported that the tissue targeting of LNP can be changed by modifying the surface charge of LNP, however, the current reports can only be used for intravenous injection, and the adjuvant effect of LNP is significantly reduced and cannot be used for nucleic acid vaccines. In the present invention, by supplementing an appropriate amount of non-ionizable cationic lipid instead of replacing the ionizable lipid, the adjuvant effect of the preparation of the present invention is still provided by the ionizable lipid and is suitable for intramuscular injection.

[0021] In addition, the present invention reveals that the stability of the liposome is enhanced after incorporating an appropriate amount of non-ionizable cationic lipid; one of its performance characteristics is: the tolerance to non-ionic surfactants is increased, and a concentration of 10 vol% or more of Triton X-100 is required to completely resolve and separate.

[0022] As a preferred technical solution: it consists of the following components: (a) mRNA; (b) an ionizable lipid, accounting for 23.01 mol% to 24.17 mol% of the total lipids; (c) a non-ionizable cationic lipid, accounting for 23.01 mol% to 24.17 mol% of the total lipids; (d) neutral phospholipids, accounting for 4.91 mol% to 9.35 mol% of the total lipids; (e) cholesterol, accounting for 42.43 mol% to 44.56 mol% of the total lipids; (f) PEG lipid, accounting for 2.20 mol% of the total lipids. In this application, it is referred to as " 45" (when the neutral phospholipid content is 4.91 mol%), and " 46" formulation (when the neutral phospholipid content is 9.35 mol%).

[0023] As a preferred technical solution, it consists of the following components: (a) at least one nucleic acid; (b) at least one ionizable lipid, accounting for 20 mol% to 35 mol% of the total lipids; (c) at least one non-ionizable cationic lipid, accounting for 15 mol% to 30 mol% of the total lipids; (d) a mixture of cholesterol or its derivatives, accounting for 40 mol% to 56 mol% of the total lipids; (e) a mixture of PEG or its derivatives, accounting for 1.5 mol% to 3 mol% of the total lipids; the nucleic acid molecule of (a) is encapsulated inside the lipid nanoparticles composed of (b), (c), (d) and (e).

[0024] As a further preferred technical solution: it consists of the following components: (a) mRNA or DNA; (b) an ionizable lipid, accounting for 25.45 mol% of the total lipids; (c) a non-ionizable cationic lipid, accounting for 25.45 mol% of the total lipids; (d) cholesterol, accounting for 46.90 mol% of the total lipids; (e) PEG lipid, accounting for 2.20 mol% of the total lipids. In this solution, it does not contain neutral phospholipids, and in addition to delivering mRNA, it can also deliver DNA. In this application, it is referred to as " 25" formulation.

[0025] In another preferred embodiment, the nucleic acid-lipid nanoparticles comprise: (a) mRNA or DNA; (b) an ionizable lipid, accounting for 30.88 mol% of the total lipids; (c) a non-ionizable cationic lipid, accounting for 15.35 mol% of the total lipids; (d) cholesterol, accounting for 42.42 mol% of the total lipids; (e) neutral phospholipids, accounting for 4.8 mol% to 9.4 mol% of the total lipids; (f) PEG lipid, accounting for 2.20 mol% of the total lipids. The nucleic acid-lipid nanoparticles in this preferred embodiment also do not contain neutral phospholipids, and in addition to delivering mRNA, it can also deliver DNA. In this application, it is generally referred to as " 74" formulation.

[0026] As a further preferred technical solution, the molar concentration of the ionizable lipid is equal to that of the non-ionizable cationic lipid.

[0027] As a further preferred technical solution, the nucleic acid comprises at least one mRNA encoding a polypeptide or an mRNA with modified nucleotides.

[0028] As a further preferred technical solution, the nucleic acid comprises DNA.

[0029] As a further preferred technical solution, the non-ionizable cationic lipid is selected from at least one of DOTAP, DOTMA, DC-chol, and DOSPA or derivatives thereof.

[0030] As a further preferred technical solution, the molar ratio of the non-ionizable cationic lipid to cholesterol is from 10:9 to 10:11.

[0031] Experiments confirmed that when the molar ratio of the cationic lipid to cholesterol in the LNP formulation is from 10:9 to 10:11, the gene delivery transfection efficiency of the LNP formulation in mice reaches the optimum.

[0032] The present invention confirmed that the neutral phospholipid component has completely different effects on the expression of mRNA and DNA in the LNP formulation. Specifically, the neutral phospholipid component enhances the expression ability of the mRNA- formulation, while the neutral phospholipid component inhibits the expression ability of the DNA- formulation. Therefore, when the present invention is used for delivering DNA, neutral phospholipids are not added.

[0033] Meanwhile, non-ionizable cationic lipids such as DOTAP can significantly enhance the expression level of the tracer gene in the intramuscular injection site of mice in the DNA- formulation.

[0034] The second object of the present invention is to provide a formulation made of the above-mentioned nucleic acid-lipid nanoparticles. The technical solution adopted is: the formulation comprises the nucleic acid-lipid nanoparticles and a pharmaceutically acceptable carrier.

[0035] As a preferred technical solution: the formulation is an injection. The present invention prepares a nucleic acid-lipid nanoparticle injection. Under intramuscular injection administration, there is an obvious dose relationship between the ionizable lipid component in the LNP and the delivery and persistent expression of the tracer gene at the intramuscular injection site. The expression intensity and duration of the tracer gene increase with the increase of the ionizable lipid dose. The ability of the non-ionizable cationic lipid to deliver the tracer gene at the intramuscular injection site is much lower than that of the ionizable lipid at the same dose, and it lacks the ability to maintain the long-term expression of the tracer gene. Therefore, maintaining a sufficient concentration and dose of the ionizable lipid is a prerequisite for stimulating the production of high-titer specific antibodies in the LNP vaccine formulation for intramuscular injection administration.

[0036] The present invention has demonstrated that, under intramuscular administration, incorporating non-ionizable cationic lipids such as DOTAP into lipid particles composed of various ionizable lipids, including ALC-0315, MC3, DHA-1, L319, SM-102, etc., can balance the adjuvant effect of ionizable lipids and reduce off-target expression of nucleic acid-lipid particles in visceral tissues of mice. By analogy with the similar effect of incorporating another non-ionizable cationic lipid DOTMA on the expression pattern of LNP formulations, it is verified that the ability of non-ionizable cationic lipids to regulate off-target expression of LNP formulations and maintain continuous expression at the intramuscular injection site is universal and applicable to various LNP types and combinations of different non-ionizable cationic lipid molecules, such as non-ionizable cationic lipids like DC-Chol, DOSPA or their derivatives, etc., which is controllable and predictable, and becomes a modular and general strategy for achieving intramuscular administration.

[0037] A third object of the present invention is to provide the use of the above-mentioned nucleic acid-lipid nanoparticles in the preparation of biological vaccines.

[0038] As a preferred technical solution: the biological vaccine is a COVID-19 vaccine, an influenza vaccine, or a tumor vaccine.

[0039] Specifically, the present invention has confirmed through experiments that within the range defined by the above lipid particle formulation, adjusting the lipid molar ratio can change the ratio of the expression levels of foreign genes in the intramuscular injection site and visceral tissues under intramuscular administration, and further adjust the proportion of humoral immunity and cellular immunity generated by the formulation to meet different needs and increase the effective utilization of the patient's immune system. For example, therapeutic tumor vaccine formulations need to activate cellular immune responses rather than humoral immune responses. For prophylactic vaccines such as COVID-19 vaccines and influenza vaccines, the neutralizing antibody titer is the main indicator for reducing severe cases, but the cellular immunity has a long onset time, and it is necessary to balance humoral immunity and cellular immunity, appropriately increase humoral immunity, and use humoral immunity to produce specific IgG antibodies to cope with acute pathogen infections.

[0040] The present invention has confirmed through an intramuscular administration-based fluorescein gene transfection experiment that, compared with the corresponding LNP formulation, the formulation reduces the transfection and expression of the marker gene in visceral tissues, especially in the liver tissue and brain tissue. Blood liver biochemical indicators can objectively, real-time, and accurately measure the liver status. The present invention detects indicators such as ALT, AST, and TBIL related to liver injury in the blood by intramuscularly injecting COVID-19 virus S protein mRNA-LNP, and the results confirm that traditional mRNA-LNP causes significant liver injury to mice within 48 hours after intramuscular injection, while No significant changes in blood liver injury indicators were detected in the mice of the preparation group. The IVIS results showed that in addition to targeting and transfecting hepatocytes, the LNP preparation also transfected the myocardium, brain tissue, and extremities to varying degrees. Based on this, it is speculated that the LNP preparation may also have caused varying degrees of tissue damage to visceral tissues, which is an important reason for the various side effects of mRNA vaccines. The gene transfection level in the liver tissue was significantly reduced under intramuscular injection of the preparation, thus avoiding damage to the liver tissue. At the same time, the preparation also significantly reduced the gene transfection level in internal tissues such as brain tissue, respiratory tract, and extremities. Based on this, it is speculated that the damaging effect of the preparation on other visceral tissues may also be further reduced, making it possible to prepare a safer mRNA-LNP preparation.

[0041] The present invention provides a preparation for treating cancer, preventing cancer, or delaying the onset or progression of cancer, or alleviating cancer-related symptoms, and administering to an individual a composition as described in the above aspects and examples. In certain embodiments, the polypeptide may encode a therapeutic enhancer, such as an immunomodulatory molecule or other factors as previously described.

[0042] The present invention also provides a method for measuring the stability of the lipid nanoparticles described herein. Specifically, the nucleic acid lipid nanoparticles of the present invention have an increased tolerance to surface detergents, and a Triton X-100 solution with a concentration of 10 vol% or more is required to completely disassemble and separate the lipid nanoparticles from the nucleic acid they encapsulate. More specifically, the nucleic acid lipid nanoparticles of the present invention remain substantially intact in a Triton X-100 solution with a concentration less than 2 vol%; in a Triton X-100 solution with a concentration of 10% or more, they are completely dissociated.

[0043] The present invention also provides a method for in vivo delivery of a preparation, which includes administering the lipid nanoparticles described herein, such as nucleic acid lipid vaccines, to mammals and subjects by intramuscular injection and subcutaneous injection.

[0044] Compared with the prior art, the advantages of the present invention are as follows: The nucleic acid-lipid nanoparticles of the present invention use lipids as nucleic acid LNP delivery carriers, which can encapsulate mRNA or plasmid DNA. They are particularly suitable for intramuscular injection, can express for a long time at the injection site, produce high-titer specific neutralizing antibodies, reduce off-target expression in visceral tissues such as the liver and spleen, and can significantly improve the thermal stability of nucleic acid-lipid nanoparticles, facilitating the transportation and distribution of mRNA vaccines and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1The effects of ionizable lipid MC3 and non-ionizable cationic lipid DOTAP components on the expression of tracer genes at the intramuscular injection site;

[0046] Figure 2 The expression pattern of tracer genes after intramuscular injection when non-ionizable cationic lipid DOTAP is added to the LNP liposomes composed of ionizable lipid ALC-0315;

[0047] Figure 3 Adding non-ionizable cationic lipid DOTAP enhances the expression level and duration of tracer genes at the intramuscular injection site of LNP liposomes;

[0048] Figure 4 The expression pattern of tracer genes after intramuscular injection when non-ionizable cationic lipid DOTMA is added to the LNP liposomes composed of ionizable lipid ALC-0315;

[0049] Figures 5 - 8 The expression patterns of tracer genes after intramuscular injection when non-ionizable cationic lipid DOTAP is added to the LNP liposomes composed of ionizable lipids MC3, DHA-1, L319, and SM-102, respectively;

[0050] Figure 9 The effects of the concentration of neutral phospholipids in the mRNA-LNP formulation on the expression of tracer genes after intramuscular injection;

[0051] Figure 10 After intramuscular injection Comparison of the expression levels of the formulation at the intramuscular injection site and in the abdominal cavity;

[0052] Figure 11 The effects of the concentration of neutral phospholipids in the DNA-LNP formulation on the expression of tracer genes after intramuscular injection;

[0053] Figure 12 The effects of the concentration of cholesterol in the mRNA-LNP formulation on the expression of tracer genes after intramuscular injection;

[0054] Figure 13 The effects of the concentration of PEG in the mRNA-LNP formulation on the expression of tracer genes after intramuscular injection;

[0055] Figure 14 ELISA test results of S-protein specific IgG antibodies in the sera of mice at 21 days after the first immunization (3wp1) and at 7 days (1wp2), 14 days (2wp2), and 21 days (3wp2) after the second immunization;

[0056] Figure 15 ELISA test results of RBD-ACE2 competitive binding neutralizing antibodies in the sera of mice at 21 days after the first immunization (3wp1) and at 7 days (1wp2), 14 days (2wp2), and 21 days (3wp2) after the second immunization;

[0057] Figure 16 Serological indexes related to the liver of Balb / C mice after intramuscular administration of the nCovS2P@LNP formulation;

[0058] Figure 17 For the present invention Structural diagram of the 46 lipid nanoparticles. Detailed implementation manners

[0059] Before describing the present invention, the following definitions are provided to assist in understanding the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0060] Ionizable lipid: Also known as ionizable cationic lipid, it is an amphiphilic molecule with a hydrophilic group and a hydrophobic group, composed of a polar head (hydrophilic group), a linker, and a hydrophobic tail. The hydrophilic head of the ionizable lipid is composed of a tertiary amine, and at different pH values, the degree of protonation is different, showing an ionizable state. The ionizable lipids used in the present invention include but are not limited to: ALC-0315, Dlin-MC3-DMA (MC3), Lipid L319, SM-102, DHA-1.

[0061] Non-ionizable cationic lipid: An amphiphilic molecule with a hydrophilic group and a hydrophobic group, composed of a polar head (hydrophilic group), a linker, and a hydrophobic tail. The hydrophilic head is a quaternary ammonium salt, which is a permanent cation and does not have ionizable characteristics. The non-ionizable lipids used in the present invention include but are not limited to: DOTAP ((2,3-dioleoyl-propyl)-trimethylammonium-chloride); DOTMA (trimethyl-2,3-dioleyloxypropylammonium chloride), DC-Chol (3β-[N-(N,N-dimethylaminoethyl)aminocarbonyl]cholesterol); DOSPA; or a derivative thereof.

[0062] Neutral phospholipid: An amphiphilic phosphatidylcholine with a hydrophilic head and a hydrophobic tail. Commonly used synthetic modified phospholipids include: DSPC, DOPE, DOPC, ePC, and their derivatives, etc.

[0063] Cholesterol: A natural lipid small molecule, which is a main component of the cell membrane.

[0064] LNP: Lipid Nanoparticle, lipid nanoparticle. A lipid nanoparticle composed of at least one ionizable lipid and at least one neutral phospholipid, encapsulating bioactive molecules such as nucleic acids. The bioactive molecules can be RNA, DNA, siRNA, miRNA, protein, and polypeptide, etc.

[0065] An LNP containing at least one non-ionizable cationic lipid and at least one ionizable lipid, which encapsulates and delivers a bioactive molecule identical to the above LNP formulation.

[0066] Nucleic acid: Refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, including DNA and RNA. RNA can be in the form of siRNA, microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, circular RNA, and combinations thereof. Nucleic acids can be synthetic, naturally occurring, and non-naturally occurring. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoroamidates, and peptide nucleic acids (PNA), and include nucleic acids containing known natural nucleotide analogs as well as artificially modified nucleotides such as pseudouridine, methylation, and methylpseudouridine modifications. DNA can be double-stranded DNA, single-stranded DNA, plasmid DNA, etc.

[0067] nCovS: The gene of the Spike protein of the novel coronavirus.

[0068] nCovS2P: A recombinant gene of the pre-fusion conformation-locked Spike protein of the novel coronavirus modified with point mutations K986P and V987P.

[0069] mRNA vaccine: An mRNA-LNP formulation based on an LNP formulation that encapsulates mRNA, generally administered by intramuscular injection, which produces a specific antigen in the subject and induces the production of specific antibodies, thereby generating immune protection.

[0070] Fluc: Firefly luciferase gene.

[0071] IV: Intravenous injection, in this invention, it is intravenous injection into the tail vein of mice.

[0072] IM: Intramuscular injection, in this invention, it is administration into the lower limb muscle tissue of mice.

[0073] mRNA: Eukaryotic messenger RNA, a single-stranded RNA composed of a 5′-m7G cap, 5′-UTR, translation initiation codon, coding region, termination codon, 3′-UTR, and polyadenylic acid, providing a template for protein sequence translation.

[0074] BNT162b2: The mRNA recombinant sequence of the Covid-19 S protein used in the Pfizer / BioNTech novel coronavirus mRNA vaccine, with S2P mutations.

[0075] IVT: In vitro transcription reaction.

[0076] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate rather than limit the present invention.

[0077] Example 1

[0078] Raw material and preparation production

[0079] 1.1 RNA preparation

[0080] In the embodiments of the present invention, the mRNA used is all obtained by in vitro transcription (IVT) reaction. The general process is the enzymatic digestion of plasmid DNA templates and column purification to obtain linearized plasmid DNA. The IVT transcription production of RNA (Thermo Fisher, Kit). After transcription is completed, RNA is purified using an RNA Cleanup Kit. Unless otherwise specified, the transcription reaction substrate UTP is replaced with N1-methylpseudouridine (m1ψ).

[0081] The capping modification reaction of mRNA is completed using the capping enzyme (Vaccinia Capping Enzyme) from Novoprotein. The capping modification reaction of mRNA is set according to the reaction system recommended by the kit, and the reaction conditions are 37°C for 1 hour. After the reaction is completed, the capped product is purified using an RNA Cleanup Kit. The purified mRNA is dissolved in sterile injection water and analyzed by RNA gel electrophoresis and Qubit concentration determination.

[0082] 1.2 Plasmid DNA preparation

[0083] In the embodiments of the present invention, the plasmid DNA used is all obtained by production with the Qiagen EndoFree Plasmid Maxi Kit.

[0084] 1.3 LNP, Preparation of the preparation

[0085] LNP, The preparation consists of ionizable lipids, non-ionizable cationic lipids, DSPC, cholesterol, and PEG2000-DMG in a certain molar ratio. The specific formulations are listed in the examples. Unless otherwise specified, each lipid component is in mmol. The lipid materials are dissolved in absolute ethanol, and the nucleic acid is dissolved in a citric acid aqueous solution (10 mM, pH 4.0). The aqueous solution and the organic solution are mixed through a microfluidic chip at a volume ratio of 3:1, and the total flow rate is greater than 3 ml / min. The LNP preparation is dialyzed overnight with 1xPBS solution, and then transferred to a glass bottle and stored at 4°C or -20°C. The final concentration of mRNA: 0.1–0.375 μg / μl. The structure of the prepared Figure 16 is as shown.

[0086] Example 2

[0087] LNP, Determination of encapsulation efficiency

[0088] The Triton concentration used in the conventional LNP encapsulation efficiency detection method cannot be resolved Formulation.

[0089] 2.1 Effect of Triton X-100 concentration on RNA / DNA fluorescence quantitative detection

[0090] Experimental description: The RNA content was measured using the Qubit HS RNA assay of Qubit 2.0, which may be affected by Triton X-100. To investigate the effect of different concentrations of Triton on the RNA quantification results, the present invention first detected the RNA quantification results in solutions with different concentrations of Triton X-100. The specific procedure was as follows: Different concentrations of Triton X-100 were mixed with the detection dilution containing 270 ng of RNA to prepare detection samples with final concentrations of 0.1%, 0.05%, 0.01%, 0.005%, 0.002%, and 0.001% Triton (all volume percentages here), and quantitative detection was performed using Qubit 2.0. The detection results are shown in Table 1.

[0091] Table 1: Effect of Triton X-100 on the detection results of Qubit HS RNA Kit

[0092]

[0093] Conclusion: The calibrated value of the RNA concentration is 267.0 ng / ml. Triton X-100 with final concentrations of 0.001% to 0.1% has no significant effect on the RNA quantification results of the Qubit HS RNA Kit, and the detection variation deviation is less than 3%. Considering the dilution factor, the range of Triton concentration applicable to RNA detection samples is 0% to 20%. The same results were repeated and verified in the quantitative detection results of plasmid DNA using the Qubit HS dsDNA Kit.

[0094] 2.2 Complete resolution of nucleic acid and lipid components in 10% Triton X-100 in

[0095] LNP, and Formulation encapsulation efficiency determination method

[0096] In the present invention, LNP and The encapsulation efficiency determination method is carried out using the Qubit RNA HS Assay Kit (Invitrogen, Q32852) and the Qubit dsDNA HS Assay Kit (Invitrogen, Q32851). The Qubit 2.0 fluorometer is used to quantitatively detect the RNA or DNA content in the nucleic acid lipid nanoparticles. The operation steps are as follows:

[0097] a) Determine the nucleic acid content of the lysed sample. Take the LNP to be detected, or add the preparation sample to an equal volume of 20% Triton X-100 solution prepared with 1×TE, mix well and centrifuge, and place at room temperature in the dark for 5 minutes. Dilute the sample 200 times, load and detect to obtain the total nucleic acid concentration (A) in the lysed sample;

[0098] b) Without adding Triton detergent, detect the content of unbound free nucleic acid in the LNP, or the sample to obtain the nucleic acid concentration (B) of the unlysed sample;

[0099] c) Calculation formula: Encapsulation efficiency (%) = ((A - B) / A) × 100

[0100] In the above calculation formula, A: the nucleic acid quantity value measured in 10% Triton at the final concentration; B: the nucleic acid quantity value measured in the assay solution without Triton.

[0101] Under normal circumstances, LNP preparations composed of ionizable lipids are completely disassembled in 1% Triton solution, and the total nucleic acid content and free nucleic acid content are detected and compared by the nucleic acid fluorescent dye colorimetry method to obtain the LNP encapsulation efficiency. After incorporating non-ionizable cationic lipids, the preparation stability is improved, and 1% Triton cannot disassemble the nucleic acid and lipid components in the preparation.

[0102] In the LNP encapsulation efficiency detection method, a 2% Triton concentration is the highest concentration used in the literature reports so far, but it still cannot completely dissociate the nucleic acid and lipid in it. The present invention tested the disassembly ability of 0%, 1%, 5%, 7.5%, and 10% Triton solutions on preparations with an RNA concentration of 0.1 μg / μl, and the detection results of the nucleic acid content of preparations with different components are shown in Table 2.

[0103] Table 2: RNA content in RNA-LNP and RNA- samples dissociated by different concentrations of Triton

[0104]

[0105] The unit of lipid component concentration in Table 2 is mmol; the concentrations of A and B are respectively (or LNP) RNA concentration before and after isolation: μg / μl.

[0106] It should be noted that in Table 2, there is only one B value and one available A value (i.e., the A value measured in 10% Triton is valid for all formulations; 1% Triton is only valid for traditional LNPs containing only ionizable lipids).

[0107] Conclusion: 1% Triton solution can completely lyse the LNP (i.e., LNP17 in Table 2) composed of ionizable lipid (ALC-0315), but cannot dissociate the formulation incorporated with cationic lipid (such as DOTAP) (i.e., other than LNP17 in Table 2 ). The lysis rate of the formulation shows an increasing trend with the increase of Triton concentration. 10% Triton solution completely lyses the total nucleic acid content measured is equivalent to the total amount of nucleic acid in the sample. Increasing the PEG concentration to 5.5 mmol, the cholesterol concentration to 111.1 mmol, or the DOTAP concentration to 69.44 mmol has no effect on the dissociation ability of 10% Triton solution. 10% Triton solution can be used to completely lyse the nucleic acid and lipid components in, and does not affect the quantitative detection of RNA by Qubit HS RNA Kit.

[0108] Using plasmid DNA- for comparative experiments, similar results are obtained. As shown in Table 3, 10% Triton solution does not affect the quantitative detection of plasmid DNA by Qubit HS dsDNA Kit.

[0109] Table 3: DNA content in DNA-LNP, DNA- formulation dissociated by different concentrations of Triton

[0110]

[0111] The concentrations of A and B in Table 3 are respectively (or LNP) DNA concentration before and after isolation: μg / μl.

[0112] In the results of this example, it is also observed that adding a high concentration of non-ionizable cationic lipid improves the encapsulation efficiency of the formulation, as shown in Table 2.

[0113] Example 3

[0114] Effect of ionizable lipid concentration on gene expression and distribution under intramuscular administration

[0115] In this example, Figure 1 The lipid nanoparticle formulation LNP1 listed in contains one ionizable lipid MC3, which is one of the marketed nucleic acid drug formulations. Without disrupting the original ratio of the four components (MC3:DSPC:Chol:PEG = 50:10:38.5:1.5), in this example, the molar ratio of MC3 was adjusted from 50% to 0%, and compensated with DOTAP. The total concentration of MC3 and DOTAP lipid in the lipid formulation remained unchanged. These lipid formulations were used to encapsulate luciferase mRNA to prepare a series of LNPs with different molar concentrations of MC3.

[0116] In this example, 7-week-old female Balb / c mice were divided into six groups of 2 each, and administered by intramuscular injection in the right hind limb at a dose of 7.5 μg / 50 μl. The luciferase expression levels in mice were tested for LNP1, 2, 3, 4, 5, and 6 and other six lipid nanoparticle formulations at different times after intramuscular administration. After 6, 24, 48, and 72 hours of administration, in vivo IVIS imaging analysis was performed. The imaging results at 6, 24, and 48 hours are shown in Figure 1 , Figure 1 . In, the molar ratio of LNP components: (MC3 + DOTAP):DSPC:Chol:PEG = 50:10:38.5:1.5. The formulation in Table 4 is the lipid dosage for encapsulating 30 μg of mRNA, and the lipid unit is mmol.

[0117] Table 4: Effect of MC3 concentration on the expression level of the tracer gene, unit: fluorescence intensity / p / s.

[0118]

[0119] This experiment found that as the molar percentage of MC3 in the LNP formulation decreased, the luciferase protein expression at the intramuscular injection site gradually decreased, showing an obvious dose-dependent delivery trend. The DOTAP concentration was not able to compensate for the amount of luciferase protein expression induced by MC3. The expression duration of the tracer gene at the intramuscular injection site of LNP formulations with an MC3 molar percentage below 10% was significantly reduced.

[0120] Conclusion: Under intramuscular administration, there is an obvious dose relationship between the ionizable lipid component in LNP and the delivery and persistent expression of the tracer gene at the intramuscular injection site. The expression intensity and duration of the tracer gene increase with the increase of the ionizable lipid dose. The ability of non-ionizable cationic lipids to deliver tracer genes at the intramuscular injection site is much lower than that of the same dose of ionizable lipids, and they lack the ability to maintain the long-term expression of tracer genes. Therefore, maintaining a sufficient concentration and dose of ionizable lipids is a prerequisite for LNP vaccines administered intramuscularly, and for vaccines, it is a prerequisite for maintaining the expression of foreign genes and directly affects the stimulation and production amount of specific antibodies.

[0121] Example 4

[0122] Under intramuscular administration, the effects of non-ionizable cationic lipids on the preparation delivery mode and gene expression level:

[0123] This invention explored the changes in the expression and distribution of tracer genes under intramuscular administration after adding additional non-ionizable cationic lipids to LNP preparations composed of ionizable lipids.

[0124] 4.1 Non-ionizable cationic lipid DOTAP changes the expression pattern of LNP liposomes

[0125] In this example, Figure 2 the liposome formulation LNP17 shown in it is one of the marketed mRNA vaccine formulations, containing an ionizable lipid ALC-0315. Replace ALC-0315 in the LNP17 formulation with the non-ionizable cationic lipid DOTAP to form 57 containing only DOTAP lipids. In addition, add DOTAP to the LNP17 formulation to form 25, 46, and 74 composed of both ionizable lipids and non-ionizable cationic lipids.

[0126] Divide 7-week-old female Balb / c mice into 7 groups, with 3 mice in each group, and administer the drug by intramuscular injection in the right lower limb, with the administration dose of 7.5 μg / 50 μl. Test the lipid nanoparticle preparations such as LNP17, 57, 25, etc. After 6, 24, 48, 72, and 96 hours of drug administration, perform in vivo IVIS imaging analysis. After 6 and 24 hours of drug administration, the in vivo IVIS imaging results of the mice are shown in Figure 2 , Figure 2 The formulations in the table of which are the lipid amounts for encapsulating 30 μg of nucleic acid, and the lipid unit is mmol. It should be noted that the formulations and lipid units in the following figures are the same unless otherwise specified.

[0127] The experimental results showed that after intramuscular administration, the tracer gene was highly and continuously expressed at the administration site in the mice of the LNP17 administration group, and the expression signal was still detected 5 days after administration. Six hours after administration, transient high expression occurred in the visceral tissues of the mice, including the liver, thoracic cavity, and brain tissues, and the expression signal disappeared within 24 hours. In the mice of the 57 administration group, the expression level of the tracer gene decreased significantly, and only weak expression was observed at the intramuscular injection site. After 72 hours, no expression of the tracer gene was observed. In the mice of the 25 administration group, the expression level of the tracer gene at the injection site was partially restored. After 48 hours, the expression signal of the tracer gene at the intramuscular injection site recovered to the same level as that of LNP17. The IVIS imaging record 6 hours after intramuscular injection showed that the 57 or In the mice of the 25 administration group, no expression of the tracer gene was observed in the abdomen, lungs, and major internal organs.

[0128] Experimental conclusion: After intramuscular administration, the persistent high expression of the tracer gene at the administration site induced by the LNP preparation and the transient expression in the visceral tissues of the mice depend on the ionizable lipid component. The ionizable cationic lipid ALC-0315 simultaneously triggers a strong adjuvant effect. The non-ionizable cationic lipid DOTAP has a weak adjuvant effect in inducing inflammation, and the LNP composed of it has a low expression level in the visceral tissues of the mice. The non-ionizable cationic lipid DOTAP can balance the adjuvant effect of the ionizable cationic lipid ALC-0315, reduce the expression level of the tracer gene of the preparation at the peritoneal site of the mice, and simultaneously enhance the persistent expression ability of the tracer gene at the intramuscular injection site to varying degrees, as shown in Figure 3 . The preparation composed of DOTAP is suitable for the nucleic acid vaccine formulation of intramuscular administration.

[0129] 4.2 Non-ionizable cationic lipid DOTMA changes the expression pattern of LNP liposomes

[0130] In this example, Figure 4 ALC-0315 in the LNP17 formulation was replaced with the non-ionizable cationic lipid DOTMA to form 56 containing only the DOTMA cationic lipid. In addition, DOTMA was added to the LNP17 formulation to form 61 composed of both ionizable lipid and non-ionizable cationic lipid. FLuc mRNA was encapsulated by the existing microfluidic process to prepare the mRNA-LNP preparation.

[0131] Seven-week-old female Balb / c mice were divided into two groups of 3 each, and administered by intramuscular injection in the right lower limb, with a dosage of 7.5 μg / 50 μl. 56, Three lipid nanoparticle formulations, such as 61. After administration for 6, 24, 48, and 72 hours, in vivo IVIS imaging analysis was performed. After administration for 6 and 24 hours, the in vivo IVIS imaging results of the mice are shown in Figure 4 . In order to reduce the number of animals, this group of experiments was carried out simultaneously with the implementation of the DOTAP group, sharing the LNP17 positive control group.

[0132] The experimental results showed that under intramuscular administration, In the 56 administration group, the expression level of the tracer gene in the mice decreased significantly, with only weak expression at the intramuscular injection site, and the expression level decreased by 622.5 times. After 72 hours, no tracer gene expression was observed. In the 61 administration group, the expression level of the tracer gene at the injection site of the mice was partially restored. After 24 hours, the tracer gene expression signal at the intramuscular injection site recovered to the same level as that of LNP17 and was maintained. The IVIS imaging record after 6 hours of intramuscular injection showed that for those incorporating DOTMA 56 or No tracer gene expression was observed in the abdomen, lungs, and major internal organs of the mice in the 61 administration group.

[0133] Experimental conclusion: Under intramuscular administration, the adjuvant effect of the non-ionizable cationic lipid DOTMA in inducing inflammation is weak, and the formulation composed of it has a low expression level in the visceral tissues of mice and better safety. DOTMA can balance the adjuvant effect of the ionizable cationic lipid ALC-0315, reduce the expression level of the formulation in the visceral tissues of mice, improve the safety of the formulation, and at the same time do not affect the continuous expression ability of the lipid particle formulation at the intramuscular injection site. The formulation composed of DOTMA is a nucleic acid vaccine formulation suitable for intramuscular administration.

[0134] 4.3 The expression pattern of non-ionizable cationic lipid particles is universal

[0135] In this example, different ionizable lipids MC3, DHA-1, L319, and SM-102 were used to replace ALC-0315 in LNP17 and the 25 formulation respectively to form different LNPs and formulations. DHA-1 is a branched ionizable cationic lipid provided by Sinobang (product number: 06040009300). Further, luciferase mRNA was encapsulated by lipid particles to prepare mRNA-LNP formulations.

[0136] Seven-week-old female Balb / c mice were divided into six groups, with 3 mice in each group, and administered by intramuscular injection into the right lower limb, and the administration dose was 7.5 μg / 50 μl. LNP53 was tested respectively, 58, LNP55, 60, LNP68, 69, LNP72, Six lipid nanoparticle formulations such as 73. After 6, 24, 48, and 72 hours of administration, in vivo IVIS imaging analysis was performed. After 6 and 24 hours of administration, the in vivo IVIS imaging results of the mice are shown in Figures 5 - 8 Figure;

[0137] The experimental results showed that under intramuscular administration, in the groups of mice administered with four ionizable lipid nanoparticle formulations such as LNP53, LNP55, LNP68, and LNP73, the tracer gene was highly and continuously expressed at the administration site, and the expression signal was still detected 3 days after administration. After 6 hours of administration, transient high expression occurred in the visceral tissues of the mice, and the expression sites included the liver, thoracic cavity, and brain tissues, and the expression signal disappeared within 24 hours. At the same time, there was an inflammatory reaction with swelling and lumps. Incorporated into the non-ionizable cationic lipid 58, 60, 69, In the groups of mice administered with four lipid nanoparticle formulations such as 73, the expression level of the tracer gene at the injection site of the mice was partially restored. After 48 hours, the expression signal of the tracer gene at the intramuscular injection site returned to the level consistent with that of the tracer gene expression signal of the corresponding positive control group of mice and was maintained synchronously all the time. The IVIS imaging record 6 hours after intramuscular injection showed that incorporated into DOTAP 58, 60, 69, No tracer gene expression was observed in the abdomen, lungs, and major internal organs of the mice in the 73 administration group.

[0138] Experimental conclusion: Under intramuscular administration, incorporating DOTAP into lipid particles composed of ionizable lipids such as MC3, DHA-1, L319, and SM-102 can balance the adjuvant effect of ionizable lipids and reduce the systemic off-target expression level, and the results are similar to those of the ALC-0315 group experiment. Combining with the similar results of the formulation after incorporating DOTMA, by analogy, the experimental results verified that the formulation composed of non-ionizable cationic lipids and ionizable lipids has the ability to reduce the systemic off-target expression level of the target gene in visceral tissues and maintain continuous expression at the intramuscular injection site.

[0139] Example 5

[0140] Under intramuscular administration, Effect of cholesterol, phospholipid, and PEG components in the formulation on gene delivery and expression pattern: This example explored the effect of the main components of the LNP formulation on the expression of the tracer gene under intramuscular administration.

[0141] 5.1 Neutral phospholipid fine-tuning of mRNA- Expression pattern

[0142] In this example, liposome formulation LNP17, 25, 45, and 46 were used to encapsulate FLuc mRNA. The contents of neutral phospholipid DSPC in the preparations were 0, 9.4, and 18.8 mmol, respectively. After encapsulating luciferase mRNA, mRNA-LNP preparations were prepared.

[0143] Seven-week-old female Balb / c mice were divided into three groups of 3 each, and administered by intramuscular injection in the right lower limb at a dose of 7.5 μg / 50 μl. LNP17, 25, 45, 46, and eLNP17 (empty liposome) and other five lipid nanoparticle preparations were tested. In vivo IVIS imaging analysis was performed 6, 24, 48, 72, 96, and 120 hours after administration. The in vivo IVIS imaging results of mice 6, 24, 48, and 72 hours after administration are shown in Figure 9 .

[0144] The experimental results showed that under intramuscular injection, the tracer gene was highly and continuously expressed at the administration site in the LNP17 administration group of mice, and the expression signal was still detected 5 days after administration. Six hours after administration, transient high expression occurred in the visceral tissues of mice, and the expression sites included the liver, thoracic cavity, and brain tissues, and the expression signal disappeared within 24 hours. At the same time, there was an inflammatory reaction with swelling and lumps. In the 25 administration group of mice incorporated with DOTAP, there was only relatively weak expression at the intramuscular injection site, and no expression of the tracer gene was observed in the visceral tissues of mice within 24 hours. 45 and 46 administration groups of mice, 6 hours after administration, the expression signals at the injection site and visceral tissues increased with the increase of the neutral phospholipid concentration, but compared with the abdominal cavity expression signal of the LNP17 group of mice, the expression levels at the intramuscular injection site decreased by 2.42 times and 1.78 times respectively, and the expression levels in the visceral tissues decreased by 16.12 times and 10.4 times respectively. Forty-eight hours later, the expression signals of the tracer gene at the intramuscular injection site of all preparation administration groups of mice recovered to the same level as that of the LNP17 group, and the expression signal levels observed in a longer time period maintained the same level as that of the LNP17 group of mice, and even some exceeded it, as shown in Figure 10 .

[0145] Experimental conclusion: Compared with LNP preparations, intramuscular injection The formulation inhibits the transient expression level of the tracer gene in the visceral tissues of mice, but does not affect its long-term expression level at the intramuscular injection site. This phenomenon is further regulated by the neutral phospholipid component. A decrease in the neutral phospholipid concentration causes The formulation causes a further decrease in the transient expression levels in the visceral tissues and intramuscular injection sites of mice, but has no direct effect on the expression level of the tracer gene at the intramuscular injection site for 24 hours or longer time periods.

[0146] 5.2 Neutral phospholipids inhibit DNA- delivery of gene expression

[0147] In this example, the lipid nanoparticle formulation LNP17, 25, 45, and 46 were used to encapsulate the FLuc plasmid DNA, as Figure 11 shown, and the content of the neutral phospholipid DSPC in the DNA- formulation was 9.4, 0, 9.4, and 18.8 mmol, respectively. The luciferase plasmid DNA was encapsulated by the microfluidic chip process to prepare the DNA-LNP formulation.

[0148] Female Balb / c mice at 7 weeks of age were divided into three groups of 3 each, and administered the drug by intramuscular injection in the right lower limb at a dose of 11.5 μg plasmid DNA / 50 μl. The DNA-LNP17, DNA- 25, DNA- 45, and DNA- 46 lipid nanoparticle formulations were tested respectively. In vivo IVIS imaging analysis was performed 6, 24, 48, and 72 hours after administration. The in vivo IVIS imaging results of the mice 6, 24, and 48 hours after administration are shown in Figure 11 .

[0149] The experimental results showed that 6 hours after intramuscular injection, the expression level of the tracer gene in the DNA-LNP17-administered group of mice was low and the persistence was poor at the administration site. In the DNA- 25-administered group of mice, the expression level was the highest at the intramuscular injection site, 3.4 times higher than that at the injection site of the LNP17 group of mice. The expression signals of the DNA- 45 and DNA- 46-administered groups of mice at the injection site were lower than those of the DNA- 25 group of mice and decreased with the increase in the neutral phospholipid concentration. 72 hours later, the expression signal of the tracer gene was observed only at the intramuscular injection site of the DNA- 25-administered group of mice. No expression signal of the tracer gene appeared in the visceral tissues of all the administered groups of mice.

[0150] Experimental conclusion: Compared with the LNP formulation encapsulating mRNA, DOTAP cationic lipid enhances the expression level of the tracer gene in the intramuscular injection site of mice in the DNA- formulation, and the neutral phospholipid component inhibits the expression ability of the DNA- formulation. The expression effects of the neutral phospholipid component on mRNA and DNA in the LNP formulation are very different.

[0151] 5.3 Suitable for intramuscular administration cholesterol concentration range

[0152] The present invention compares the effect of cholesterol concentration on the gene delivery ability of the formulation. In the mRNA-LNP formulation used in this example, the molar ratio of the cationic lipid (including ionizable lipid and non-ionizable cationic lipid) to cholesterol is designed to be between 10:7 and 10:12. The mRNA-LNP formulation is prepared by encapsulating luciferase mRNA using a microfluidic process.

[0153] Female Balb / c mice at 7 weeks of age were divided into six groups, three in each group, and administered by intramuscular injection in the right lower limb, with a dosage of 7.5 μg / 50 μl. LNP17, 29, 25, 33, 34, and 35 and other six lipid nanoparticle formulations were tested. After 6, 24, 48, 72, and 96 hours of administration, in vivo IVIS imaging analysis was performed, and the results are shown in Figure 12 .

[0154] The experimental results show that after 6 hours of intramuscular administration, compared with the LNP17 group, the change in cholesterol concentration has a certain effect on the expression level of the formulation. After 96 hours of administration, the formulation with the molar ratio of cholesterol designed in the range of 10:9 to 10:11 still has a relatively high expression at the intramuscular injection site.

[0155] Experimental conclusion: Cholesterol concentration has an impact on the expression persistence of the formulation. When the molar ratio of the cationic lipid (including ionizable lipid and non-ionizable cationic lipid) to cholesterol in the formulation is in the concentration range of 10:9 to 10:11, it is beneficial to the gene expression and maintenance delivered by the formulation.

[0156] 5.4 Effect of PEG concentration on the expression pattern

[0157] The present invention compares the effect of PEG concentration on the Effect on the gene delivery ability of the formulation. In the mRNA-LNP formulation used in this example, the PEG concentration was designed to be 0.23%, 0.46%, 0.91%, 1.64%, 1.66%, 1.78%, 1.93%, 2.20%, 2.47%, 2.73%, and 3.0% of the total lipid molar amount. The luciferase mRNA was encapsulated by the microfluidic process to prepare the mRNA-LNP formulation.

[0158] Female Balb / c mice at 7 weeks of age were divided into twelve groups, with three mice in each group. The mice were administered by intramuscular injection in the right hind limb, and the administration dose was 7.5 μg / 50 μl. LNP17, 28, 25, 30, 31, and 32 and other twelve lipid nanoparticle formulations were respectively tested. In vivo IVIS imaging analysis was performed 6, 24, 48, 72, and 96 hours after administration. The results are shown in Figure 13 , Figure 13 where the amount of PEG is expressed as the percentage value of PEG in the total lipid molar amount.

[0159] The experimental results showed that 6 hours after intramuscular administration, compared with the LNP17 group, all PEG concentrations had no obvious effect on the expression level of the formulation. 72 hours after administration, PEG with a molar ratio of 1.93% to 3.0% of the total lipid continued to maintain the expression level of the formulation, and the expression level of the formulation with a PEG concentration lower than 1.93% decreased significantly.

[0160] Experimental conclusion: PEG with a molar ratio of 2.20% to 3.0% maintains the expression level of the formulation, which is beneficial to the gene expression and maintenance delivered by the formulation.

[0161] Example 6

[0162] nCovS2P mRNA- Intramuscular administration induces the production of specific antibodies and neutralizing antibodies against the SARS-CoV-2 S protein in Balb / C mice

[0163] 6.1mRNA- The formulation stimulates a high-level immune response and coordinates the humoral immune level

[0164] In this example, 7-week-old female BalB / C mice were randomly divided into 5 groups. A lipid nanoparticle complex encapsulating mRNA encoding the SARS-CoV-2 spike protein (pre-fusion conformation S2P locked) was used as the vaccine and administered by intramuscular injection. The nCovS2P mRNA coding sequence is identical to the coding sequence of the Pfizer / BioNTech recombinant SARS-CoV-2 spike protein BNT162b2 mRNA. The particle size and encapsulation efficiency of the lipid nanoparticles are shown in Table 5. Each group of animals was injected twice at 3-week intervals. On the 21st day after the first immunization (3wp1), and on the 7th, 14th, and 21st days after the second immunization (1wp2, 2wp2, 3wp2), the mice were anesthetized and blood was collected to measure the IgG antibody and SARS-CoV-2 spike protein neutralizing antibody titers in the serum samples.

[0165] Table 5: LNP, Particle size, particle size distribution, and EE% test results

[0166]

[0167] ELISA test results for serum IgG antibodies showed that: 7 days after the second immunization, compared with the blank group, the levels of spike protein-specific IgG antibodies in the sera of mice in the nCovS2P@LNP17 administration group, the nCovS2P@ 46, the nCovS2P@ 25 administration group, and the nCovS2P@ 74 administration group increased significantly (p < 0.001). The IgG antibody level in the LNP17 administration group reached the antibody titer level reported in the literature and was significantly higher than that in other administration groups ( Figure 14 ). 46, 25, and 74 administration groups had serum antibodies that were 1 / 20 to 1 / 100 of those in the LNP17 group. It is worth mentioning that compared with the immune effect of traditional vaccines, the levels of spike protein-specific antibodies in the sera of all formulation groups of mice were at extremely high expression levels. 14 days after the second immunization, the specific antibody titer in the sera of mice in the LNP17 administration group began to decline, decreasing by about 5-fold, while 46, and 74 formulation groups of mice had continuously increasing serum antibodies, showing a stable upward trend. 21 days after the second immunization, 46, and 74 administration groups of mice had a significant reduction in the difference in the content of spike protein-specific IgG antibodies in their sera compared with the LNP17 administration group, between 50% and 75% of its content.

[0168] 6.2mRNA- formulation maximally enhances the neutralizing antibody level

[0169] The mouse serum samples obtained in Experiment 6.1 were diluted 1000-fold, and then ELISA detection of RBD competitive neutralizing antibodies was performed. The results showed that 7 days after the second immunization, compared with the blank group, the neutralizing antibody titers in the sera of the mice in the LNP17 administration group, 46, @ 25 administration group, and 74 administration group were significantly increased (p < 0.001). The neutralizing antibody level in the nCovS2P@LNP17 administration group was close to the highest peak value ( Figure 15 ). 46, 25, and The neutralizing antibodies in the sera of the mice in the 74 administration group were 79.17% to 91.72% of those in the LNP17 group, and the expression levels were extremely high. On the 14th and 21st days after the second immunization, 46, and The neutralizing antibodies in the sera of the mice in the 74 formulation group showed a stable increasing trend and reached the peak level of the control group mice. The neutralizing antibody level in the sera of the mice in the 25 administration group showed a decreasing trend, and the overall level was 40% of the highest peak value.

[0170] Combined with the results analysis of the S protein-specific antibody and the RBD-ACE2 binding neutralizing antibody, compared with the control LNP formulation, 46 and 74 formulations induced the same level of neutralizing antibodies under the condition of stimulating the production of lower levels of IgG antibodies. The antigen expressed at the intramuscular injection site had the effect of stimulating neutralizing antibodies. Compared with 46 formulation, 25 formulation was only expressed at the intramuscular injection site, and the antibody level stimulated was lower, while maintaining a relatively high neutralizing antibody titer. Therefore, by adjusting the formulation, regulating the dynamic expression of the antigen gene at the intramuscular injection site and in visceral tissues, the ratio of antibodies (humoral immunity) and neutralizing antibodies generated by the immune system can be regulated.

[0171] Example 7

[0172] nCovS2P mRNA- Serological indexes of acute toxicological reactions induced in Balb / C mice

[0173] In this example, 7-week-old female BalB / C mice were randomly divided into 5 groups, and liposome complexes encapsulating nCovS2P were used as the formulation for intramuscular injection. The administration dose was: 20 μg mRNA (or an equivalent amount of liposomes). Grouping information: 1, blank control (1xPBS); 2, eLNP17; 3, nCovS2P@LNP17; 4, nCovS2P@ 25; 5, nCovS2P@ 46. After 6 hours, 24 hours, and 48 hours of administration, sera were collected and liver-related biochemical indices in blood samples were detected.

[0174] The test results are shown in Figure 16 , and the result analysis is as follows:

[0175] 1. ALB: The ALB levels of the mice in the four groups were consistent with those of the negative control group and showed no increase.

[0176] 2. ALT: nCovS2P@ 25, nCovS2P@ The ALT levels of the mice in the three groups of 46 and the negative control were basically consistent at the three time points, with slight fluctuations and no significant differences. The ALT level of the mice in the LNP17 group was significantly higher than that of the control group, especially at 24 and 48 hours, showing an obvious increase.

[0177] 3. TBIL: Generally speaking, nCovS2P@ 25, nCovS2P@ At 24 and 48 hours, the TBIL levels of the mice in the three groups of 46 and the negative control were slightly higher than those of the negative control group, and the levels of the three groups were basically consistent. However, at 24 hours, the TBIL concentration in the blood of the LNP17 group increased significantly.

[0178] 4. AST: nCovS2P@ The AST levels of the mice in the 25 group were consistent with those of the negative control group at the three time points and showed no obvious changes. nCovS2P@ The AST level of the mice in the 46 group increased significantly at 24 hours. The AST level of the mice in the LNP17 group increased significantly at 24 hours and decreased at 48H, but the expression was obvious. The AST levels were observed to increase significantly at the three time points in the blank liposome group.

[0179] Result analysis: Hemolysis and redness and swelling inflammation at the injection site may occur during the experiment, which mainly lead to an increase in AST and have little effect on other indices. It cannot be excluded that the slight increase in AST level is related to the above phenomena. However, combined with ALT and TBIL with higher liver specificity, the levels of these two key indices of the mice in the LNP17 group were higher than those of the negative control group and the formulation group at each time point. Slight signs of liver injury may occur at 6 hours, obvious liver injury occurred at 24 and 48 hours, the injury was the most serious at 24 hours, and showed a downward trend at 48 hours. While no obvious signs of liver injury were seen in the blank control group and nCovS2P@ 25, nCovS2P@ 46 formulation groups.

[0180] Possible mechanism: Fluorescence experiments showed that the mRNA-LNP17 formulation was highly expressed in the liver 6 hours and 24 hours after injection, and gradually subsided to the negative control level around 48 hours. Liver damage may be caused by a strong immune response induced by the expression of mRNA in the liver, resulting in the secretion of a large amount of immune factors by cells, overactivation of immune cells, and attack on normal liver cells. Therefore, the damage continued within 24 hours after injection, leading to a continuous increase in liver enzymes. However, when the liver gene expression gradually decreased after 24 hours and was only expressed in the muscle at 48 hours, the attack caused by this immune activation stopped, the liver was repaired, the liver enzymes were gradually metabolized, and the indicators improved. In the control group and the immune stimulation after injection in the formulation group only occurred in the muscle and spread slightly to the lower abdomen, so it had little stimulation to the liver and there was little change in liver enzyme levels, which was also consistent with the conclusions obtained in this experiment.

Claims

1. A nucleic acid-lipid nanoparticle suitable for intramuscular administration, characterized in that, It consists of the following components: (a) mRNA; (b) an ionizable lipid, accounting for 23.01 mol% to 24.17 mol% of the total lipids; (c) a non-ionizable cationic lipid, accounting for 23.01 mol% to 24.17 mol% of the total lipids; (d) neutral phospholipids, accounting for 4.91 mol% to 9.35 mol% of the total lipids; (e) cholesterol, accounting for 42.43 mol% to 44.56 mol% of the total lipids; (f) PEG lipid, accounting for 2.20 mol% of the total lipids. Among them, the ionizable lipid is selected from one of ALC-0315, MC3, DHA-1, L319, and SM-102; the non-ionizable cationic lipid is selected from one of DOTAP, DOTMA, DC-chol, or DOSPA.

2. A nucleic acid-lipid nanoparticle suitable for intramuscular administration, characterized in that, It consists of the following components: (a) mRNA or DNA; (b) an ionizable lipid, accounting for 25.45 mol% of the total lipids; (c) a non-ionizable cationic lipid, accounting for 25.45 mol% of the total lipids; (d) cholesterol, accounting for 46.90 mol% of the total lipids; (e) PEG lipid, accounting for 2.20 mol% of the total lipids; Among them, the ionizable lipid is selected from one of ALC-0315, MC3, DHA-1, L319, and SM-102; the non-ionizable cationic lipid is selected from one of DOTAP, DOTMA, DC-chol, or DOSPA.

3. The nucleic acid-lipid nanoparticle suitable for intramuscular administration according to claim 1 or 2, wherein The nucleic acid contains at least one mRNA encoding a polypeptide or an mRNA with modified nucleotides.

4. The nucleic acid-lipid nanoparticle suitable for intramuscular administration according to claim 1 or 2, characterized in that, The nucleic acid contains DNA.

5. The nucleic acid-lipid nanoparticle suitable for intramuscular administration according to claim 1 or 2, wherein The molar ratio of the sum of the ionizable lipid and the non-ionizable cationic lipid to cholesterol is 10:9 to 10:

11.

6. The nucleic acid-lipid nanoparticle suitable for intramuscular administration according to claim 1 or 2, wherein The molar concentrations of the ionizable lipid and the non-ionizable cationic lipid are equal.

7. A preparation made of the nucleic acid-lipid nanoparticle according to claim 1 or 2, characterized in that: The preparation includes the nucleic acid-lipid nanoparticles and a pharmaceutically acceptable carrier.

8. The preparation according to claim 7, characterized in that: The preparation is an injection.

9. Use of the nucleic acid-lipid nanoparticle according to claim 1 or 2 in the preparation of a biological vaccine, characterized in that, The vaccine is a COVID-19 vaccine, and the COVID-19 vaccine is a vaccine encapsulating a lipid complex encoding the SARS-CoV-2 spike protein mRNA.

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