Lipid nanoparticle formulations for vaccines

Abstract

Lipid formulations capable of forming lipid-based nanoparticles are provided, the lipid formulations comprising a molar ratio of ionizable lipid to phospholipid bound to a nucleic acid payload of 0.1-1.30, and in some embodiments a stabilizer. In embodiments, the nucleic acid payload is a vaccine genetic element.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 357,094, filed on June 30, 2022, which is incorporated herein by reference. Technical Field

[0003] The field of the invention relates to methods and lipid formulations suitable for forming RNA-based vaccines in lipid nanoparticles. Background Art

[0004] Nucleic acid-based vaccines have advantages over traditional vaccines in terms of safety and efficacy. RNA vaccines are degraded by exonucleases and endonucleases in the body without a delivery system, so they require a carrier.

[0005] At present, lipid nanoparticles (LNPs) are one of the most commonly used carriers for RNA delivery in vivo. Lipid nanoparticles or LNPs are usually composed of different lipids, each of which has different functions. These LNPs can have a lipid or aqueous core and can contain a double-layer structure, depending on the abundance of each type of lipid.

[0006] The components of LNP formulations are typically: ionizable cationic lipids, which spontaneously encapsulate negatively charged mRNA through a combination of attractive electrostatic and hydrophobic interactions with RNA; neutral phospholipids, which are used to reduce charge-related toxicity and maintain the structure of the LNP; and cholesterol, which is used to stabilize the LNP and aid in cellular entry, as well as lipid-conjugated polyethylene glycol (PEG).

[0007] Individual LNPs have different properties. Diffusion or bulk mixing can result in LNPs with variable composition. Therefore, rapid mixing of the ethanol-lipid phase with the mRNA in excess water is key to synthesizing small and uniform LNPs. For this purpose, Precision Nanoystems ULC Series of mixers.

[0008] The recognized challenges in creating a successful LNP vaccine are safety, manufacturability, stability and efficacy. Current mRNA vaccines must be kept frozen at low temperatures for storage. Some cases of adverse events have been reported for LNP vaccines, which has led some people to resist vaccination.

[0009] There remains a need for better vaccines in LNP format with increased stability. Summary of the invention

[0010] According to one embodiment of the present invention, a lipid formulation is provided that can form a lipid-based nanoparticle suitable for a vaccine, the lipid formulation comprising an ionizable lipid and phospholipid ratio of 0.10–1.30 mol:mol, in some embodiments, a ratio of 0.33–1.20 mol:mol, in some embodiments, a ratio of 0.10–0.70 mol:mol, and in some embodiments, a ratio of 0.40–0.70 mol:mol. In embodiments, the lipid formulation further includes a nucleic acid payload. In embodiments, the nucleic acid payload is a nucleic acid vaccine element. In some embodiments, the lipid formulation further includes a stabilizer. In some embodiments, the lipid formulation further includes a sterol.

[0011] In embodiments, the molar ratio of ionizable lipid to phospholipid is about 1.30, or about 1.29, or about 1.28, or about 1.27, or about 1.26, or about 1.25, or about 1.24, or about 1.23, or about 1.22, or about 1.21, or about 1.20, or about 1.19, or about 1.19, or about 1.18, or about 1.17, or about 1.16, or about 1.15, or about 1.14, or about 1.13, or about 1.12, or about 1.11, or about 1.10, or about 1.09, or about 1.08, or about 1.07, or about 1.06, or about 1.05, or about 1.04, or about 1.03, or about 1.04. about 1.02, or about 1.01, or about 1.00, or about 0.99, or about 0.98, or about 0.97, or about 0.96, or about 0.95, or about 0.94, or about 0.93, or about 0.92, or about 0.91, or about 0.90, or about 0.89, or about 0.88, or about 0.87, or about 0.86, or about 0.85, or about 0.84, or about 0.83, or about 0.82, or about 0.81, or about 0.80, or about 0.79, or about 0.78, or about 0.77, or about 0.76, or about 0.75, or about 0.74, or about 0.73, or about 0.72, or about 0.71.

[0012] In embodiments, the molar ratio of ionizable lipid to phospholipid is about 0.70, or about 0.69, or about 0.68, or about 0.67, or about 0.66, or about 0.65, or about 0.64, or about 0.63, or about 0.62, or about 0.61, or about 0.60, or about 0.59, or about 0.58, or about 0.57, or about 0.56, or about 0.55, or about 0.54, or about 0.53, or about 0.52.

[0013] In embodiments, the ionizable lipid to phospholipid ratio is about 0.51, or about 0.50, or about 0.49, or about 0.48, or about 0.47, or about 0.46, or about 0.45, or about 0.44, or about 0.43, or about 0.42, or about 0.41, or about 0.40.

[0014] In embodiments, the ionizable lipid to phospholipid ratio is about 0.39, or about 0.38, or about 0.37, or about 0.36, or about 0.35, or about 0.34, or about 0.33, or about 0.32, or about 0.31, or about 0.30.

[0015] In embodiments, the ionizable lipid to phospholipid ratio is about 0.29, or about 0.28, or about 0.27, or about 0.26, or about 0.25, or about 0.24, or about 0.23, or about 0.22, or about 0.21, or about 0.20.

[0016] In embodiments, the ionizable lipid to phospholipid ratio is about 0.19, or about 0.18, or about 0.17, or about 0.16, or about 0.15, or about 0.14, or about 0.13, or about 0.12, or about 0.11, or about 0.10.

[0017] According to one embodiment of the present invention, there is provided a lipid preparation, wherein the ionizable lipids account for about 20-40 mol % of the total composition.

[0018] According to one embodiment of the present invention, a lipid preparation is provided, wherein the ionizable lipid comprises a mixture of ionizable lipids. In an embodiment, the phospholipids account for about 25-60 mol% of the total preparation.

[0019] According to one embodiment of the invention, the sterol, such as cholesterol or cholesterol hemisuccinate, comprises 15-25 mol% of the total composition.

[0020] According to one embodiment of the present invention, a lipid preparation is provided, wherein the stabilizer accounts for 0.0-2.5 mol % of the total amount of the lipid preparation.

[0021] According to the present invention, a lipid formulation for encapsulating a nucleic acid payload in a nanoparticle is provided, the formulation comprising an ionizable lipid, a sterol and a phospholipid, wherein the phospholipid content is between 25-60% of the nanoparticle, and the ionizable lipid to phospholipid molar ratio is 0.33 to 1.2. In an embodiment, the nanoparticle is a vaccine.

[0022] In embodiments, the stabilizer is a PEG lipid, in some embodiments PEG-DMPG. In embodiments, the phospholipid is DSPC. In embodiments, the ionizable lipid is selected from PNI 516, PNI 550, PNI 127, PNI 560, PNI 580, PNI 659, PNI 721, PNI 722, PNI 726, PNI 728, and PNI 730. In embodiments, the sterol is cholesterol.

[0023] According to the present invention, a vaccine is provided, comprising a lipid formulation for encapsulating a nucleic acid payload, the vaccine comprising an ionizable lipid, a sterol and a phospholipid, wherein the phospholipid content is 25-60% of the vaccine, and the ionizable lipid to phospholipid molar ratio is 0.33-1.2. In an embodiment, the nucleic acid payload encodes an antigen selected from a coronavirus spike protein and an influenza hemagglutinin protein.

[0024] In an embodiment, the nucleic acid payload is derived from influenza virus mRNA. In an embodiment, the nucleic acid payload is derived from a coronavirus.

[0025] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic representation of the dose response of SARsCOV-2 protein expression after transfection of Vaccmixb PNI516 and V46-PNI 516 in BHK 570 cells. The dose response curve was created using RNA doses ranging from 1000 to 0.49 ng / mL;

[0027] Figure 2 is a schematic diagram of the dose response of SARsCOV-2 protein expression after LNP transfection in BHK570 cells using various PNI516 LNP formulations containing 1000 to 0.49 ng / mL of nCoVPNI A5 saRNA;

[0028] Figure 3 is an ELISA scatter plot of SARsCOV-2 spike protein-specific IgG expression in BALB / c mouse serum 21 days after 1 ug / mouse intramuscular LNP injection; and

[0029] Figure 4 Replicas of Cryo-TEM images of A) Vaccmixb PNI 516, B) V46-PNI 516, and C) V47-PNI 516 LNPs.

[0030] Figure 5 Schematic diagram of the in vitro transfection capacity of saRNA LNPs with Vaccmixb, V46-PNI 516, V47-PNI516, and V22-PNI 516 after adding GFP saRA LNPs to Jurkat cells.

[0031] Figure 6 is a schematic diagram of the in vitro transfection ability of mRNA LNPs with V47-PNI 516, V46-PNI 516, V02-PNI 516, and V22-PNI 516 after adding GFP mRNA LNPs to Jurkat cells.

[0032] Figure 7 Schematic representation of the transfection capacity of EGFP saRNA LNPs using expansion medium supplemented with activator / recombinant human IL-2.

[0033] Figure 8 It is a photo of mice into which LNPs with luciferase mRNA payload were injected intravenously at 0.1 mg / kg. The upper two photos show the fluorescence of LNPs containing PNI 516 30%, DPSC 56%, cholesterol 12.5% ​​and DMP-PEG 1.5% after 4 hours of administration. The lower two photos are brighter than the upper two photos, showing the fluorescence of LNPs containing PNI 580 30%, DSPC 56%, cholesterol 12.5% ​​and DMG-PEG 1.5% after 4 hours of administration. DETAILED DESCRIPTION

[0034] According to an embodiment of the present invention, a lipid mixture formulation for producing a more effective lipid-based formulation of a nucleic acid cargo (including a vaccine) and other oligomers (e.g., a peptide), and a method for preparing a vaccine using these lipid mixtures and the resulting formulations are provided. The lipid mixture has an unusual ionizable lipid to phospholipid ratio compared to the established optimal ratio of 0.2, but is surprisingly measured to be more effective.

[0035] Table 1: Exemplary lipid mixtures from the prior art

[0036]

[0037] In another aspect, the lipid mixture formulation of the present invention is provided for mixing with nucleic acid vaccine elements to produce lipid nucleic acid particles, which enhance the delivery of nucleic acids to target cells or tissues, and lipid nucleic acid particles, such as those made from commercially available lipid mixtures (e.g., Lipofectamine TM Compared with lipid-nucleic acid particles made of poly(acrylonitrile) transfection agents, they have lower toxicity and greater manufacturing convenience.

[0038] In another aspect, the present invention provides a lipid mixture formulation comprising an ionizable lipid, one or more phospholipids, cholesterol, and optionally a stabilizer.

[0039] In another aspect, there is provided a lipid mixture formulation according to the invention for use in formulating a vaccine.

[0040] In another aspect, the present invention provides a lipid mixture formulation for formulating mRNA LNPs.

[0041] Compared with conventional mRNA vaccines such as Moderna mRNA-1273 (Spikevax) and Pfizer-Biontech's BNT162b2 (Comirnaty) (see Table 1), self-amplifying mRNA (saRNA) has the advantages of extended translation time and high yield of target antigen. An example of a nucleic acid vaccine element is the self-amplifying mRNA described in PCT Publication No. WO23057979 of Abraham et al.

[0042] This review of the genetics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) includes the spike protein genetics. The spike protein has proven to be a useful target for SARS-CoV-2 vaccines and contributes to viral variants that continue to cause disease. This article uses a modified spike protein gene sequence.

[0043] In this disclosure, the word "comprising" is used in a non-limiting sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. It is understood that in embodiments that include or may include specified features or variables or parameters, alternative embodiments may consist of or consist essentially of such features or variables or parameters. Reference to an element by the indefinite article "a" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element.

[0044] In the present disclosure, descriptions of numerical ranges by endpoints include all numbers contained within the range, including all whole numbers, all integers, and all decimal intermediate numbers (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In the present disclosure, the singular forms "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds.

[0045] In this disclosure, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0046] As used herein, the term "about" is defined to mean plus or minus 12.5% ​​of the stated number. It is used to indicate that the desired target concentration may be, for example, 40 Mol%, but due to inconsistent mixing, the actual percentage may vary by + / - 5 Mol%.

[0047] As used herein, the term "substantially" is defined as plus or minus 5% of the stated figure. It is used to indicate that the desired target concentration may be, for example, 40 Mol%, but due to inconsistent mixing, the actual percentage may vary by + / - 2 Mol%.

[0048] As used herein, the term "nucleic acid cargo" is defined as a substance intended to have a direct effect of alleviating or preventing a disease or to be used as a research reagent. In a preferred embodiment, the nucleic acid cargo is mRNA or saRNA. In a preferred embodiment, the therapeutic agent is a nucleic acid therapeutic agent, such as an RNA polynucleotide. In a preferred embodiment, the therapeutic agent is messenger RNA (mRNA) or self-amplifying RNA (saRNA). In a preferred embodiment, the therapeutic agent is a double-stranded circular DNA (plasmid), a linearized plasmid DNA, a mini-circle or msDNA (multi-copy single-stranded DNA).

[0049] In this disclosure, the word "comprising" is used in a non-limiting sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. It is understood that in embodiments that include or may include specified features or variables or parameters, alternative embodiments may consist of or consist essentially of such features or variables or parameters. Reference to an element by the indefinite article "a" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element.

[0050] In this disclosure, "transfection" means the transfer of nucleic acids into cells for the purpose of inducing the expression of a specific gene of interest in both laboratory and clinical settings. It generally involves an ionizable lipid associated with the nucleic acid, as well as a phospholipid. TM and LIPOFECTAMINE TM are established commercial transfection reagents sold by ThermoFisher Scientific. These research reagents contain permanent cationic lipids and are not suitable for in vivo or ex vivo use.

[0051] In the present disclosure, descriptions of numerical ranges by endpoints include all numbers contained within the range, including all whole numbers, all integers, and all decimal intermediates (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In the present disclosure, the singular forms "an" and "the" include plural objects unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing a "compound" includes a mixture of two or more compounds. In the present disclosure, the term "or" is generally used in its sense including "and / or" unless the context clearly dictates otherwise.

[0052] "nCoV PNI A5 saRNA" is a saRNA expressing the SARSCov2 spike protein incorporated into the Venezuelan equine encephalitis virus TC83 replicon, which has a subgenomic promoter and contains a multiple cloning site for insertion of any GOI as described in PCT Publication No. WO23057979 to Abraham et al.

[0053] "Lipid" refers to a group of structurally diverse organic compounds that are either fatty acid derivatives or sterols, or may be lipid-like materials such as lipidoids (eg, C12-200), and are characterized by being insoluble in water but soluble in many organic solvents.

[0054] "Lipid mixture formulation". A lipid mixture formulation refers to the type of components, the ratio of components, and the ratio of total components to nucleic acid payload. For example, a lipid mixture formulation of 30 mol% ionizable lipids, 50 mol% phospholipids, 20 mol% sterols, and 1.5 mol% stabilizers is a lipid mixture formulation. In a preferred embodiment, the lipid mixture formulation is 28.7 mol% IL / 49.8 mol% DSPC / 20 mol% cholesterol / 1.5 mol% PEG-DMG.

[0055] "Lipid particles" or "lipid nanoparticles" or "LNPs". The present invention provides lipid particles made from lipid mixture formulations described above and shown below. Lipid particles represent the physical organization of lipid mixture formulations with therapeutic agents and between the components. Lipid nanoparticles are lipid particles with a diameter of less than 200 microns. Lipid particles are typically spherical assemblies of lipids, nucleic acids, cholesterol, and stabilizers. The positive and negative charges, ratios, and hydrophilicity and hydrophobicity determine the physical structure of the lipid particles in terms of size and component orientation. The structural organization of these lipid particles may result in an aqueous interior with one or more bilayers (such as in liposomes), or it may have a solid interior (such as in solid nucleic acid lipid nanoparticles). There may be a single or multiple phospholipid monolayer or bilayer. The diameter of the lipid particles is between 1 and 1000 microns.

[0056] When referring to cells in vitro, "viability" means the ability to continue to grow, divide, and continue to grow and divide as is normal for the cell type or tissue culture strain. Cell viability is affected by harsh conditions or treatments. Cell viability is critical in ex vivo therapy or parenteral administration.

[0057] "Ionizable lipid". The composition of the present invention comprises an ionizable lipid as a component. The term "ionizable lipid" used herein refers to a lipid that is cationic or becomes ionizable (protonated) when the pH is reduced to below the pKa of the ionizable group of the lipid, but is more neutral at a higher pH value. At a pH value below pKa, the lipid is able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that present a positive charge when the pH is reduced from physiological pH, and any of many lipid species with a net positive charge at a selective pH. Examples of suitable ionizable lipids can be found in PCT Publication Nos. WO20252589 and WO21000041. The ionizable lipid is preferably present in a ratio of about 10 to about 40Mol% in a lipid formulation according to other embodiments of the present invention ("Mol%" means the total molar percentage belonging to a specific component). The term "about" in this paragraph represents a positive or negative range of 5Mol% at an increment of 0.1. For example, 28.7Mol% is within the scope of the claimed embodiment. DODMA or 1,2-dioleyloxy-3-dimethylaminopropane is an alternative ionizable lipid, as is DLin-MC3-DMA or O-(Z,Z,Z,Z-triacontahedral-6,9,26,29-tetraene-19-yl)-4-(N,N-dimethylamino) ("MC3"). LNP can be produced by lipid preparations including ionizable lipids of the present invention.

[0058] Phospholipids, also referred to as "helper lipids" or "neutral lipids" as used herein, are incorporated into lipid preparations and lipid particles of the present invention in embodiments. Lipid preparations and lipid particles of the present invention include about 25 to 60Mol% of one or more phospholipids of the composition. Suitable phospholipids contribute to the formation of particles during the manufacturing process. Phospholipids refer to any of several lipid species that exist in anionic, uncharged or neutral zwitterionic forms at physiological pH. Representative phospholipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, and although not strictly "phospholipids" in a technical sense, it is intended to include sphingomyelin, dihydrosphingomyelin, cephalin and cerebrosides.

[0059] Exemplary phospholipids include zwitterionic lipids such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-methyl In a preferred embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC). In a preferred embodiment, the phospholipid is DOPE. In a preferred embodiment, the phospholipid is DSPC.

[0060] In another embodiment, the phospholipid is any lipid with negative charge at physiological pH. These lipids include phosphatidylglycerol, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid and other anionic modified groups connected to neutral lipids. Other suitable phospholipids include glycolipids (e.g., monosialoganglioside GM1).

[0061] "Stabilizer" or stabilizing agent is a term used to identify agents added to form ionizable lipids, phospholipids and sterols in lipid formulations according to the present invention. Examples of non-ionic stabilizers include: polyethylene glycol (PEG), polysorbate (Tweens), TPGS (vitamin E polyethylene glycol succinate), Brij TM S20 (polyoxyethylene (20) stearyl ether), Brij TM 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij TM S10 (polyethylene glycol stearyl ether, polyoxyethylene (10) stearyl ether) and Myrj TM 52 (polyoxyethylene (40) stearate).

[0062] In some embodiments, the stabilizer comprises a pegylated lipid, including PEG-DMG 2000 ("PEG-DMG"). Other polyethylene glycol-conjugated lipids may also be used. The stabilizers may be used alone or in combination with one another.

[0063] In some embodiments, there is no stabilizer. In other embodiments, the stabilizer accounts for about 0.1 to 3Mol% of the total lipid mixture. In some embodiments, the stabilizer accounts for about 0.5 to 2.5Mol% of the total lipid mixture. In some embodiments, the stabilizer is present at more than 2.5Mol%. In some embodiments, the stabilizer is present at 5Mol%. In some embodiments, the stabilizer is present at 10 to 15Mol%. In some embodiments, the stabilizer is present at 2.5 to 10 mol%. In some embodiments, the stabilizer is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is 2.6-10 Mol % of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 Mol % of the lipid mixture.

[0064] Sterol is included in the preferred lipid mixture preparation for some application, and the lipid particles made therefrom include cholesterol, beta-sitosterol and 20-alpha-hydroxysterol and plant sterol.In lipid mixture of the present invention, sterol exists with about 15 to 25Mol% of final lipid mixture in some embodiments.In some embodiments, there is the sterol of modification or the sterol of synthetic origin.

[0065] Nucleic acid. The lipid mixture formulation and lipid particles of the present invention can be used for systemic or local delivery of nucleic acid. In the case of vaccines, delivery is located in the skin or muscle. As used herein, the term "nucleic acid" means any oligonucleotide or polynucleotide including its delivery into cells to cause a desired effect. This definition includes diagnostic agents and research reagents that follow the same physical principles provided by the present invention. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, and longer RNAs are referred to as polynucleotides. In a specific embodiment, the length of the oligonucleotides of the present invention is 20-50 nucleotides. In an embodiment of the present invention, the length of the polynucleotides is 996 to 4500 nucleotides, as in the case of messenger RNA. In a specific embodiment, the polynucleotides of the present invention include up to 14000 nucleotides.

[0066] The term "nucleic acid" also refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and may be single-stranded, double-stranded, or comprise portions of both double-stranded and single-stranded sequences, as appropriate. Messenger RNA (mRNA) may be modified or unmodified, base-modified, and may include different types of capping structures, such as Cap1. In some embodiments, nucleic acid refers to self-amplifying RNA ("saRNA"). In some embodiments, nucleic acid refers to a plasmid comprising self-amplifying RNA.

[0067] As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) connected by internucleotide phosphodiester bonds, such as 3'-5' and 2'-5', inverted bonds, such as 3'-3' and 5'-5', branched structures, or internucleotide analogs. Polynucleotides have associated counterions, such as H+, NH4+, trialkylammonium, Mg2+, Na+, etc. Polynucleotides can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides may include internucleotides, nucleobases, and / or sugar analogs.

[0068] The term "polypeptide" herein includes "oligopeptides" and "proteins" and their tertiary and quaternary structures, which are therapeutic agents in some embodiments. Oligopeptides are usually composed of 2 to 20 amino acids. Polypeptides are single linear chains of many amino acids of any length held together by amide bonds. Proteins are composed of one or more and may include structural proteins, energy catalysts, albumins, hemoglobins, immunoglobulins and enzymes.

[0069] At present, nucleic acid cargoes include deoxyribonucleic acid, complementary deoxyribonucleic acid, complete genes, ribonucleic acid, oligonucleotides and ribozymes for gene therapy for a variety of diseases, such as cancer, infectious diseases, genetic disorders and neurodegenerative diseases. As described herein, nucleic acid therapeutics (NAT) are incorporated into lipid particles in the process of forming lipid particles with the compounds of the present invention. More than one nucleic acid therapeutic agent can be incorporated in this way. They can come from natural sources, or more commonly synthesized or grown in culture. Examples of nucleic acid cargoes include, but are not limited to, antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozymes, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-concentrated DNA, plasmids or pDNA or aptamers. Nucleic acid agents are used to silence genes (e.g., with siRNA), express genes (e.g., with mRNA), edit genomes (e.g., with CRISPR / Cas9) and reprogram cells to return to the source organism (e.g., ex vivo cell therapy to reprogram immune cells for cancer therapy; autologous transfer or allogeneic transfer).

[0070] Nucleic acids present in the lipid particles according to the present invention include any known form of nucleic acids. The nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replication systems, such as viruses or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference agents. Single-stranded nucleic acids include antisense oligonucleotides, guide RNA (including CRISPR-Cas9gRNA), ribozymes, microRNA, mRNA and triplex formation oligonucleotides. More than one nucleic acid can be incorporated into the lipid particles, such as mRNA and guide RNA together, or different types of each, or in combination with proteins.

[0071] In some cases, the nucleic acid encodes a genetically engineered receptor that specifically binds to a ligand, such as a recombinant receptor, and a molecule or a functional part thereof that participates in a metabolic pathway. Alternatively, the molecule that participates in a metabolic pathway is a recombinant molecule, including an exogenous entity. The genetically engineered receptor and the molecule that participates in a metabolic pathway can be encoded by one nucleic acid or two or more different nucleic acids. In some instances, a first nucleic acid can encode a genetically engineered receptor that specifically binds to a ligand, and a second nucleic acid can encode a molecule that participates in a metabolic pathway.

[0072] As used herein, "therapeutic agent" includes the nucleic acid cargoes described herein.

[0073] An apparatus for measuring particle size in solution (e.g., Malvern TM ZetasizerTM ) to assess the size of the lipid particles of the present invention. The average particle size of the particles is generally 15nm to 1000nm. A subgroup of lipid particles is "lipid nanoparticles" or LNPs, and their average diameter is about 15 to about 300nm. In some embodiments, the average particle size is greater than 300nm. In some embodiments, the diameter of the lipid particles is about 300nm or less, 250nm or less, 200nm or less, 150nm or less, 100nm or less, or 50nm or less. In one embodiment, the diameter of the lipid particles is about 50 to about 150nm. Compared with larger particles, smaller particles generally show increased circulation life in vivo. Smaller particles have an increased ability to reach tumor sites than larger nanoparticles. In one embodiment, the diameter of the lipid particles is about 15 to about 50nm.

[0074] Lipid particles according to embodiments of the present invention can be prepared by standard T-tube mixing techniques, turbulent mixing, grinding mixing, stirring to promote sequential self-assembly or passive mixing of all ingredients to self-assemble the ingredients into nanoparticles. A variety of methods have been developed to prepare lipid nanoparticles (LNPs) containing gene drugs. Suitable methods are disclosed in, for example, U.S. Patent No. 5,753,613, U.S. Patent No. 6,734,171 and U.S. Patent No. 7,901,708. These methods include mixing preformed lipid particles with nucleic acid therapeutic agents (NAT) in the presence of ethanol, or mixing lipids dissolved in ethanol with an aqueous medium containing NAT, and obtaining lipid particles with a NAT encapsulation efficiency of 65-99%. All of these methods rely on the presence of ionizable lipids to achieve the encapsulation of NAT, and rely on the presence of stabilizers to suppress aggregation and the formation of large structures. The characteristics of the lipid particle system produced, including size and NAT encapsulation efficiency, are sensitive to various lipid mixture formulation parameters (such as ionic strength, lipid and ethanol concentrations, pH, NAT concentrations and mixing rates).

[0075] Microfluidic two-phase droplet technology has been applied to produce monodisperse polymer microparticles for drug delivery or to produce large vesicles for encapsulation of cells, proteins or other biomolecules. The use of hydrodynamic flow focusing has also been demonstrated to produce monodisperse liposomes of controlled size.

[0076] Parameters, such as the relative lipid and NAT concentrations during mixing and the mixing rate, are difficult to control using current formulation procedures, leading to variability in the properties of the resulting NATs within and between preparations. This is what makes the new formulation so unique, as the ratio of ionizable lipids to phospholipids is surprisingly low. Automated micromixing instruments, such as The instrument (PrecisionNanoSystems ULC, Vancouver, Canada) enables rapid and controlled production of nanomedicines (liposomes, lipid nanoparticles, and polymer nanoparticles). The instrument achieves controlled molecular self-assembly of nanoparticles through microfluidic mixing columns that allow millisecond mixing of nanoparticle components at nanoliter, microliter, or larger scales, and customization or parallelization. Rapid mixing at small scales allows reproducible control of particle synthesis and quality, which is not possible in larger instruments.

[0077] Preferred methods incorporate instrumentation, such as a microfluidic mixing device, e.g. Spark TM 、Ignite TM 、Benchtop TM and Blaze TM , so that nearly 100% of the nucleic acid used in the formation process is encapsulated in the particle in one step. In a preferred embodiment, the lipid particles are prepared by a process that encapsulates about 75% to about 100% of the nucleic acid used in the formation process in the particle.

[0078] U.S. Patent Nos. 9,758,795 and 9,943,846 describe methods using small volume mixing techniques and new formulations derived therefrom. U.S. Patent No. 10,159,652 describes more advanced methods for formulating different materials using small volume mixing techniques and products. U.S. Patent No. 9,943,846 discloses a microfluidic mixer with different paths and holes for the components to be mixed. PCT Publication No. WO 2017117647 discloses a microfluidic mixer with disposable sterile paths. U.S. Patent No. 10,076,730 discloses bifurcated annular micromixing geometry and its application in microfluidic mixing. PCT Publication No. WO2018006166 discloses a programmable automatic micromixer and its mixing chip. U.S. Design Nos. D771834, D771833, D772427, D803416, D800335, D800336, and D812242 disclose mixing columns with microchannels and mixing geometry for use in mixer instruments sold by Precision NanoSystems ULC.

[0079] In embodiments of the present invention, the device for biological microfluid mixing is used to prepare the lipid granules according to embodiments of the present invention.Described device comprises the first and second reagent streams, and it is fed in the microfluid mixer, and the lipid granules are collected from the outlet, or enter aseptic environment.

[0080] The first stream includes a therapeutic agent in a first solvent. Suitable first solvents include solvents in which the therapeutic agent is soluble and miscible with the second solvent. Suitable first solvents include aqueous buffers. Representative first solvents include citrate and acetate buffers, or optional other low pH buffers.

[0081] The second stream includes a lipid mixture material in a second solvent. Suitable second solvents include solvents in which the ionizable lipids according to embodiments of the present invention are soluble and miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acid and alcohol. Representative second solvents include 90% ethanol aqueous solution or anhydrous ethanol.

[0082] In one embodiment of the invention, a suitable device includes one or more microchannels (i.e., channels with a maximum dimension less than 2 mm). In one example, the diameter of the microchannel is about 20 to about 300 μm. In another example, the diameter of the microchannel is about 300 to about 1000 μm. In an example, at least one region of the microchannel has a main flow direction, and one or more surfaces defining at least one groove or protrusion therein, wherein the groove or protrusion has an orientation (e.g., a staggered herringbone mixer) that forms an angle with the main direction, as described in U.S. Patent No. 9,943,846, or a bifurcated annular flow, as described in U.S. Patent No. 10,076,730. In order to achieve maximum mixing rate, it is advantageous to avoid excessive fluid resistance before the mixing zone. Therefore, an example of a device has a non-microfluidic channel having a size greater than 1000 μm to deliver fluid to a single mixing channel.

[0083] Less complex mixing methods and apparatus, such as those disclosed in US Published Patent Application No. 20040262223, can also be used to produce the lipid particle formulations of the present invention.

[0084] The lipid mixture of the present invention can be used for delivering therapeutic agents to cells in vitro or in vivo. In a specific embodiment, the therapeutic agent is a nucleic acid, which is delivered to cells using the nucleic acid-lipid particles of the present invention. The nucleic acid can be siRNA, miRNA, LNA, plasmid, replicon (including a carrier with antigen mRNA), self-amplification RNA, mRNA, guide RNA, transposon or a single gene.

[0085] In other embodiments, the therapeutic agent is an oligopeptide, a polypeptide or a protein, which is delivered to cells using the peptide-lipid particles of the present invention. In other embodiments, the therapeutic agent is a mixture of nucleic acid and protein components (e.g., Cas9). The method and lipid mixture formulation can be easily adapted to deliver any suitable therapeutic agent for treating any disease or condition that will benefit from such treatment.

[0086] In certain embodiments, the invention provides a method for introducing nucleic acid into cells (i.e., transfection). Transfection is a common technique in molecular biology, for introducing nucleic acid cargo (or NAT) into intracellular space from extracellular space, for the purpose of transcription, translation and expression of nucleic acid therapeutic agents (NAT) delivered, to produce certain gene products or to lower the expression of disease-related genes. Transfection efficiency is generally defined as i) showing the percentage of cells expressing the positive expression of the delivered gene in the total population of treatment, such as by living cell or fixed cell imaging (for detecting fluorescent protein) and flow cytometry measurement, or ii) by the intensity or quantity of the protein expressed by the cells of treatment, such as by living cell or fixed cell imaging or flow cytometry analysis, or iii) using protein quantification techniques, such as ELISA or Western blot. These methods can be achieved by contacting the lipid granules of the present invention or lipid mixture preparations with cells for the time sufficient for intracellular delivery.

[0087] Typical applications include using well-known procedures to provide intracellular delivery of siRNA to knock down or silence specific cell targets in vitro and in vivo. Alternatively, applications include delivering DNA or mRNA sequences encoding useful polypeptides for treatment. In this way, therapy is provided for genetic diseases by providing defective or missing gene products. The method of the present invention can be implemented in vitro, in vitro or in vivo. For example, the lipid mixture formulation of the present invention can also be used to deliver nucleic acids to cells in vivo using methods known to those skilled in the art. In another example, the lipid mixture formulation of the present invention can be used to deliver nucleic acids to a sample of patient cells, which is in vitro and then returned to the patient.

[0088] Delivery of nucleic acid cargo via the lipid particles of the present invention is described below.

[0089] For in vivo administration, the pharmaceutical composition is preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseously, intramuscularly, or intratumorally). In a specific embodiment, the pharmaceutical composition is administered intravenously, intramuscularly, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include topical (skin, eyes, mucosa), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0090] For in vitro applications, the pharmaceutical composition is preferably administered to a biological sample that has been removed from an organism, and the cells are then washed and returned to the organism. The organism may be a mammal, and in particular may be a human. This process is used, for example, for cell reprogramming, genetic restoration, or immunotherapy.

[0091] In one embodiment, the invention provides a method for regulating the expression of a target polynucleotide or polypeptide. These methods generally include contacting a cell with a lipid granule of the present invention, which is associated with a nucleic acid capable of regulating the expression of a target polynucleotide or polypeptide. The term "regulate" as used herein refers to changing the expression of a target polynucleotide or polypeptide. Regulating may mean increasing or enhancing, and may also mean reducing or decreasing.

[0092] In a related embodiment, the present invention provides a method for treating a disease or condition characterized by overexpression of a polypeptide in a subject, comprising providing the subject with a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from siRNA, microRNA, antisense oligonucleotide and a plasmid capable of expressing siRNA, microRNA or antisense oligonucleotide, and wherein the siRNA, microRNA or antisense RNA comprises a polynucleotide or its complement that specifically binds to a polynucleotide encoding the polypeptide.

[0093] In a related embodiment, the present invention provides a method of treating a disease or condition characterized by underexpression of a polypeptide in a subject, comprising providing the subject with a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from mRNA, self-amplifying RNA (saRNA) or a plasmid, including a nucleic acid therapeutic agent that specifically encodes or expresses the underexpressed polypeptide, or its complement. Examples include RNA vaccines, more particularly self-amplifying mRNA vaccines.

[0094] Methods of delivering bioactive agents for treating diseases include, in one embodiment, the compounds, compositions, methods and uses of the present invention are used to deliver bioactive agents to liver cells (e.g., hepatocytes). In one embodiment, the compounds, compositions, methods and uses of the present invention are used to deliver bioactive agents to tumors or tumor cells (e.g., primary tumors or metastatic cancer cells). In another embodiment, the compounds, compositions, methods and uses are used to deliver bioactive agents to skin fat, muscle and lymph nodes (subcutaneous administration).

[0095] In order to deliver the bioactive agent to the liver or liver cells, in one embodiment, the formulation of the present invention is contacted with the liver or liver cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, portal vein injection, catheterization, stenting) to facilitate delivery. In order to deliver the bioactive agent to the kidney or kidney cells, in one embodiment, the formulation of the present invention is contacted with the kidney or kidney cells of the patient by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting) to facilitate delivery. In order to deliver the bioactive agent to a tumor or tumor cell, in one embodiment, the formulation of the present invention is contacted with the patient's tumor or tumor cell by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting) to facilitate delivery.

[0096] In order to deliver the bioactive agent to the CNS or CNS cells, in one embodiment, the formulation of the present invention is contacted with the patient's CNS or CNS cells (e.g., brain cells and / or spinal cord cells) by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting, osmotic pump administration (e.g., intrathecal or intraventricular)) to facilitate delivery. In order to deliver the bioactive agent to the peripheral nervous system (PNS) or PNS cells, in one embodiment, the formulation of the present invention is contacted with the patient's PNS or PNS cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection) to facilitate delivery. In order to deliver the bioactive agent to the lungs or lung cells, in one embodiment, the formulation of the present invention is contacted with the patient's lungs or lung cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct pulmonary administration to lung tissue and cells) to facilitate delivery.

[0097] In order to deliver the bioactive agent to the vascular system or vascular cells, in one embodiment, the formulation of the present invention is contacted with the patient's vascular system or vascular cells by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., clamping, catheterization, stenting) to facilitate delivery.

[0098] In order to deliver the bioactive agent to the skin or skin cells (e.g., dermal cells and / or follicular cells), in one embodiment, the formulation of the present invention is contacted with the patient's skin or skin cells (e.g., dermal cells and / or follicular cells) by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct skin application, iontophoresis) to facilitate delivery. In order to deliver the bioactive agent to the eye or eye cells (e.g., macula, fovea, cornea, retina), in one embodiment, the formulation of the present invention is contacted with the patient's eye or eye cells (e.g., macula, fovea, cornea, retina) by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eye drops, implants) to facilitate delivery. To deliver a bioactive agent to the ear or cells of the ear (e.g., cells of the inner ear, middle ear, and / or outer ear), in one embodiment, the formulation of the invention is contacted with the patient's ear or cells of the ear (e.g., cells of the inner ear, middle ear, and / or outer ear) as is generally known in the art, e.g., by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection) to facilitate delivery. To deliver a bioactive agent (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen presenting cells, including specialized antigen presenting cells), in one embodiment, the formulation of the invention is delivered intramuscularly, after which immune cells can infiltrate the delivery site and process the delivered RNA and / or process the encoded antigen produced by non-immune cells (e.g., muscle cells). Such immune cells may include macrophages (e.g., bone marrow-derived macrophages), dendritic cells (e.g., bone marrow-derived plasmacytoid dendritic cells and / or bone marrow-derived myeloid dendritic cells), T cells, and monocytes (e.g., human peripheral blood mononuclear cells), etc. (e.g., see WO2012 / 006372).

[0099] Immunization. For the purpose of immunization, the formulations of the present invention are generally prepared as injections, pulmonary aerosols or nasal aerosols, or prepared in a delivery device (e.g., a syringe, atomizer, sprayer, inhaler, skin patch, etc.). The delivery device can be used to administer the pharmaceutical formulation to a subject (e.g., a human) for immunization.

[0100] According to the present invention, for immunization purposes, in some embodiments, the present invention includes delivering RNA encoding an immunogen. This immunogen triggers an immune response that recognizes the immunogen to provide immunity to pathogens or allergens or tumor antigens. Preferably, the disease and / or infection caused by the pathogen is immunized.

[0101] The RNA is delivered with the lipid formulation of the present invention (for example, formulated as liposome or LNP). In some embodiments, the present invention utilizes LNP, wherein the RNA encoding immunogen is encapsulated. Encapsulation in LNP can protect RNA from RNAse digestion. Encapsulation efficiency does not have to be 100%. It is acceptable to have external RNA molecules (for example, on the outer surface of liposome or LNP) or "naked" RNA molecules (RNA molecules not associated with liposome or LNP). Preferably, for the preparation comprising lipid and RNA molecules, at least half of the RNA molecules (for example, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least about 96%, at least about 97%, at least about 98% or at least 99% RNA molecules) are encapsulated in LNP or compounded with LNP.

[0102] Some lipid nanoparticles can include lipid cores (e.g., the preparation can include a mixture of LNP and nanoparticles with lipid cores). In this case, RNA molecules can be encapsulated by LNPs with one or more aqueous cores, and compounded with LNPs with lipid cores by non-covalent interactions (e.g., ionic interactions between negatively charged RNA and cationic lipids). Encapsulation and compounding with LNPs (whether lipid or aqueous core) can protect RNA from RNAse digestion. Encapsulation / compounding efficiency does not have to be 100%. The presence of "naked" RNA molecules (RNA molecules not associated with LNPs) is acceptable. Preferably, for preparations comprising LNP colonies and RNA molecule colonies, at least half of the RNA molecule colonies (e.g., at least, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% RNA molecules) are encapsulated in LNPs, or compounded with LNPs.

[0103] Some lipid nanoparticles have multilayer components.

[0104] For delivery of immunogen encoding RNA, preferred LNP diameters range from 60-180 nm, and in more specific embodiments, from 80-160 nm. The LNP may be part of a composition comprising a population of LNPs, and the LNPs in the population may have a range of diameters. For compositions comprising a population of LNPs having different diameters, preferably (i) at least 80% of the number of LNPs have a diameter within the range of 60-180 nm, such as within the range of 80-160 nm, (ii) the average diameter of the population (by intensity, e.g., Z-average) is ideally within the range of 60-180 nm, such as within the range of 80-160 nm; and / or the diameters within the majority have a polydispersity index <0.2. In order to obtain LNPs having a desired diameter, mixing may be performed using the following process: two RNA aqueous solution feed streams are combined with one lipid ethanol solution stream in a single mixing zone, all at the same flow rate (e.g., in a microfluidic channel). See, for example, a composition comprising a population of LNPs having different diameters, and preferably (i) at least 80% of the number of LNPs have a diameter within the range of 60-180 nm, such as within the range of 80-160 nm, (ii) the average diameter of the population (by intensity, e.g., Z-average) is ideally within the range of 60-180 nm, such as within the range of 80-160 nm; and / or the diameters within the majority have a polydispersity index <0.2. Additional Description of Microfluidic Mixers.

[0105] RNA molecules.After the immune composition (" vaccine carrier LNP ") is administered in vivo, the RNA delivered is released and translated in the cell to provide an immunogen in situ. In certain embodiments, RNA is positive (" + ") strand, so it can be translated by the cell without any intermediate replication step, such as reverse transcription. In certain embodiments, the RNA is a self-replicating RNA. When a self-replicating RNA molecule (replicon) is delivered to a vertebrate cell, even without any protein, it can also lead to the production of multiple sub-RNAs by self-transcription (antisense copies produced by it from itself). Therefore, in certain embodiments, a self-replicating RNA molecule is: a (+) strand molecule that can be directly translated after being delivered to the cell, and the translation provides an RNA-dependent RNA polymerase, which then produces antisense and sense transcripts from the RNA delivered. Therefore, the RNA delivered causes the generation of multiple sub-RNAs. These sub-RNAs and colinear subgenomic transcripts can be self-translated to provide the in situ expression of the encoded immunogen, or can be transcribed to provide more transcripts with the same meaning as the RNA delivered, and these transcripts are translated to provide the in situ expression of the immunogen. The overall result of this transcription sequence is that the amount of the introduced replicon RNA is amplified so that the encoded immunogen becomes the major polypeptide product of the host cell.

[0106] A suitable system for realizing self-replication is to use an RNA replicon based on alphavirus. These (+) chain replicons are translated after being delivered to cells to produce replicase (or replicase-transcriptase). Replicase is translated into polyprotein, which is self-cleaved to provide a replication complex, and the complex produces a genome (-) chain copy of the (+) chain delivery RNA. These (-) chain transcripts themselves can be transcribed to provide more copies of the (+) chain parent RNA, and also provide a subgenomic transcript encoding an immunogen. Therefore, the translation of the subgenomic transcript causes the infected cell to express the immunogen in situ. Suitable alphavirus replicons can use replicase from Sindbis virus, Semliki Forest virus, eastern equine encephalitis virus or more preferably Venezuelan equine encephalitis virus, etc. The system can be a hybrid or chimeric replicase in some embodiments. A preferred embodiment is a replicon according to an embodiment of the present invention, which shows that it can be self-amplified in mammalian cells, and by assembling the mRNA expression immunogenic protein such as the PNI-V101 replicon of the Sars-COV-2 spike protein.

[0107] The RNA molecule can have a 5' cap (e.g., 7-methylguanosine). The cap can enhance the in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the present invention can have a 5' triphosphate group. In the capped RNA, this can be connected to the 7-methylguanosine through a 5' to 5' bridge. The 5' triphosphate can enhance RIG-I binding, thereby promoting the adjuvant effect. The RNA molecule can have a 3' poly A tail. It can also include a poly A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecules that can be used for immunization purposes of the present invention are generally single-stranded. Single-stranded RNA can generally induce an adjuvant effect by binding to TLR7, TLR8, RNA helicase and / or PKR.

[0108] RNA molecules can be easily prepared by in vitro transcription (IVT). IVT can use (cDNA) templates produced and amplified in bacteria as plasmids, or (cDNA) templates produced synthetically (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). As described in WO2011 / 005799, self-replicating RNA can include (except any 5' cap structures) one or more nucleotides with modified nucleobases. For example, self-replicating RNA can include one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5 methylcytosine residues. However, in some embodiments, the RNA does not include modified nucleobases, and modified nucleotides may not be included, that is, all nucleotides in the RNA are standard A, C, G and U ribonucleotides (except any 5' cap structures, which may include 7' methylguanosine). In other embodiments, the RNA may include a 5' cap comprising 7' methylguanosine, and the 1st, 2nd or 3rd 5' ribonucleotides may be methylated at the 2' position of ribose. Ideally, the RNA used for the present invention for immune purposes only includes the phosphodiester bond between nucleosides, but in some embodiments, it includes phosphoramidate, phosphorothioate and / or methylphosphonate bond. The present invention includes the embodiment that the RNA of wherein multiple kinds is prepared with lipid formulations provided by the invention, for example two, three, four or more kinds of RNA, including different classes of RNA (for example mRNA, siRNA, self-replicating RNA and combination thereof).

[0109] In some embodiments, the immunogen RNA molecules used for immunization purposes of the present invention encode polypeptide immunogens. In these embodiments, after administration, the RNA is translated in vivo, and the immunogen can trigger an immune response in the recipient. The immunogen can trigger an immune response against pathogens (e.g., bacteria, viruses, fungi or parasites), but in some embodiments, it triggers an immune response against allergens or tumor antigens. The immune response may include an antibody response (usually including IgG) and / or a cell-mediated immune response. Polypeptide immunogens usually trigger an immune response that recognizes the corresponding pathogen (or allergen or tumor) polypeptide, but in some embodiments, the polypeptide can act as a mimetic epitope to trigger an immune response that recognizes carbohydrates. Immunogens are usually surface polypeptides, such as adhesins, hemagglutinins, envelope glycoproteins, spike glycoproteins, etc. RNA molecules can encode single polypeptide immunogens or multiple polypeptides. Multiple immunogens can be provided as single polypeptide immunogens (fusion polypeptides) or separate polypeptides. If the immunogen is expressed from a replicon as a separate polypeptide, one or more of them can be provided with an upstream IRES or additional viral promoter elements. Alternatively, multiple immunogens can be expressed from a polyprotein encoding individual immunogens fused to a short autocatalytic protease (e.g., foot-and-mouth disease virus 2A protein), or as inteins. In certain embodiments, polypeptide immunogens (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more immunogens) can be used alone or in combination with an RNA molecule (e.g., a self-replicating RNA) encoding one or more immunogens (the same or different from the polypeptide immunogen).

[0110] In some embodiments, the immunogen elicits an immune response against: Coronavirus species, whose immunogens include, but are not limited to, those derived from SARS CoV-1, SARS-CoV-2 (12); human influenza virus, and Neisseria meningitidis, whose useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding proteins. A combination of three useful polypeptides is disclosed in Giuliani et al. (Proc Natl Acad Sci US A. 2006; 103(29): 10834-9. Epub 2006 / 07 / 06. doi: 10.1073 / pnas.0603940103. PubMed PMID: 16825336; PubMed Central PMCID: PMC2047628); Streptococcus pneumoniae, useful polypeptide immunogens thereof are disclosed in WO2009 / 016515, including RrgB pilus subunit, β-N-acetylhexosaminidase precursor (spr0057), spr0096, general stress protein GSP-781 (spr2021, SP2216), serine / threonine kinase StkP (SP1732) and pneumococcal surface adhesin PsaA; Hepatitis virus, immunogens thereof may include hepatitis B virus surface antigen (HBsAg), hepatitis C virus, hepatitis D virus, hepatitis E virus or hepatitis G virus antigen; Rhabdovirus: immunogens include but are not limited to those derived from rhabdovirus, such as lyssavirus (e.g., rabies virus) and vesiculovirus (VS V); Caliciviridae, whose immunogens include, but are not limited to, those derived from Caliciviridae, such as Norwalk virus (Norovirus) and Norwalk-like viruses, such as Hawaii virus and Snow Mountain virus; Avian infectious bronchitis (IBV), mouse hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV); Retroviruses, whose immunogens include those derived from tumor viruses, lentiviruses (such as HIV-1 or HIV-2) or foamy viruses; Reoviruses: immunogens include, but are not limited to, those derived from orthoreovirus, rotavirus, orbivirus or Coltivirus; Parvoviruses, whose immunogens include those derived from parvovirus B19; Herpesviruses, whose immunogens include those derived from human herpesviruses, such as herpes simplex virus (HSV) (e.g., HSV 1 and 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8); papillomavirus, immunogens thereof include those derived from papillomaviruses and adenoviruses.

[0111] In some embodiments, the immunogen elicits an immune response against Chikungunya virus; in other embodiments, the immunogen elicits an immune response against Zika virus.

[0112] In some embodiments, the immunogen elicits an immune response against a virus that infects fish.

[0113] Fungal immunogens can be derived from dermatophytes and other opportunistic organisms.

[0114] In some embodiments, the immunogen elicits an immune response against parasites from the genus Plasmodium, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale. Thus, the present invention can be used for malaria vaccination. In some embodiments, the immunogen elicits an immune response against parasites from the family Ichthyidae, particularly those from the genera Ichthyidae and Ichthyidae, such as sea lice, such as Ichthyidae salmonis or Caligus rogercresseyi.

[0115] In some embodiments, the immunogen is mRNA specific for a neoantigen in a cancer cell or solid tumor.

[0116] In some embodiments, the immunogen is a tumor antigen selected from the group consisting of: (a) cancer testis antigens, such as NY-ESO-1, SSX2, SCPI, and RAGE, BAGE, GAGE, and MAGE family polypeptides, such as GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to treat melanoma, lung, head and neck, NSCLC, breast, gastrointestinal tract, and bladder tumors; (b) mutated antigens, such as p53 (associated with various solid tumors, such as colorectal, lung, head and neck cancer), p21 / Ras (associated with, for example, melanoma, pancreatic cancer, and colorectal cancer), CDK4 (associated with, for example, melanoma), MUMI (associated with, for example, melanoma), caspase-8 (associated with, for example, head and neck cancer), CIA 0205 (associated with, for example, bladder cancer), HLA-A2-R1701, β-catenin (associated with, for example, melanoma), TCR (associated with, for example, T-cell non-Hodgkin's lymphoma), BCR-abl (associated with, for example, chronic myeloid leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLRFUT; (c) overexpressed antigens, such as galectin 4 (associated with, for example, colorectal cancer), galectin 9 (associated with, for example, Hodgkin's disease), proteinase 3 (associated with, for example, chronic myeloid leukemia, etc.), WT I (associated with, for example, various leukemias), carbonic anhydrase (associated with, for example, renal cancer), aldolase A (associated with, for example, lung cancer), PRAME (associated with, for example, melanoma), HER-2 / neu (associated with, for example, breast, colon, lung and ovarian cancers), mammaglobin, alpha-fetoprotein (associated with, for example, hepatoma), KSA (associated with, for example, colorectal cancer), gastrin (associated with, for example, pancreatic and gastric cancers), telomerase catalytic protein, MUC-I (associated with, for example, breast and ovarian cancers), G-250 (associated with, for example, renal cell carcinoma), p53 (associated with, for example, breast, colon cancer), and carcinoembryonic antigen (associated with, for example, breast cancer, lung cancer, and gastrointestinal cancers, such as colorectal cancer); (d) shared antigens, such as melanoma-melanocyte antigens, such as MART-1 / Melan A, gp100, MCIR, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase-related protein-I / TRPI and tyrosinase-related protein-2 / TRP2 (associated with, for example, melanoma); (e) prostate-associated antigens, such as PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, associated with, for example, prostate cancer; (f) immunoglobulin idiotype (associated with, for example, myeloma and B-cell lymphoma).In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, human papillomavirus (HPV) antigens (including E6 and E7), hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23HI, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29 & BCAA), CA 195, CA 242, CA-50, CAM43, CD68&KPI, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-I, RCASI, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, etc.

[0117] Pharmaceutical compositions for vaccines. The pharmaceutical compositions of the present invention, particularly those useful for immunization, may include one or more small molecule immunopotentiators. The pharmaceutical compositions of the present invention may include one or more preservatives, such as thimerosal or 2-phenoxyethanol. Mercury-free and preservative-free vaccines may be prepared.

[0118] Compositions include effective amounts of lipid preparations described herein (e.g., LNP), and any other components as required.Immunologically effective amount refers to an effective amount for treatment (e.g., preventive immune response to pathogens) used to an individual in a single dose or as a part for a series of doses. This amount is based on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primates, primates, etc.), the ability of the individual immune system to synthesize antibodies, the required degree of protection, the preparation of the vaccine, the attending physician to the assessment of medical conditions and other related factors and changes. It is estimated that this amount will fall within a relatively wide range, which can be determined by routine tests.

[0119] The RNA and pharmaceutical compositions prepared by LNP described herein are used for in vivo use to induce an immune response to a target immunogen. The invention provides a method of inducing an immune response in a vertebrate, comprising administering an effective amount of RNA or pharmaceutical compositions prepared by LNP as described herein. The immune response is preferably protective, and preferably includes antibodies and / or cell-mediated immunity. The composition can be used for primary immunization and boosting purposes. Alternatively, the primary immunization-boosting immunization scheme can be a mixture of RNA and a corresponding polypeptide immunogen (e.g., RNA primary immunization, protein boosting).

[0120] The present invention also provides a LNP or a pharmaceutical composition thereof for inducing an immune response in a vertebrate. The present invention also provides the use of LNP or a pharmaceutical composition thereof in the manufacture of a medicament for inducing an immune response in a vertebrate. By inducing an immune response in a vertebrate through these uses and methods, the vertebrate can be protected from various diseases and / or infections, for example, bacterial and / or viral diseases as described above. Vaccines according to the present invention can be preventive (i.e., preventing infection) or therapeutic (i.e., treating infection), but are generally preventive. The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (e.g., horse, cattle, deer, sheep, llama, goat, pig).

[0121] The composition of the present invention will usually be directly applied to the patient. Direct delivery can be completed by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal or injected into the interstitial space of the tissue). Optional delivery routes include rectal, oral (e.g., tablets, drops, sprays), buccal, sublingual, vaginal, topical, transdermal or percutaneous, intranasal, eye, ear, lung or other mucosal administration. Intradermal and intramuscular administration are two preferred approaches. Injection can be performed by a needle (e.g., a hypodermic needle), but needleless injection can also be used. A typical intramuscular dose is 0.5 ml. The present invention can be used to induce systemic and / or mucosal immunity, preferably triggering enhanced systemic and / or mucosal immunity. Administration can be performed by a single dose regimen or a multiple dose regimen. Multiple doses can be used in primary immunization regimens and / or booster immunization regimens.

[0122] In a multiple dose regimen, each dose can be given by the same or different routes, for example, parenteral priming and mucosal boosting, mucosal priming and parenteral boosting, etc. Multiple doses are usually administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, multiple doses can be administered at about 6 weeks, 10 weeks and 14 weeks after birth, for example, at 6 weeks, 10 weeks and 14 weeks of age, as commonly used in the World Health Organization's Expanded Program of Immunization ("EPI"). In an alternative embodiment, two primary doses are administered at intervals of about two months, for example, two primary doses are administered at intervals of about seven, eight or nine weeks, followed by one or more booster doses at about six months to one year after the second primary dose, for example, at about six, eight, ten or twelve months after the second primary dose. In a further embodiment, three primary doses are administered at intervals of about two months, for example, three primary doses are administered at intervals of about seven, eight or nine weeks, followed by one or more booster doses at about six months to one year after the third primary dose.

[0123] In embodiments, the pharmaceutical compositions described herein may be prepared by any method known or later developed in the art of pharmacology. Generally, such preparation methods include the step of bringing the active ingredient into association with an excipient and / or one or more other auxiliary ingredients.

[0124] Pharmaceutical compositions according to the present disclosure can be prepared, packaged and / or sold in bulk form, as a single unit dose and / or as multiple single unit doses. As used herein, "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient can generally be equal to the dosage of the active ingredient to be administered to the subject and / or a convenient fraction of such dosage, including but not limited to one-half or one-third of such dosage.

[0125] The relative amounts of active ingredient, pharmaceutically acceptable excipient and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary, depending on the identity, size and / or condition of the subject being treated, and further on the route of administration of the composition. For example, the composition may contain 0.1% to 99% (w / w) of active ingredient.

[0126] The pharmaceutical preparation may also include a pharmaceutically acceptable excipient, as used herein, including but not limited to any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, etc., as appropriate for the specific dosage form desired. Various excipients for preparing pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006). The use of conventional excipient media is contemplated herein, unless any conventional excipient media may be incompatible with the substance or its derivatives, for example by producing any unwanted biological effects or otherwise interacting in a harmful manner with any other component of the pharmaceutical composition.

[0127] In some embodiments, the particle size of the lipid particles can be increased and / or decreased. Changes in particle size can help counteract biological responses, such as but not limited to inflammation, or can increase the biological effect of NAT delivered to a mammal by changing biodistribution. Size can also be used to determine target tissues, with larger particles being cleared quickly and smaller particles reaching different organ systems.

[0128] Pharmaceutically acceptable excipients for making pharmaceutical compositions comprising LNPs include, but are not limited to, inert diluents, surfactants and / or emulsifiers, preservatives, buffers, lubricants and / or oils. Such excipients may optionally be included in the pharmaceutical formulations of the present invention.

[0129] The following is a description of representative lipid particles (LNPs) prepared with nucleic acids, how they are prepared, evidence of their advantages, and methods of using them to provide therapeutic benefit.

[0130] Example

[0131] General Considerations: Unless otherwise stated, all solvents and reagents are commercial products and were used as received. Temperatures are given in degrees Celsius.

[0132] abbreviation

[0133] EPO = erythropoietin

[0134] GFP = Green Fluorescent Protein

[0135] ug = μg = microgram

[0136] pg = picogram

[0137] ng = nanogram

[0138] g = grams

[0139] h = hours

[0140] HPLC = High Performance Liquid Chromatography

[0141] MFI = median fluorescence intensity

[0142] min = minutes

[0143] mL = milliliters

[0144] mmol = millimole

[0145] N / P ratio = the ratio of positively charged polymer amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups

[0146] PBS = Phosphate Buffered Saline

[0147] wt = weight

[0148] Deg.C = degrees Celsius

[0149] "Gene of interest" (GOI) means one or more genetic elements intended for expression to achieve a therapeutic goal, including immunity. A5 SARS Cov-2 antigen encoding element and epidermal growth factor (EPO) are examples of GOIs illustrating the present invention, but GOIs are not limited to this demonstrated example.

[0150] IL = ionizable lipid, a lipid that is cationic at higher pH and converts to uncharged at lower pH. ILs are often used to formulate nucleic acid cargoes.

[0151] Stabilizer = Any stabilizer including polyethylene glycol derivatives, including PEG-DMG 2000 and other suitable polymers, the purpose of which is to extend cycle life, etc.

[0152] The components of the lipid mixture include ionizable lipids, phospholipids, cholesterol and stabilizers. A low pH buffer (3-6) can be used. For ionizable amino lipids, the pH of the buffer is usually lower than the pKa of the lipid.

[0153] PNI 516 is an ionizable lipid (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 4-(dimethylamino)butyrate found in PCT Publication WO20252589 A1 by Jain N, Thomas A and Brown A.

[0154] PNI 560 is an ionizable lipid (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 1,4-dimethylpiperidine-4-carboxylate, found in PCT Publication WO20252589 to Jain et al.

[0155] PNI 127 is an ionizable lipid found in PCT Publication WO21000041 A1 by Thomas A, Jain N and Brown A. Its structural formula is (2R,3S,4S)-2-(((1,4-dimethylpiperidine-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate).

[0156] Also found in PCT Publication WO2100041 by Thomas et al.:

[0157] PNI 550, 3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)cyclopentyl 4-(dimethylamino)butyrate;

[0158] PNI 580: (2R,3S,4S)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate); PNI 659: ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl 4-(dimethylamino)butanoate; PNI 721: (2R,3S,4S)-2-((((2-(dimethylamino)ethyl)carbamoyl)oxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate); PNI 722: 2-(((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methoxy)-N,N-dimethylethane-1-amine; PNI PNI 726: (2R,3S,4S)-2-((3-(dimethylamino)propoxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate); PNI 728: ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl (2-(dimethylamino)ethyl)carbamate; and PNI 730:

[0159] (2R,3S,4S)-2-((2-(dimethylamino)ethoxy)methyl)tetrahydrofuran-3,4-diyl bis(2-hexyldecanoate).

[0160] Example 1

[0161] Methods for self-amplifying mRNA synthesis

[0162] Restriction digests of circular plasmids encoding the SARS Covid spike protein were performed according to the manufacturer's instructions for BspQI (New England BioLabs Inc., catalog number R0712S) or PmeI (New England BioLabs Inc., catalog number R0560S) in the buffer specified by the supplier.

[0163] Linearized vector was purified using phenol / chloroform / isoamyl alcohol (25:24:1) and sodium acetate precipitation. Briefly, an equal volume of phenol / chloroform / isoamyl alcohol solution was added to the linearized vector, vortexed for 20 seconds, and incubated at room temperature for 2 minutes. The mixture was spun, and the top aqueous phase containing the linearized vector was carefully pipetted into a clean RNase / DNase-free tube and precipitated. Three volumes of 100% ethanol were added, mixed thoroughly and carefully removed, and the DNA pellet was air-dried and resuspended in nuclease-free water. The DNA was purified using a NanoDrop TM The concentration and purity of the linearized vector were checked by spectrophotometer (VWR).

[0164] Using HiScribe TM T7 High Yield RNA Synthesis Kit (New England BioLabs, Inc., catalog number E2040S) was used for in vitro transcription, and then TURBO TM DNase (Thermofisher Scientific, catalog number AM2238) was used for linear DNA template digestion and the Vaccina Capping System was used. TM The final in vitro transcribed self-amplified RNA (saRNA) was capped (New England Biolabs Inc., catalog number M2080S). All of these processes were performed according to the manufacturer's protocol to generate self-amplified RNA using DNA templates obtained from vector linearization strategies. The capped saRNA was purified using a standard salt precipitation method, followed by a 70% ethanol wash and resuspending the RNA pellet in RNA storage solution (Thermofisher). This is described in PCT Publication No. WO23057979 by Geall et al.

[0165] Example 2

[0166] Microfluidic mixing of nucleic acids into lipid nanoparticles (LNPs)

[0167] The lipid mixture formulation of lipid particles is generated by rapidly mixing a lipid-ethanol solution with an aqueous buffer within a microfluidic mixer that is designed to induce chaotic advection and provide a controlled mixing environment at intermediate Reynolds numbers (24 < Re < 1000). The microfluidic channels have herringbone features or are configured in the manner shown in PCT Publication No. WO2017117647 or U.S. Patent No. 10,835,878.

[0168] The particle size and “polydispersity index” (PDI) of lipid particles are measured by dynamic light scattering (DLS). The PDI represents the width of the particle distribution. This is a parameter calculated from the cumulative analysis of the intensity autocorrelation function measured by (DLS), assuming a single-particle size mode and a single-exponential fit of the autocorrelation function. From a biophysical perspective, a PDI below 0.1 indicates that the sample is monodisperse. Assuming all other variables are neutral, particles generated by a mechanical micro-mixer such as Spark TM and Ignite TM (PrecisionNanoSystems ULC) are substantially uniform in size. A lower PDI indicates a more homogeneous population of lipid particles. The Spark TM instrument is used for screening settings to identify the dominant composition. Once the composition is selected, the Ignite instrument can be used to fine-tune the lipid particles. Once the process parameters flow rate ratio and total flow rate for a specific nanoparticle formulation are identified, the nanoparticle technology can be scaled up using the same process parameter values.

[0169] The self-amplifying RNA or nucleic acid therapeutic agent (NAT) as described above is diluted to the desired concentration using sodium acetate buffer. Then, as described, a lipid nucleic acid particle (LNP) sample is prepared by running two fluids using the Ignite instrument. Briefly, 63 μg of nucleic acid in 0.75 mL of sodium acetate buffer is mixed with 0.25 mL of a 12.5 mM lipid mixture solution as required by an N / P ratio of 8, and then the LNP is diluted by online dilution in PBS at a ratio of 2:1.

[0170] The prepared lipid nucleic acid particles (LNPs) are immediately diluted in the aqueous outlet with 48 μL of 1X PBS (pH 7.4) without Ca++ and Mg++. These LNPs are immediately collected into a microcentrifuge tube containing 96 μL of the same buffer (pH 7.4). By a modified Ribogreen TM assay (Quanti-iT RiboGreen TM ​​RNA Assay Kit, Fisher) to measure encapsulation efficiency. This information is used to determine the required dose.

[0171] The lipid particles are also mixed by a larger microfluidic instrument Ignite TM Manufactured for testing. Briefly, 350 μL of mRNA was diluted to a desired concentration of 0.2 to 0.3 mg / mL using 100 mM sodium acetate buffer. A 12.5 or 25 mM lipid mixture solution was typically used. LNPs were then prepared by running two fluids, nucleic acids in an aqueous solvent and a lipid mixture in ethanol, in a microfluidic mixer at a flow rate ratio of 3:1 and a total flow rate of 12 ml / min. After mixing in the microfluidic device, the post-column lipid nucleic acid particle sample was diluted into an RNase-free tube containing 3 to 40 volumes of PBS (pH 7.4). The final concentration was determined by dialysis in PBS (pH 7) or using an Amicon TM Remove ethanol by centrifugal filter (Millipore, USA) or using TFF system. Once the desired concentration is reached, the lipid nucleic acid particles are sterilized by filtration using a 0.2 μm filter under sterile conditions. Assay, Quant-iT TM RNA reagents and kits (Invitrogen) were used following the manufacturer's instructions and the final encapsulation efficiency was measured. Self-amplified mRNA plasmid NAT was prepared as described below. For mRNA, the observed particle properties were generally sized from 50 to 200 nm, depending on the lipid composition.

[0172] Table 1 above lists the LNP preparations ONPATTRO that have been marketed. TM 、SPIKEVAX TM and COMIRNATY TM The vaccine LNP formulation "Vaccmixb-PNI 516" is listed in Table 2, as well as formulations according to the present invention identified with prefixes starting with V46. These new vaccine formulations have a significantly different ratio of ionizable lipids to phospholipids than the composition of Table 1 above, and offer surprising advantages in terms of safety, since ionizable lipids can be irritating and PEG-lipids are known to cause allergic reactions.

[0173] Table 2: Lipid mixture for LNP

[0174]

[0175]

[0176]

[0177] For Tables 3 and 4, all formulations were made of saRNA expressing Covid spike protein. Both PNI 516 and MC3 (ionizable lipids) were formulated at an N / P of 8, and the RNA concentration of each formulation was kept constant at 84ug / mL.

[0178] Table 3: Composition, size, PDI, EE% and RNA concentration

[0179]

[0180] Table 4: Dimensions and PDI for MC3 and PNI 516

[0181]

[0182] Example 3

[0183] Characterization and Encapsulation of Lipid Nucleic Acid Particles or "LNPs"

[0184] After lipid particles were prepared as described above, the ZetaSizer TM Nano ZS TM (Malvern Instruments, UK) LNP particle size (hydrodynamic diameter of the particles) was determined by dynamic light scattering (DLS). A He / Ne laser with a wavelength of 633 nm was used as the light source. Data were measured based on scattering intensity data performed in backscattering detection mode (measurement angle = 173). The measurements were averaged over 10 runs of two cycles for each sample. The Z-average size was reported as the particle size and was defined as the harmonic intensity average particle diameter. The modified Ribogreen TM Assay (Quanti-iTRiboGreen TM The encapsulation efficiency was measured by RNA Assay Kit (Fisher). Good encapsulation was observed in all formulations (compositions shown in Table 2) with polydispersities (PDI) below 0.19.

[0185] The post-encapsulation step is tangential flow filtration (TFF) concentration for about 40 minutes, and diafiltration. These steps have little impact on LNP properties. Results are not shown. The lipid mixture of the present invention and LNP and the LNP properties of the comparison are described in Table 5 and Table 6.

[0186] Table 5: Effect on LNP (LNP formulated with SARS Cov2 spike protein saRNA and PNI 516 ionizable lipids), N / P ratio of 8

[0187]

[0188] Table 6: Effect of phospholipid ratio on encapsulation (LNPs formulated with SARS Cov2 saRNA and PNI 516 ionizable lipids). Note: N / P ratio = 8

[0189]

[0190] Example 4

[0191] High-throughput LNP potency assay in BHK cells

[0192] BHK 570 cells were purchased from ATCC, cultured normally, and transfected with saRNAA5-loaded LNPs at 1000 to 0.49 ng / mL in 96-well plates. LNPs containing V46-PNI 516 showed EC 50 The V46 lipid mixture showed an EC 50 An unexpected 12x improvement.

[0193] SARsCOV-2 protein expression was measured after transfection of BHK 570 cells with various PNI 516LNP formulations containing nCoVPNIA5 saRNA, as disclosed in PCT Publication WO23057979 by Abraham et al.

[0194] Table 7: Comparison of EC50 of two lipid mixtures

[0195]

[0196] Visual demonstration of SARsCOV-2 protein expression after transfection in BHK 570 cells was possible, with green fluorescence representing SARsCOV-2 protein stained with a fluorescently labeled anti-SARsCOV-2 spike protein antibody (FAB105403G) after BHK 570 cells were transfected with Vaccmixb PNI 516 or V46-PNI 516 at 31.25 ng / RNA well. BHK 570 cells were plated in 96-well plates (20k cells / well) for 48 hours before transfection. After transfection, cells were incubated for an additional 24 hours and then fixed with 4% PFA for 20 minutes. Cells were then permeabilized in 0.1% Triton X-100 and stained with FAB105403G antibody at a dilution of 1:50. The chromatograms were taken at Cytation 7 TM Cells were imaged on a Cell Imaging Multimode Reader (BioTek, Agilent, Santa Clara, CA).

[0197] Dose response of SARsCOV-2 protein expression after Vaccmixb PNI 516 and V46-PNI 516 transfection in BHK 570 cells Figure 1 As shown. Dose response studies were performed by testing decreasing amounts of RNA from 1000 to 0.49 ng / mL. BHK 570 cells were plated in 96-well plates (20k cells / well) for 48 hours before transfection. After transfection, cells were incubated for an additional 24 hours and then fixed with 4% PFA for 20 minutes. Cells were then permeabilized in 0.1% Triton X-100 and stained with FAB105403G antibody at a dilution of 1:50. TM Cells were imaged on a Sigma-Aldrich (BioTek, Agilent, Santa Clara, CA) multimode reader.

[0198] SARsCOV-2 protein expression was measured after transfection of BHK 570 cells with various PNI516 LNP formulations containing nCoVPNIA5 saRNA. Dose response studies were performed by testing decreasing amounts of RNA from 1000 to 0.49 ng / mL. BHK 570 cells were plated in 96-well plates (20k cells / well) for 48 hours before transfection. After transfection, cells were incubated for an additional 24 hours and then fixed with 4% PFA for 20 minutes. Cells were then permeabilized in 0.1% Triton X-100 and stained with FAB105403G antibody at a dilution of 1:50. The expression of 400 μg / mL RNA was detected by ELISA on Cytation 7. TM The results of the study are as follows: Figure 2 shown.

[0199] For mRNA testing, use CLEANCAP with a similar testing protocol as above. TM EGFP mRNA (Trilink Biotechnologies, catalog number L-7601). The last column of Table 8 (N / P is 8) and Table 9 (N / P is 12) shows the various EC 50 readout, where lower levels indicate greater therapeutic efficacy. This finding demonstrates the precise interplay of ionizable lipid chemistry and optimized ratios of components. “IL” is the ionizable lipid used. EC 50 "NP" in a column means the reading was too high for a meaningful measurement (negative result).

[0200] Table 8: EC50 ng / L test of mRNA in BHK cells for different ionizable lipids with N / P ratio of 8

[0201]

[0202]

[0203] In Table 8, there are large differences in the efficacy of the formulations of the invention depending on the ionizable lipid used. 50 Is a measure of the efficacy of the LNP in transfecting cells and the LNP contents in expressing the GOI. The lower this value, the more potent the formulation.

[0204] Table 9: EC50 ng / L test of mRNA in BHK cells with N / P ratio of 12 for different ionizable lipids

[0205]

[0206] Table 10: Combined optimal ionizable lipids for each composition at N / P 8 and N / P 12

[0207]

[0208]

[0209] Example 5

[0210] In vivo studies

[0211] SARS-CoV-2 spike protein-specific IgG expression in mouse serum was measured after intramuscular LNP injection into BALB / c mice at 1 ug / mouse. Enzyme-linked immunosorbent assay (ELISA) was performed according to an established method for measuring SARS-CoV-2 spike protein-specific IgG antibodies in serum samples collected from animals on days 21 and 42 after immunization.

[0212] Reagents used for ELISA: DPBS 1X Sterile (pH 7.4), without Ca2+ and Mg2+, Corning; Assay Diluent B (5X); Purified anti-SARS-CoV-2 S protein S1 antibody rat; ELISA wash buffer (20X); HRP goat anti-rat IgG (minimal cross-reactivity) antibody; and TMB substrate solution (all from BioLegend, San Diego, CA); Isopropanol (IPA) 70% and water (nuclease-free) (from VWR); Nunc MaxiSorp TM Flat-bottom 96-well plates and stop solution (ThermoFisher); and SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His recombinant protein (Sino Biologicals). Figure 3 The IgG levels of the different groups are illustrated at day 21. At day 42, the results were advantageously about 10 times higher (not shown), which is a very good result.

[0213] Example 6

[0214] Three-month stability study

[0215] This study was performed on V46 using PNI 516 as an ionizable lipid. Ten aliquots of frozen LNPs (50 ug / mL saRNA) in pH 7.4 buffer were thawed to room temperature and then stored in the dark for 1 hour, 2 hours, 4 hours, 24 hours, 2 days, 3 days, 4 days and 7 days, 1 month and 3 months. At each time point, LNP particle size was measured by dynamic light scattering and quantified using Quant-it TM RiboGreen RNA Assay Kit TM (Thermo Fisher) was used to determine the saRNA encapsulation percentage. The results are shown in Table 11.

[0216] Table 11: Room temperature stability of V46-PNI 516LNP

[0217]

[0218]

[0219] Example 7

[0220] Cryo-transmission electron microscopy (CryoTEM) structure of LNP

[0221] LNPs containing PNI 516, DSPC, cholesterol and PEG-DMG encapsulating saRNA were cryopreserved, sectioned and prepared for Cryo-TEM examination. Cryo-TEM of LNPs containing the standard IL:DSPC ratio of 3.8 resulted in mostly dense unilamellar vesicles and some multilamellar vesicles, while LNPs containing IL:DSPC plus DMG-PEG ratio of 0.58 showed multilamellar vesicles and a greater number of multilamellar vesicles.

[0222] The PEG-free LNPs with an IL:DSPC ratio of 0.58 also showed more multilamellar vesicles, but the particles were closer together than the PEGylated version of this composition. Figure 4 Exemplary images of A, Vaccmixb-PNI 516; B, V46-PNI 516; and C, V47-PNI 516 are shown.

[0223] Example 8

[0224] Stability and activity of LNPs after lyophilization

[0225] In this experiment, 500 μL of the LNP preparation described below was filled into a 2 mL glass vial, each preparation using PNI516 as an ionizable lipid, and then the LNP sample was balanced to a temperature of 5 ° C and a pressure of 750 Torr. Afterwards, at 400-500 Torr, the temperature was reduced from 5 ° C to -50 ° C. The sample was stored at -50 ° C for 3 hours. Subsequently, the pressure was reduced to 0.062 Torr at -50 ° C to start preliminary drying for 120 minutes, and then the shelf temperature was raised to -40 ° C under a pressure of 0.062 Torr, and kept under these conditions for 12 hours (720 minutes). Finally, secondary drying was carried out at 10 ° C, 0.062 Torr for 180 minutes.

[0226] After lyophilization, the samples were sealed and stored at 4°C. The next day, the samples were reconstituted with 500 μL of molecular biology grade water. The rehydrated LNPs were then analyzed for particle size, SARNA encapsulation, and bioactivity as described above.

[0227] The EC of the preparations was determined in vitro using a dose response by testing decreasing amounts of RNA from 1000 to 0.49 ng / mL. 50 BHK 570 cells were plated in 96-well plates (20k cells / well) for 48 hours before transfection. After transfection, cells were incubated for another 24 hours and then fixed with 4% PFA for 20 minutes. Cells were then permeabilized in 0.1% TritonX-100 (Sigma Aldrich) and stained with FAB105403G antibody (Bio-Techne, Toronto, Ontario) at a dilution of 1:50. TM Cells were imaged on a Sigma-Aldrich (Agilent, Santa Clara, CA) reader.

[0228] The lyophilized forms of V46-PNI 516LNP and V47-PNI 516 showed no significant change in particle size after rehydration, in contrast to Vaccmixb PNI516, which showed a significant increase in size. Furthermore, in BHK 570 cell transfections, Vaccmixb PNI516 showed a 7-fold decrease in activity compared to V46-PNI 516LNP and V47-PNI 516.

[0229] Table 12: Size, PDI, encapsulation percentage and EC50 before and after lyophilization

[0230]

[0231] As described above, the ionizable lipid combination is carried out in the high phospholipid composition according to the present invention. TMThe assay was used to measure LNP particle size (hydrodynamic diameter of the particles) and encapsulation efficiency. Good encapsulation size and PDI were found in all formulations (compositions are shown in Table 13), with polydispersity (PDI) less than 0.25 and size <117 nm. EC was determined in BHK 570 cells using an in vitro dose response as described above by testing decreasing amounts of RNA from 1 to 0.00049 μg / well. 50 .

[0232] Table 13 illustrates the composition and ratio of various mixtures of ionizable lipids, and the effect of LNPs prepared therefrom on GFPsaRNA EC50 and the difference between the EC50 results and the Vaccmixb (control) EC50. V76 and V68, representing ionizable lipid combinations, showed very good EC50 (pg / ml) scores. V75, V69, V46, V74, and V76, all representing ionizable lipid mixtures, were also very good compared to the control.

[0233] Table 12: EC compared to “benchmark” Vaccmixb 50 (PNI GFP saRNA) compared with the composition and EC50

[0234]

[0235]

[0236] The physical properties of LNPs were also tested for mixed ionizable lipid mixtures. The results are shown in Table 13.

[0237] Table 13: Physical properties of novel compositions containing mixtures of ionizable lipids

[0238]

[0239]

[0240] Example 9

[0241] This example demonstrates the in vitro transfection ability of saRNA LNPs prepared with different lipid mixtures in Jurkat cells. After transfection with 0.32ug / mL of V02 Vaccmixb, V46-PNI516, V47-PNI516, and V22-PNI516 in Jurkat cells, flow cytometry was used to assess % GFP positivity. LNPs were tested by adding Jurkat cells (50k / well) to GFPsaRNA LNPs to a final saRNA concentration of 3.2ug / mL. After incubation for 24 hours, the cells were resuspended in BSA staining buffer and stained using BECKMAN COULTER TMThe results were analyzed by Cytoflex S flow cytometer. Figure 5 shown.

[0242] Example 10

[0243] This example demonstrates the in vitro transfection ability of mRNA LNP prepared with different lipid mixtures in Jurkat cells. After transfection of V46-PNI516, V02-PNI516 and V22-PNI516 in Jurkat cells, % GFP positivity was evaluated in a dose response experiment. The dose response was tested by adding Jurkat cells (50k / well) to decreasing amounts of GFPmRNA LNP from 3 to 0.001465ug / well. After incubation for 24 hours, the cells were resuspended in BSA staining buffer and analyzed using a BeckmanCoulter Cytoflex S flow cytometer. The results are shown in Table 1. Figure 6 shown.

[0244] Embodiment 11

[0245] This example demonstrates the transfection ability of EGFP saRNA LNPs prepared with different lipid mixtures in T cells isolated from PBMCs. Human PBMCs were activated for 3 days in ImmunoCult-XF T cell expansion medium supplemented with activator / recombinant human IL-2. After activation, cells were diluted with ApoE to a concentration of 0.25x106 cells / ml. LNP formulations containing EGFP saRNA (such as Figure 7 ) were added to the cell culture medium and transfected for 24 hours. CD4+ and CD8+ T cells were gated using specific antibodies and the % GFP positivity in T cells was assessed using standard flow cytometry analysis. Dead cells were omitted from the analysis using viability staining. Dead cells are prone to nonspecific staining with fluorescent antibodies and are usually excluded by gating. The results are shown in Figure 7 shown.

[0246] While specific embodiments of the present invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not limiting of the invention as construed according to the appended claims.

[0247] Example 12

[0248] This example demonstrates the efficacy of the LNPs of the invention in an intravenous protein replacement model.

[0249] Two different LNPs according to embodiments of the present invention were injected intravenously into mice at 0.1 mg / kg. The payload was luciferase mRNA (Trilink-CLEANCAP TMFLuc mRNA (5 moU, catalog number L-7202). Figure 8 The first row of photographs in shows fluorescence 4 hours after administration achieved by intravenous administration of LNPs containing PNI 516 30%, DPSC 56%, cholesterol 12.5% ​​and DMG-PEG 1.5%. Figure 8 The brighter bottom row in the figure is achieved by LNPs containing PNI 580 30%, DSPC 56%, cholesterol 12.5% ​​and DMG-PEG 1.5%. This shows that the LNPs of the present invention successfully express proteins in vivo after intravenous administration. In this model, PNI 580 appears to be slightly more effective than PNI 516 in achieving non-native protein expression.

Claims

1. A lipid formulation capable of forming lipid-based nanoparticles suitable for use in vaccines, the lipid formulation comprising an ionizable lipid to phospholipid ratio of 0.1 Mol:Mol-1.3 Mol:Mol.

2. The lipid formulation of claim 1, further comprising a nucleic acid payload.

3. A lipid formulation according to claim 2, wherein the nucleic acid payload is a nucleic acid vaccine element.

4. according to the lipid preparation described in any one of claims 1-3, further comprising a stabilizer.

5. according to any one of claims 1-4 lipid preparations, further comprising sterol.

6. according to the lipid preparation described in any one of claims 1-5, wherein the ionizable lipid and phospholipid lipid ratio is 0.4Mol:Mol–0.70Mol:Mol.

7. according to the lipid preparation described in any one of claims 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

70.

8. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

69.

9. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

68.

10. The lipid formulation of any one of claims 1-6, wherein the ionizable lipid to phospholipid ratio is about 0.

67.

11. according to the lipid preparation described in any one of claims 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

66.

12. according to the lipid preparation described in any one of claims 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

65.

13. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

64.

14. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

63.

15. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

62.

16. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

61.

17. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

60.

18. according to the lipid preparation described in any one of claims 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

59.

19. according to the lipid preparation described in any one of claims 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

58.

20. The lipid formulation of any one of claims 1-6, wherein the ionizable lipid to phospholipid ratio is about 0.

57.

21. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

56.

22. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

55.

23. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

54.

24. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

53.

25. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

52.

26. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

51.

27. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

50.

28. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

49.

29. according to the lipid preparation described in any one of claims 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

48.

30. The lipid formulation of any one of claims 1-6, wherein the ionizable lipid to phospholipid ratio is about 0.

47.

31. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

46.

32. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

45.

33. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

44.

34. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

43.

35. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

42.

36. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

41.

37. according to the lipid preparation described in any one of claim 1-6, wherein said ionizable lipid and phospholipid ratio is about 0.

40.

38. according to the lipid preparation described in any one of claims 1-5, wherein said ionizable lipid and phospholipid ratio is about 0.30 to 0.

39.

39. according to the lipid preparation described in any one of claims 1-5, wherein said ionizable lipid and phospholipid ratio is about 0.20 to 0.

29.

40. The lipid formulation of any one of claims 1-5, wherein the ionizable lipid to phospholipid ratio is about 0.10 to 0.

19.

41. The lipid preparation of claim 1, wherein the ionizable lipids account for 20-40 mol% of the total lipids.

42. according to the lipid preparation described in any one of claims 1-41, wherein said ionizable lipid comprises the mixture of ionizable lipid.

43. The lipid preparation according to claim 1, wherein the phospholipids account for 40-60 mol%.

44. A lipid formulation according to any one of claims 5-43, wherein the sterol accounts for 15-25 mol%.

45. according to any one of claims 5-44 lipid formulations, wherein the sterol is cholesterol.

46. ​​A lipid formulation according to any one of claims 5-44, wherein the sterol is cholesterol hemisuccinate.

47. A lipid formulation according to any one of claims 4-46, wherein the stabilizer accounts for 0.0-2.5 mol%.

48. according to the lipid formulation described in any one of claims 4-47, wherein the stabilizer is a PEG lipid.

49. according to the lipid preparation described in any one of claim 1-48, wherein said phospholipid is DSPC.

50. The lipid formulation of any one of claims 1-49, wherein the ionizable lipid is selected from PNI 127, PNI 516, PNI 550, PNI 560, PNI 580, PNI 659, PNI 721, PNI 722, PNI 726, PNI 728, and PNI730.

51. The lipid formulation of claim 50, wherein the ionizable lipid is selected from PNI 516, PNI 550 and PNI 127.

52. A lipid formulation for encapsulating nucleic acid payloads in vaccine nanoparticles, the formulation comprising ionizable lipids, sterols and phospholipids, wherein the phospholipid content is between 25-60%, and the molar ratio of ionizable lipids to phospholipids is 0.33 to 1.

2.

53. A vaccine comprising a lipid formulation according to any one of claims 1-52, wherein the nucleic acid vaccine element encodes an antigen selected from a coronavirus spike protein and an influenza hemagglutinin protein.

54. A vaccine comprising a lipid formulation according to any one of claims 1-53, wherein the nucleic acid vaccine element is derived from an influenza virus.

55. A vaccine comprising a lipid formulation according to any one of claims 1-54, wherein the nucleic acid vaccine element is derived from a coronavirus.

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