Lipid nanoparticle formulations for vaccines

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 357,094, filed June 30, 2022, which is incorporated by reference.

[0002] The field of the invention relates to methods and lipid formulations suitable for forming RNA-based vaccines in lipid nanoparticles. [Background technology]

[0003] Nucleic acid-based vaccines offer advantages over conventional vaccines in terms of safety and efficacy. RNA vaccines require a carrier because, without a delivery system, they are subject to in vivo degradation by exonucleases and endonucleases.

[0004] Currently, lipid nanoparticles (LNPs) are one of the most commonly used vectors for in vivo RNA delivery. Lipid nanoparticles or LNPs generally consist of multiple different lipids, each of which performs a different function. These LNPs can have a lipid core or an aqueous core, and may contain a bilayer structure depending on the amount of each type of lipid present.

[0005] The components of LNP formulations generally include: ionizable cationic lipids that spontaneously encapsulate negatively charged mRNA through a combination of attractive electrostatic and hydrophobic interactions with the RNA; neutral phospholipids to mitigate charge-related toxicity and maintain the structure of the LNP; and cholesterol and lipid-conjugated polyethylene glycol (PEG) to stabilize the LNP and aid in cellular entry.

[0006] The properties of individual LNPs vary. Diffusional or bulk mixing can result in LNPs with different compositions. Therefore, rapid mixing of the ethanol-lipid phase with mRNA in excess water is important for the synthesis of small, uniform LNPs. Precision NanoSystems ULC's NanoAssemblr® series mixers are recommended for this purpose.

[0007] Recognized challenges to successfully creating LNP vaccines are safety, manufacturability, stability, and efficacy. Current mRNA vaccines require cryogenic freezing for storage. Adverse events have been reported in some cases with LNP vaccines, resulting in some reluctance to vaccinate. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] PCT Publication No. WO 23057979 [Patent Document 2] PCT Publication No. WO 20252589 [Patent Document 3] PCT Publication Number WO 021000041 [Patent Document 4] U.S. Patent No. 5,753,613 [Patent Document 5] U.S. Patent No. 6,734,171 [Patent Document 6] U.S. Patent No. 7,901,708 [Patent Document 7] U.S. Patent No. 9,758,795 [Patent Document 8] U.S. Patent No. 9,943,846 [Patent Document 9] U.S. Patent No. 10,159,652 [Patent Document 10] PCT Publication Number WO 2017117647 [Patent Document 11] U.S. Patent No. 10,076,730 [Patent Document 12] PCT Publication Number WO 2018006166 [Patent Document 13] U.S. Design Registration No. D771834 [Patent Document 14] U.S. Design Registration No. D771833 [Patent Document 15] U.S. Design Registration No. D772427 [Patent Document 16] U.S. Design Registration No. D803416 [Patent Document 17] U.S. Design Registration No. D800335 [Patent Document 18] U.S. Design Registration No. D800336 [Patent Document 19] U.S. Design Registration No. D812242 [Patent Document 20] U.S. Published Patent Application No. 20040262223 [Patent Document 21] WO 2012 / 006372 [Patent Document 22] WO 2011 / 005799 [Patent Document 23] WO 2009 / 016515 [Patent Document 24] U.S. Patent No. 10,835,878 [Non-patent literature]

[0009] [Non-Patent Document 1] 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 [Non-patent document 2] Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006 Summary of the Invention [Problem to be solved by the invention]

[0010] There remains a need for better LNP forms of vaccines with improved stability. [Means for solving the problem]

[0011] According to one embodiment of the present invention, there is provided a lipid formulation capable of forming lipid-based nanoparticles suitable for vaccines, the lipid formulation comprising an ionizable lipid to phospholipid ratio of 0.10 to 1.30 mol:mol, in some embodiments, a 0.33 to 1.20 mol:mol ratio, in some embodiments, a 0.10 to 0.70 mol:mol ratio, and in some embodiments, a 0.40 to 0.70 mol:mol ratio. In some embodiments, the lipid formulation further comprises a nucleic acid payload. In some embodiments, the nucleic acid payload is a nucleic acid vaccine component. In some embodiments, the lipid formulation further comprises a stabilizer. In some embodiments, the lipid formulation further comprises a sterol.

[0012] In various 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 1.02, 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.

[0013] In various 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.

[0014] In various embodiments, the ratio of ionizable lipid to phospholipid 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.

[0015] In embodiments, the ratio of ionizable lipid to phospholipid 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.

[0016] In embodiments, the ratio of ionizable lipid to phospholipid 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.

[0017] In embodiments, the ratio of ionizable lipid to phospholipid 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.

[0018] According to one embodiment of the present invention, there is provided a lipid formulation in which an ionizable lipid constitutes approximately 20 to 40 mol % of the entire formulation.

[0019] According to one embodiment of the present invention, there is provided a lipid formulation in which the ionizable lipid comprises a mixture of ionizable lipids, in various embodiments, the phospholipids comprising about 25-60 mol % of the total formulation.

[0020] According to one embodiment of the present invention, the sterol, for example cholesterol or cholesteryl hemisuccinate, constitutes 15-25 mol % of the total formulation.

[0021] According to one embodiment of the present invention, there is provided a lipid formulation in which the stabilizer constitutes 0.0 to 2.5 mol % of the total lipid formulation.

[0022] According to the present invention, there is provided a lipid formulation for encapsulating a nucleic acid payload in a nanoparticle, the lipid formulation comprising an ionizable lipid, a sterol, and a phospholipid, wherein the phospholipid content of the nanoparticle is 25-60% and the molar ratio of ionizable lipid to phospholipid is 0.33-1.2. In various embodiments, the nanoparticle is a vaccine.

[0023] In some embodiments, the stabilizer is a PEG lipid, and in some embodiments, is PEG-DMPG. In some embodiments, the phospholipid is DSPC. In some embodiments, the ionizable lipid is selected from the group consisting of PNI 516, PNI 550, PNI 127, PNI 560, PNI 580, PNI 659, PNI 721, PNI 722, PNI 726, PNI 728, and PNI 730. In some embodiments, the sterol is cholesterol.

[0024] The present invention provides a vaccine comprising a lipid formulation for encapsulating a nucleic acid payload, the lipid formulation comprising an ionizable lipid, a sterol, and a phospholipid, wherein the phospholipid content of the vaccine is 25-60% and the molar ratio of ionizable lipid to phospholipid is 0.33-1.2. In some embodiments, the nucleic acid payload encodes an antigen selected from a coronavirus spike protein and an influenza hemagglutinin protein.

[0025] In embodiments, the nucleic acid payload is derived from influenza virus mRNA. In embodiments, the nucleic acid payload is derived from a coronavirus.

[0026] Other aspects and features of the present invention will become apparent to those skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. [Brief explanation of the drawings]

[0027] [Figure 1]1 shows the dose response of SARS-COV-2 protein expression after transfection with Vaccmixb PNI516 and V46-PNI516 in BHK 570 cells. Dose-response curves were generated using RNA doses ranging from 1000 to 0.49 ng / mL. [Figure 2] Figure 10 is a graph depicting the dose response of SARS-COV-2 protein expression after LNP transfection in BHK 570 cells using various PNI516 LNP formulations containing 1000 to 0.49 ng / mL of nCoV PNI A5 saRNA. [Figure 3] Scatter plot graph of ELISA showing SARS-COV-2 spike protein-specific IgG expression in BALB / c mouse serum 21 days after intramuscular LNP injection at 1 ug / mouse. [Figure 4] FIG. 11 is a reproduction of Cryo-TEM imaging for A) Vaccmixb PNI 516, B) V46-PNI 516, and C) V47-PNI 516 LNPs. [Figure 5] 10 is a graph showing the in vitro transfection ability of saRNA LNPs containing Vaccmixb, V46-PNI 516, V47-PNI 516, and V22-PNI 516 after addition of Jurkat cells to GFP saRNA LNPs. [Figure 6] 1 is a graph depicting the in vitro transfection capacity of mRNA LNPs containing V47-PN1516, V46-PN1516, V02-PN1516, and V22-PN1516 following addition of Jurkat cells to GFP mRNA LNPs. [Figure 7] This is a graph showing the transfection ability of EGFP saRNA LNPs using expansion medium supplemented with activator / recombinant human IL-2. [Figure 8]These are photographs of mice intravenously injected with 0.1 mg / kg of LNPs containing a luciferase mRNA payload. The top two photographs show fluorescence 4 hours after administration of LNPs containing 30% PNI 516, 56% DSPC, 12.5% cholesterol, and 1.5% DMG-PEG. The bottom two photographs, which are brighter than the two above, show fluorescence 4 hours after administration of LNPs containing 30% PNI 580, 56% DSPC, 12.5% cholesterol, and 1.5% DMG-PEG. DETAILED DESCRIPTION OF THE INVENTION

[0028] According to embodiments of the present invention, lipid mixture formulations for use in making more effective lipid-based formulations of nucleic acid cargo (including vaccines) and other oligomers, such as peptides, and methods for using these lipid mixtures and the resulting formulations to prepare vaccines are provided. The lipid mixtures, which have an unusual ratio of ionizable lipid to phospholipid compared to the established optimal ratio of 0.2, are surprisingly evaluated to be effective.

[0029] [Table 1]

[0030] In another aspect, lipid mixture formulations of the present invention are provided for mixing with nucleic acid vaccine components to create lipid nucleic acid particles that are less toxic and easier to manufacture than lipid nucleic acid particles such as those produced from commercially available lipid mixtures, e.g., Lipofectamine™ transfection agent, and that enhance delivery of nucleic acids to target cells or tissues.

[0031] In another aspect, the present invention provides lipid mixture formulations comprising an ionizable lipid, one or more phospholipids, cholesterol, and optionally a stabilizer.

[0032] In another aspect, a lipid mixture formulation according to the present invention is provided for formulating a vaccine.

[0033] In another aspect, the present invention provides lipid mixture formulations for formulating mRNA LNPs.

[0034] Self-amplifying mRNA (saRNA) has the advantage of long-term translation and high yield of target antigen compared to conventional mRNA vaccines such as Moderna's mRNA-1273 (Spikevax) and Pfizer-BioNTech's BNT162b2 (Comirnaty) (see Table 1). One example of a nucleic acid vaccine component is the self-amplifying mRNA described by Abraham et al. in PCT Publication No. WO 23057979.

[0035] A review of the genetic properties of "Severe Acute Respiratory Syndrome Coronavirus 2" (SARS-CoV-2) includes the genetic properties of the spike protein. The spike protein has proven to be a useful target for SARS-CoV-2 vaccines and is of concern for variants of this virus that continue to cause disease. Modified spike protein gene sequences were used herein.

[0036] In this disclosure, the word "comprising" is used in an open-ended sense, meaning that items following the word are included, but items not specifically mentioned are not excluded. In embodiments that include or may include a particular feature or variable or parameter, it will be understood that alternative embodiments may consist of or consist essentially of such feature or variable or parameter. Reference to an element by the indefinite article "a" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one, and only one, of the element be present.

[0037] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range, including all numbers, integers, and all fractional intermediate values (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In this disclosure, the singular forms "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds.

[0038] In this disclosure, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0039] As used herein, the term "about" is defined to mean ±12.5% of the recited number. This is used to mean that the desired target concentration may be, for example, 40 Mol%, but due to inconsistencies in mixing, the actual percentage may vary by + / - 5 Mol%.

[0040] As used herein, the term "substantially" is defined as being ±5% of the recited number. This is used to mean that the desired target concentration may be, for example, 40 Mol%, but due to inconsistencies in mixing, the actual percentage may vary by + / - 2 Mol%.

[0041] As used herein, the term "nucleic acid cargo" is defined as a substance intended to have a direct effect in the alleviation or prevention of disease or to act as a research reagent. In preferred embodiments, the nucleic acid cargo is mRNA or saRNA. In preferred embodiments, the therapeutic agent is a nucleic acid therapeutic agent, such as an RNA polynucleotide. In preferred embodiments, the therapeutic agent is messenger RNA (mRNA) or self-amplifying RNA (saRNA). In preferred embodiments, the therapeutic agent is double-stranded circular DNA (plasmid), linearized plasmid DNA, minicircle, or msDNA (multicopy single-stranded DNA).

[0042] In this disclosure, the word "comprising" is used in an open-ended sense, meaning that items following the word are included, but items not specifically mentioned are not excluded. In embodiments that include or may include a particular feature or variable or parameter, it will be understood that alternative embodiments may consist of or consist essentially of such feature or variable or parameter. Reference to an element by the indefinite article "a" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one, and only one, of the element be present.

[0043] In this disclosure, "transfection" refers to the transfer of nucleic acids into cells for the purpose of inducing expression of a specific gene of interest, both in laboratory and clinical settings. This typically involves an ionizable lipid and a phospholipid associated with the nucleic acid. LIPOFECTIN™ and LIPOFECTAMINE™ are established commercial transfection reagents sold by Thermo Fisher Scientific. These research reagents contain lipids that are permanently cationic and are not suitable for in vivo or ex vivo use.

[0044] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range, including all numbers, integers, and fractional intermediate values (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In this disclosure, the singular forms "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "one compound" includes a mixture of two or more compounds. In this disclosure, the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise.

[0045] "nCoV PNI A5 saRNA" is a SARS Cov2 spike protein expressing saRNA integrated into a Venezuelan Equine Encephalitis virus TC83 replicon with a subgenomic promoter containing multiple cloning sites for insertion of any GOI, as described in PCT Publication No. WO 23057979 by Abraham et al.

[0046] "Lipid" refers to a structurally diverse group of organic compounds that may be fatty acid derivatives or sterols, or lipid-like substances such as lipidoids (e.g., C12-200), and are characterized by being insoluble in water but soluble in many organic solvents.

[0047] "Lipid mixture formulation." Lipid mixture formulation refers to the type of components, the ratio of components, and the ratio of all components to nucleic acid payload. For example, a lipid mixture formulation consisting of 30 mol% ionizable lipid, 50 mol% phospholipid, 20 mol% sterol, and 1.5 mol% stabilizer 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.

[0048] "Lipid Particles" or "Lipid Nanoparticles" or "LNPs." The present invention provides lipid particles produced from the lipid mixture formulations described above and exemplified below. Lipid particles are a physical organization of the lipid mixture formulation with a therapeutic agent and among the components. Lipid nanoparticles are lipid particles with a diameter of less than 200 microns. Lipid particles are generally spherical aggregates of lipids, nucleic acids, cholesterol, and stabilizers. The positive and negative charges, ratios, and hydrophilic and hydrophobic properties determine the physical structure of the lipid particles in terms of the size and orientation of the components. The structural organization of these lipid particles can result in an aqueous interior with one or more bilayers, as in liposomes, or a solid interior, as in solid nucleic acid-lipid nanoparticles. Single or multiple phospholipid monolayers or bilayers can be present. The lipid particles are 1 to 1000 microns in diameter.

[0049] "Viability" when referring to cells in vitro means the ability to continue to grow, divide, and continue to grow and divide as is normal for that cell type or tissue culture line. Cell viability is affected by harsh conditions or treatments. Cell viability is important in ex vivo therapy or parenteral administration.

[0050] "Ionizable Lipids." The compositions of the present invention include an ionizable lipid as an ingredient. As used herein, the term "ionizable lipid" refers to a lipid that becomes cationic or ionizable (protonated) when the pH drops below the pKa of the lipid's ionizable group, but becomes more neutral at higher pH values. At pH values below the pKa, the lipid can associate with then-negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that become positively charged when the pH drops below physiological pH, and any of several lipid species that assume a net positive charge at select pHs. Examples of suitable ionizable lipids can be found in PCT Publication Nos. WO 20252589 and WO 021000041. The ionizable lipid is preferably present in lipid formulations according to other embodiments of the present invention at a ratio of about 10 to about 40 mol% ("mol%" refers to the percentage of the total moles of a particular component). The term "about" in this paragraph refers to a range of + / - 5 mol% in increments of 0.1. For example, 28.7 mol% is within the claimed range of embodiments. DODMA or 1,2-dioleyloxy-3-dimethylaminopropane is an alternative ionizable lipid, as is DLin-MC3-DMA or O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) ("MC3"). LNPs can be made from lipid formulations containing the ionizable lipids of the present invention.

[0051] As used herein, phospholipids, also known as "helper lipids" or "neutral lipids," are incorporated in the lipid formulations and lipid particles of the present invention in various embodiments. The lipid formulations and lipid particles of the present invention comprise one or more phospholipids at approximately 25-60 mol % of the composition. Suitable phospholipids assist in particle formation during manufacturing. Phospholipid refers to any one of several lipid species that exist in either anionic, uncharged, or neutral zwitterionic form at physiological pH. Representative phospholipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, and diacylphosphatidylglycerol; although not strictly "phospholipids" in the technical sense, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides are also intended to be included.

[0052] Exemplary phospholipids include zwitterionic lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). 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.

[0053] In another embodiment, the phospholipid is any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached to neutral lipids. Other suitable phospholipids include glycolipids (e.g., monosialoganglioside GM1).

[0054] "Stabilizer" or stabilizing agent is a term used to identify agents added to the ionizable lipids, phospholipids, and sterols that form the lipid formulations according to the present invention. Examples of non-ionic stabilizers include polyethylene glycol (PEG), polysorbate (Tween), TPGS (vitamin E polyethylene glycol succinate), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™ 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij™ S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), and Myrj™ 52 (polyoxyethylene (40) stearate).

[0055] In some embodiments, the stabilizing agent comprises a PEGylated lipid, including PEG-DMG 2000 ("PEG-DMG"). Other polyethylene glycol-conjugated lipids can also be used. The stabilizing agents can be used alone or in combination with each other.

[0056] In some embodiments, the stabilizer is absent. In other embodiments, the stabilizer comprises about 0.1-3 mol% of the total lipid mixture. In some embodiments, the stabilizer comprises about 0.5-2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at greater than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10-15 mol%. In some embodiments, the stabilizer is present at 2.5-10 mol%. In some embodiments, the stabilizer is present at 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 present at 2.6-10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 Mol % of the lipid mixture.

[0057] Sterols are included in preferred lipid mixture formulations for certain applications, and lipid particles made therefrom include cholesterol, beta-sitosterol, and 20-alpha-hydroxysterols, and plant sterols. In the lipid mixtures of the present invention, sterols are present in some embodiments at about 15-25 mol% of the final lipid mixture. In some embodiments, modified sterols or synthetically derived sterols are present.

[0058] Nucleic Acids. The lipid mixture formulations and lipid particles of the present invention are useful for systemic or localized delivery of nucleic acids. In the case of vaccines, delivery is limited to the skin or muscle. As used herein, the term "nucleic acid" is meant to include any oligonucleotide or polynucleotide whose delivery to a cell causes a desired effect. This definition includes diagnostic 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, while longer RNAs are referred to as polynucleotides. In certain embodiments, oligonucleotides of the present invention are 20-50 nucleotides in length. In various embodiments of the present invention, polynucleotides are 996-4500 nucleotides in length, as in the case of messenger RNA. In certain embodiments, polynucleotides of the present invention contain up to 14,000 nucleotides.

[0059] 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 contain portions of both double-stranded and single-stranded sequence, as appropriate. Messenger RNA (mRNA) may be modified or unmodified, may be base-modified, and may contain various 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 containing self-amplifying RNA.

[0060] As used herein, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single- and double-stranded polymers of nucleotide monomers comprising 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bonds, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'-3' and 5'-5', branched structures, or internucleotide analogs. Polynucleotides have associated counterions such as H+, NH4+, trialkylammonium, Mg2+, Na+, etc. Polynucleotides may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides may contain internucleotide nucleobase and / or sugar analogs.

[0061] As used herein, the term "polypeptide" includes "oligopeptides" and "proteins" and their tertiary and quaternary structures, which in some embodiments are therapeutic agents. Oligopeptides generally consist of 2 to 20 amino acids. Polypeptides are single linear chains of any length of many amino acids held together by amide bonds. Proteins may consist of one or more and may include structural proteins, energy catalysts, albumin, hemoglobin, immunoglobulins, and enzymes.

[0062] Currently, nucleic acid cargoes include deoxyribonucleic acids, complementary deoxyribonucleic acids, complete genes, ribonucleic acids, oligonucleotides, and ribozymes for gene therapy targeting various diseases, such as cancer, infectious diseases, genetic disorders, and neurodegenerative diseases. As described herein, nucleic acid therapeutic agents (NATs) are incorporated into lipid particles during their formation using the compounds of the present invention. More than one type of nucleic acid therapeutic agent can be incorporated in this manner. They may be derived from natural sources, or more commonly, they may be synthesized or grown in culture. Examples of nucleic acid cargoes include, but are not limited to, antisense oligonucleotides, ribozymes, microRNAs, mRNAs, ribozymes, tRNAs, tracrRNAs, sgRNAs, snRNAs, siRNAs, shRNAs, ncRNAs, miRNAs, mRNAs, pre-condensed DNA, plasmids or pDNAs, or aptamers. Nucleic acid reagents are used to silence genes (e.g., using siRNA), express genes (e.g., using mRNA), edit genomes (e.g., using CRISPR / Cas9), and reprogram cells back to their original organism (e.g., ex vivo cell therapy, autologous or allogeneic transplantation for reprogramming immune cells for cancer treatment).

[0063] The nucleic acid present in the lipid particle of the present invention includes any known form of nucleic acid. The nucleic acid used herein can be single-stranded DNA or RNA, double-stranded DNA or RNA, or DNA-RNA hybrid. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral DNA or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, guide RNAs including CRISPR-Cas9 gRNAs, ribozymes, microRNAs, mRNAs, and triplex-forming oligonucleotides. For example, more than one nucleic acid can be incorporated into the lipid particle, such as mRNA and guide RNA together, or in combination with different types of mRNA and guide RNA, or with proteins.

[0064] In some cases, the nucleic acid encodes a ligand, such as a recombinant receptor, and an engineered receptor that specifically binds to a molecule involved in a metabolic pathway, or a functional portion thereof. Alternatively, the molecule involved in a metabolic pathway is a recombinant molecule comprising an exogenous entity. The engineered receptor and the molecule involved in a metabolic pathway can be encoded by one nucleic acid or two or more different nucleic acids. In some examples, a first nucleic acid can encode an engineered receptor that specifically binds to a ligand, and a second nucleic acid can encode a molecule involved in a metabolic pathway.

[0065] As used herein, a "therapeutic agent" includes a nucleic acid cargo as described herein.

[0066] The size of the lipid particles of the present invention can be assessed using a device that measures the size of particles in solution, such as a Malvern™ Zetasizer™. The particles generally have an average particle size of 15 nm to 1000 nm. A subgroup of lipid particles are "lipid nanoparticles" or LNPs, which have an average diameter of about 15 to about 300 nm. In some embodiments, the average particle size is greater than 300 nm. In some embodiments, the lipid particles have a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In one embodiment, the lipid particles have a diameter of about 50 to about 150 nm. Smaller particles generally exhibit an increased circulatory lifetime in vivo compared to larger particles. Smaller particles have a greater ability to reach tumor sites than larger nanoparticles. In one embodiment, the lipid particles have a diameter of about 15 to about 50 nm.

[0067] Lipid particles according to embodiments of the present invention can be prepared into nanoparticles by standard T-tube mixing techniques, turbulent mixing, trituration mixing, stirring to promote ordered self-assembly, or passive mixing of all components with subsequent self-assembly of the components. Various methods have been developed to formulate lipid nanoparticles (LNPs) containing genetic drugs. Suitable methods are disclosed, for example, in U.S. Patent Nos. 5,753,613, 6,734,171, and 7,901,708. These methods involve mixing preformed lipid particles with nucleic acid therapeutics (NATs) in the presence of ethanol, or mixing lipids dissolved in ethanol with an aqueous medium containing NATs, resulting in lipid particles with NAT encapsulation efficiencies of 65-99%. All of these methods rely on the presence of ionizable lipids to achieve NAT encapsulation and stabilizers to prevent aggregation and the formation of large structures. The properties of the resulting lipid particle systems, including size and NAT encapsulation efficiency, are sensitive to various lipid mixture formulation parameters, such as ionic strength, lipid and ethanol concentrations, pH, NAT concentration, and mixing rate.

[0068] Microfluidic two-phase droplet techniques have been applied to generate monodisperse polymer microparticles for drug delivery or to generate large vesicles for encapsulation of cells, proteins, or other biomolecules. The use of hydrodynamic flow focusing has also been demonstrated to create monodisperse liposomes of controlled size.

[0069] Parameters such as the relative lipid and NAT concentrations during mixing and mixing speed are difficult to control using current formulation procedures, resulting in variability in the properties of the resulting NAT both within and between preparations. What makes the new formulation so unique is the surprisingly low ratio of ionizable lipids to phospholipids. Automated micromixing instruments, such as the NanoAssemblr® instrument (Precision NanoSystems ULC, Vancouver, Canada), enable the rapid and controlled production of nanomedicines (liposomes, lipid nanoparticles, and polymer nanoparticles). The NanoAssemblr® instrument achieves controlled molecular self-assembly of nanoparticles via a microfluidic mixing cartridge that allows millisecond mixing of nanoparticle components at nanoliter, microliter, or larger scales with customization or parallelization. Rapid mixing on a small scale allows for reproducible control of particle synthesis and quality that is not possible with larger instruments.

[0070] A preferred method incorporates equipment such as microfluidic mixing devices like the NanoAssemblr® Spark™, Ignite™, Benchtop™, and NanoAssemblr® Blaze™ to achieve particle encapsulation of nearly 100% of the nucleic acid used in the formation process in a single step. In a preferred embodiment, the lipid particles are prepared by a process in which about 75 to about 100% of the nucleic acid used in the formation process is encapsulated in the particles.

[0071] U.S. Patent Nos. 9,758,795 and 9,943,846 describe methods using small-volume mixing technology and novel formulations obtained thereby. U.S. Patent No. 10,159,652 describes more advanced methods using small-volume mixing technology and products for formulating various materials. U.S. Patent No. 9,943,846 discloses a microfluidic mixer with different channels and wells for the components to be mixed. PCT Publication No. WO 2017117647 discloses a microfluidic mixer with a disposable, sterile channel. U.S. Patent No. 10,076,730 discloses branched toroidal micromixing geometries and their application to microfluidic mixing. PCT Publication No. WO 2018006166 discloses a programmable automated micromixer and a mixing chip therefor. US Design Nos. D771834, D771833, D772427, D803416, D800335, D800336 and D812242 disclose mixing cartridges having microchannels and mixing geometries for stirring devices sold by Precision NanoSystems ULC.

[0072] In embodiments of the present invention, a device for biological microfluidic mixing is used to prepare lipid particles according to embodiments of the present invention. The device includes first and second streams of reagents that are fed into a microfluidic mixer, and the lipid particles are collected from an outlet or exit into a sterile environment.

[0073] The first stream contains a therapeutic agent in a first solvent. Suitable first solvents include those in which the therapeutic agent is soluble and which are miscible with the second solvent. Suitable first solvents include aqueous buffers. Exemplary first solvents include citrate and acetate buffers, or optionally other low pH buffers.

[0074] The second stream contains the lipid mixture material in a second solvent. Suitable second solvents include those in which the ionizable lipids according to embodiments of the present invention are soluble and are miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols. Exemplary second solvents include 90% aqueous ethanol or absolute ethanol.

[0075] In one embodiment of the present invention, a suitable device comprises one or more microchannels (i.e., channels whose largest dimension is less than 2 millimeters). In one example, the microchannel has a diameter of about 20 to about 300 μm. In another example, the microchannel has a diameter of about 300 to about 1000 μm. In an example, at least one region of the microchannel has a primary flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having a direction that forms an angle with the primary direction (e.g., a staggered herringbone agitator), as described in U.S. Pat. No. 9,943,846, or a diverging toroidal flow, as described in U.S. Pat. No. 10,076,730. To achieve maximum mixing rates, it is advantageous to avoid excessive fluidic resistance before the mixing region. Thus, one example of a device has non-microfluidic channels with dimensions greater than 1000 μm to deliver fluids to a single mixing channel.

[0076] Less complicated mixing methods and equipment, such as those disclosed in US Published Patent Application No. 20040262223, are also useful in making the lipid particle formulations of the present invention.

[0077] The lipid mixture of the present invention can be used to deliver therapeutic agents to cells in vitro or in vivo.In certain embodiments, the therapeutic agent is a nucleic acid that 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 vectors with antigenic mRNA), self-amplifying RNA, mRNA, guide RNA, transposon, or single gene.

[0078] In other embodiments, the therapeutic agent is an oligopeptide, polypeptide, or protein delivered to cells using the peptide-lipid particles of the present invention. In other embodiments, the therapeutic agent is a mixture of a nucleic acid and a protein component, such as Cas9. The present methods and lipid mixture formulations can be readily adapted for the delivery of any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.

[0079] In certain embodiments, the present invention provides a method for introducing nucleic acids into cells (i.e., transfection). Transfection is a technique commonly used in molecular biology to introduce nucleic acid cargo (or NAT) from the extracellular space into the intracellular space for transcription, translation, and expression of the delivered nucleic acid therapeutic (NAT), for the production of some gene products or for downregulating the expression of disease-related genes. Transfection efficiency is generally defined as either i) the percentage of cells in the entire treated population that show positive expression of the delivered gene, as measured by live or fixed cell imaging (for detection of fluorescent proteins) and flow cytometry, or ii) the intensity or amount of protein expressed by the treated cells, as analyzed by live or fixed cell imaging or flow cytometry, or iii) using protein quantification techniques, such as ELISA or Western blot. These methods can be carried out by contacting the lipid particle or lipid mixture formulation of the present invention with cells for a period of time sufficient for intracellular delivery to occur.

[0080] Typical applications include providing intracellular delivery of siRNA using well-known procedures to knockdown or silence specific cellular targets in vitro and in vivo. Alternatively, applications include delivery of DNA sequences or mRNA sequences encoding therapeutically useful polypeptides. In this way, providing defective or missing gene products provides treatment for genetic diseases. The method of the present invention can be carried out in vitro, ex vivo, 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 ex vivo, and then return them to the patient.

[0081] Delivery of nucleic acid cargo by lipid particles of the present invention is described below.

[0082] 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 certain embodiments, the pharmaceutical composition is administered intravenously, intramuscularly, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include topical (cutaneous, ocular, mucosal), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0083] For ex vivo applications, the pharmaceutical composition is preferably administered to a biological sample removed from an organism, and the cells are then washed and returned to the organism. The organism may be a mammal, particularly a human. This process is used, for example, for cell reprogramming, gene repair, or immunotherapy.

[0084] In one embodiment, the present invention provides a method for modulating the expression of a target polynucleotide or polypeptide. These methods generally involve contacting a cell with a lipid particle of the present invention associated with a nucleic acid that can modulate the expression of the target polynucleotide or polypeptide. As used herein, the term "modulate" refers to changing the expression of a target polynucleotide or polypeptide. Modulation can mean increasing or enhancing, or decreasing or alleviating.

[0085] In a related embodiment, the invention provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing the siRNA, microRNA, or antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide, or a complement thereof.

[0086] In a related embodiment, the present invention provides a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent comprises a nucleic acid therapeutic agent selected from mRNA, self-amplifying RNA (saRNA), or a plasmid that specifically encodes or expresses the underexpressed polypeptide, or its complement. Examples include RNA vaccines, more particularly self-amplifying mRNA vaccines.

[0087] Methods for delivery of biologically active agents for the treatment of disease include, in one embodiment, the compounds, compositions, methods, and uses of the present invention for delivering biologically active agents to liver cells (e.g., hepatocytes). In one embodiment, the compounds, compositions, methods, and uses of the present invention are for delivering biologically active agents to tumors or tumor cells (e.g., primary tumor cells or metastatic cancer cells). In another embodiment, the compounds, compositions, methods, and uses are for delivering biologically active agents to skin, fat, muscle, and lymph nodes (subcutaneous administration).

[0088] For delivery of biologically active agents to the liver or hepatocytes, in one embodiment, a formulation of the present invention is contacted with the liver or hepatocytes via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, portal vein injection, catheterization, stent placement) to facilitate delivery. For delivery of biologically active agents to kidney or renal cells, in one embodiment, a formulation of the present invention is contacted with the patient's kidney or renal cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stent placement) to facilitate delivery. For delivery of biologically active agents to tumor or tumor cells, in one embodiment, a formulation of the present invention is contacted with the patient's tumor or tumor cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stent placement) to facilitate delivery.

[0089] For delivery of a biologically active agent to the CNS or CNS cells, in one embodiment, a formulation of the invention is contacted with a patient's CNS or CNS cells (e.g., brain cells and / or spinal cord cells) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stent placement, osmotic pump administration (e.g., intrathecal or ventricular)) to facilitate delivery. For delivery of a biologically active agent to the peripheral nervous system (PNS) or PNS cells, in one embodiment, a formulation of the invention is contacted with a patient's PNS or PNS cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection). For delivery of a biologically active agent to the lung or lung cells, in one embodiment, a formulation of the invention is contacted with a patient's lung or lung cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct pulmonary administration to lung tissue and cells) to facilitate delivery.

[0090] For delivery of biologically active agents to the vasculature or vascular cells, in one embodiment, the formulations of the present invention are contacted with the vasculature or vascular cells of a patient via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., clamping, catheterization, stent placement) to facilitate delivery.

[0091] For delivery of a biologically active agent to skin or skin cells (e.g., dermal cells and / or follicular cells), in one embodiment, a formulation of the present invention is contacted with a patient's skin or skin cells (e.g., dermal cells and / or follicular cells) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct skin application, iontophoresis) to facilitate delivery. For delivery of a biologically active agent to the eye or ocular cells (e.g., macula, fovea, cornea, retina), in one embodiment, a formulation of the present invention is contacted with a patient's eye or ocular cells (e.g., macula, fovea, cornea, retina) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, intraocular injection, periocular injection, subretinal injection, iontophoresis, use of eye drops, implant) to facilitate delivery. For delivery of a biologically active agent to an ear or aural cells (e.g., inner, middle, and / or outer ear cells), in one embodiment, to facilitate delivery, a formulation of the invention is contacted with a patient's ear or aural cells (e.g., inner, middle, and / or outer ear cells) via, for example, parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection), as generally known in the art. For delivery of a biologically active agent (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen-presenting cells, including professional antigen-presenting cells), in one embodiment, a 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, such as 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 monocytes) (see, e.g., WO2012 / 006372).

[0092] Immunization. For purposes of immunization, the formulations of the invention are generally prepared as injections, pulmonary or nasal aerosols, or in delivery devices (e.g., syringes, nebulizers, sprayers, inhalers, skin patches, etc.), which can be used to administer the pharmaceutical formulation to a subject, e.g., a human, for immunization.

[0093] According to the present invention, for purposes of immunization, in some embodiments, the invention encompasses the delivery of RNA encoding an immunogen that elicits an immune response that recognizes the immunogen and provides immunity against a pathogen, an allergen, or a tumor antigen. Immunization against disease and / or infection caused by a pathogen is preferred.

[0094] RNA is delivered with the lipid formulations of the present invention (e.g., formulated as liposomes or LNPs). In some embodiments, the present invention utilizes LNPs in which RNA encoding an immunogen is encapsulated. Encapsulation within LNPs can protect the RNA from RNase digestion. Encapsulation efficiency need not be 100%. The presence of external RNA molecules (e.g., on the outer surface of liposomes or LNPs) or "naked" RNA molecules (RNA molecules not associated with liposomes or LNPs) is acceptable. Preferably, for formulations comprising lipids and RNA molecules, at least half of the RNA molecules (e.g., 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%) are encapsulated in or complexed with the LNPs.

[0095] Some lipid nanoparticles may contain a lipid core (e.g., a formulation may contain a mixture of LNPs and nanoparticles with lipid cores). In such cases, RNA molecules may be encapsulated by one or more aqueous-core LNPs and complexed with the lipid-core LNPs through non-covalent interactions (e.g., ionic interactions between negatively charged RNA and cationic lipids). Encapsulation and complexation with LNPs (either lipid or aqueous core) can protect the RNA from RNase digestion. The efficiency of encapsulation / complexation does not need to be 100%. The presence of "naked" RNA molecules (RNA molecules not associated with LNPs) is acceptable. Preferably, for a formulation comprising a population of LNPs and a population of RNA molecules, at least half of the population of RNA molecules (e.g., at least, e.g., 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%) are either encapsulated in or complexed with the LNPs.

[0096] Some lipid nanoparticles have multilayered components.

[0097] For delivery of RNA encoding an immunogen, a preferred range of LNP diameters is 60-180 nm, and in more specific embodiments, 80-160 nm. LNPs can be part of a composition comprising a population of LNPs, where the LNPs within the population can have a variety of diameters. For compositions comprising a population of LNPs with varying diameters, it is preferred that (i) at least 80% of the LNPs by number have diameters in the 60-180 nm range, e.g., 80-160 nm range, and (ii) the mean diameter (by intensity, e.g., Z-average) of the population is ideally in the 60-180 nm range, e.g., 80-160 nm range, and / or the diameters of the majority have a polydispersity index of less than 0.2. To obtain LNPs with the desired diameter, mixing can be performed using a process in which two feed streams of aqueous RNA solution are combined in a single mixing zone, e.g., in a microfluidic channel, with one stream of ethanolic lipid solution, all at the same flow rate. See elsewhere for a description of the NanoAssemblr® microfluidic mixer sold by Precision NanoSystems ULC, Vancouver, Canada.

[0098] RNA molecule. After in vivo administration of the immunization composition ("vaccine vector LNP"), the delivered RNA is released and translated inside the cell, resulting in an immunogen in situ. In certain embodiments, the RNA is positive ("+") strand and therefore can be translated by the cell without the need for any intervening replication step, such as reverse transcription. In certain embodiments, the RNA is self-replicating RNA. A self-replicating RNA molecule (replicon), when delivered to a vertebrate cell, can direct the production of multiple daughter RNAs by transcription from itself (via antisense copies generated from itself) even in the absence of proteins. Thus, a self-replicating RNA molecule, in certain embodiments, is a (+) strand molecule that can be directly translated after delivery to a cell; this translation leads to an RNA-dependent RNA polymerase, which subsequently produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA directs the production of multiple daughter RNAs. These daughter RNAs, as well as colinear subgenomic transcripts, can themselves be translated, resulting in the in situ expression of the encoded immunogen, or can be transcribed to give rise to additional transcripts with the same sense as the delivered RNA, which can also be translated to give the in situ expression of the immunogen. The overall result of this series of transcriptions is an amplification of the number of introduced replicon RNAs, so that the encoded immunogen becomes the major polypeptide product of the host cell.

[0099] One suitable system for achieving self-replication is the use of alphavirus-based RNA replicons. These (+)-strand replicons are translated after delivery to cells to generate a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein, which self-cleaves to generate a replication complex, which generates a genomic (-)-strand copy of the delivered (+)-strand RNA. These (-)-strand transcripts can themselves be transcribed to generate additional copies of the (+)-strand parent RNA and also to generate subgenomic transcripts encoding immunogens. Translation of the subgenomic transcripts thus leads to in situ expression of the immunogen by infected cells. Suitable alphavirus replicons can use replicases from viruses such as Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, or, more preferably, Venezuelan equine encephalitis virus. This system may, in some embodiments, employ hybrid or chimeric replicases. A preferred embodiment is a replicon according to one embodiment of the present invention, which represents the PNI-V101 replicon, which is capable of self-amplifying in mammalian cells and expressing an immunogenic protein, e.g., the Sars-COV-2 spike protein, via the assembled mRNA.

[0100] The RNA molecule may have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the present invention may have a 5' triphosphate group. In capped RNA, this may be linked to the 7-methylguanosine via a 5'-5' bridge. The 5' triphosphate can enhance RIG-I binding and therefore promote adjuvant effect. The RNA molecule may have a 3' polyA tail. It may also contain a polyA polymerase recognition sequence (e.g., AAUAAA) near its 3' end. RNA molecules useful in the present invention for immunization purposes are typically single-stranded. Single-stranded RNA can generally initiate adjuvant effect by binding to TLR7, TLR8, RNA helicase, and / or PKR.

[0101] RNA molecules can be conveniently prepared by in vitro transcription (IVT). IVT can use (cDNA) templates produced and propagated in bacteria in plasmid form or synthetically produced (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). As discussed in WO 2011 / 005799, self-replicating RNA can contain one or more nucleotides with modified nucleobases (in addition to any 5' cap structure). For example, the self-replicating RNA can contain one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues. However, in some embodiments, the RNA does not contain modified nucleobases or modified nucleotides at all, i.e., all nucleotides in the RNA are standard A, C, G, and U ribonucleotides (except for any 5' cap structure, which may contain 7'-methylguanosine). In other embodiments, the RNA may include a 5' cap containing a 7' methylguanosine, and the first one, two, or three 5' ribonucleotides may be methylated at the 2' position of the ribose. RNA used with the present invention for immunization purposes ideally contains only phosphodiester internucleoside linkages, but in some embodiments, contains phosphoramidate, phosphorothioate, and / or methylphosphonate linkages. The present invention includes embodiments in which multiple species of RNA, such as two, three, four, or more species of RNA, including different classes of RNA (e.g., mRNA, siRNA, self-replicating RNA, and combinations thereof), are formulated with the lipid formulations provided by the present invention.

[0102] Immunogenic RNA molecules used with the present invention for immunization purposes, in some embodiments, encode polypeptide immunogens. In these embodiments, after administration, the RNA is translated in vivo, and the immunogen can elicit an immune response in the recipient. The immunogen can elicit an immune response against a pathogen (e.g., a bacterium, virus, fungus, or parasite), but in some embodiments, an immune response against an allergen or tumor antigen. The immune response can include an antibody response (usually including IgG) and / or a cell-mediated immune response. Polypeptide immunogens typically elicit an immune response that recognizes the corresponding pathogen (or allergen or tumor) polypeptide, but in some embodiments, the polypeptide can act as a mimotope to elicit an immune response that recognizes saccharides. Immunogens are typically surface polypeptides, such as adhesins, hemagglutinins, envelope glycoproteins, spike glycoproteins, etc. The RNA molecule can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If the immunogens are expressed as separate polypeptides from a replicon, one or more of them may be equipped with an upstream IRES or additional viral promoter elements. Alternatively, multiple immunogens may be expressed from a polyprotein encoding individual immunogens fused to a short autocatalytic protease (e.g., the 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 RNA molecules, such as self-replicating RNAs, encoding one or more immunogens (either the same or different from the polypeptide immunogens).

[0103] In some embodiments, the immunogen elicits an immune response against coronavirus species, including, but not limited to, those derived from SARS-CoV-1 and SARS-CoV-2 (12). Useful immunogens against human influenza viruses and Neisseria meningitidis include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding proteins. Three useful polypeptide combinations are disclosed in Giuliani et al. (Proc Natl Acad Sci U S A. 2006; 103(29): 10834-9, Epub 2006 / 07 / 06. doi: 10.1073 / pnas.0603940103. PubMed PMID: 16825336; PubMed Central PMCID: PMC2047628). Useful polypeptide immunogens for Streptococcus pneumoniae, including the RrgB pilus subunit, β-N-acetyl-hexosaminidase precursor (spr0057), spr0096, general stress protein GSP-781 (spr2021, SP2216), serine / threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA, are disclosed in WO 02009 / 016515.

[0104] Hepatitis virus immunogens include hepatitis B virus surface antigen (HBsAg), hepatitis C virus, hepatitis delta virus, hepatitis E virus, or hepatitis G virus antigen. Rhabdovirus immunogens include, but are not limited to, those derived from rhabdoviruses such as lyssaviruses (e.g., rabies virus) and vesiculoviruses (VSV). Calicivirus immunogens include, but are not limited to, those derived from caliciviruses, such as Norwalk virus (norovirus), and Norwalk-like viruses, such as Hawaii virus and Snow Mountain virus. Infectious avian bronchitis virus (IBV), mouse hepatitis virus (MHV), and transmissible porcine gastroenteritis virus (TGEV). Retrovirus immunogens include those derived from oncoviruses, lentiviruses (e.g., HIV-1 or HIV-2), or spumaviruses. Reovirus immunogens include, but are not limited to, those derived from orthoreovirus, rotavirus, orbivirus, or coltivirus. Parvovirus immunogens include those derived from parvovirus B19. Herpesvirus immunogens include those derived from human herpesviruses, such as herpes simplex virus (HSV) (e.g., HSV types I 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). Papovavirus immunogens include those derived from papillomavirus and adenovirus.

[0105] In some embodiments, the immunogen induces an immune response against Chikungunya virus, hi other embodiments, the immunogen induces an immune response against Zika virus.

[0106] In some embodiments, the immunogen elicits an immune response against a virus that infects the fish.

[0107] Fungal immunogens can be derived from dermatophytes and other opportunistic fungi.

[0108] In some embodiments, the immunogen induces an immune response against a parasite from the genus Plasmodium, such as P. falciparum, P. vivax, P. malariae, or P. ovale. Thus, the present invention can be used for immunization against malaria. In some embodiments, the immunogen induces an immune response against a parasite from the family Caligidae, particularly those from the genera Lepeophtheirus and Caligus, such as sea lice, such as Lepeophtheirus salmonis or Caligus rogercresseyi.

[0109] In some embodiments, the immunogen is an mRNA specific for a neo-antigen in a cancer cell or solid tumor.

[0110] In some embodiments, the immunogen is a tumor antigen selected from: (a) cancer-testis antigens, such as NY-ESO-I, SSX2, SCPI, and polypeptides of the RAGE, BAGE, GAGE, and MAGE families, 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 to address, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors); (b) mutant antigens, such as p53 (associated with various solid tumors, such as colorectal, lung, and head and neck cancers), p21 / Ras (associated with, for example, melanoma, pancreatic, 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 (e.g., associated with bladder cancer), HLA-A2-R1701, beta-catenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin's lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLRFUT; (c) overexpressed antigens, e.g., galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT I (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung, and ovarian cancer), mammaglobin, alpha-fetoprotein (e.g., associated with hepatocellular carcinoma), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast and colon cancer), and carcinoembryonic antigen (e.g., associated with breast, lung, and gastrointestinal cancers, e.g., colorectal cancer);(d) common antigens, e.g., melanoma-melanocyte antigens, e.g., MART-1 / MelanA, gp100, MCIR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-I / TRPI, and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) prostate-related antigens, e.g., PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, which are associated with prostate cancer; (f) immunoglobulin idiotypes (e.g., associated with melanoma and B-cell lymphoma). In certain embodiments, tumor immunogens include 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 virus antigens and hepatitis 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, 791 Tgp72, beta-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.;

[0111] Pharmaceutical compositions for vaccines. Pharmaceutical compositions of the present invention, particularly those useful for immunization, may contain one or more small molecule immunostimulants. Pharmaceutical compositions of the present invention may contain one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free, preservative-free vaccines can be prepared.

[0112] The composition comprises an effective amount of the lipid formulation (e.g., LNP) described herein, as well as any other components, as needed. An immunologically effective amount refers to an amount administered to an individual, either as a single dose or as part of a series of doses, that is effective for treatment (e.g., a preventive immune response against a pathogen). This amount will vary depending on the health and physical condition, age, taxonomic group (e.g., non-human primate, primate, etc.) of the individual being treated, the ability of the individual's immune system to synthesize antibodies, the desired degree of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that this amount will fall within a relatively broad range that can be determined through routine testing.

[0113] The LNP-formulated RNA and pharmaceutical compositions described herein are used in vivo to induce an immune response against a target immunogen. The present invention provides a method for inducing an immune response in a vertebrate, comprising administering an effective amount of an LNP-formulated RNA or pharmaceutical composition as described herein. The immune response is preferably protective and preferably includes antibodies and / or cell-mediated immunity. The composition can be used for both priming and boosting purposes. Alternatively, a prime-boost immunization schedule can be a mixture of RNA and the corresponding polypeptide immunogen (e.g., prime with RNA, boost with protein).

[0114] The present invention also provides LNPs or pharmaceutical compositions thereof for use in inducing an immune response in a vertebrate. The present invention also provides the use of LNPs or pharmaceutical compositions thereof in the manufacture of a medicament for inducing an immune response in a vertebrate. Inducing an immune response in a vertebrate by these uses and methods can protect the vertebrate against various diseases and / or infectious diseases, for example, bacterial and / or viral diseases as discussed above. Vaccines according to the present invention can be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but are typically prophylactic. The vertebrate is preferably a mammal, such as a human or a large domestic mammal (e.g., horse, cow, deer, sheep, llama, goat, pig).

[0115] The compositions of the present invention are generally administered directly to patients. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or into the interstitial space of tissues). Alternative delivery routes include rectal, oral (e.g., tablets, drops, sprays), buccal, sublingual, vaginal, topical, transdermal or transdermal, intranasal, ocular, otic, pulmonary, or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. Injection may be via a needle (e.g., a hypodermic needle), although needleless injection may alternatively be used. A typical intramuscular dose is 0.5 ml. The present invention can be used to induce systemic and / or mucosal immunity, preferably to elicit enhanced systemic and / or mucosal immunity. Dosing can be by a single-dose schedule or a multiple-dose schedule. Multiple doses can be used in a primary immunization schedule and / or a booster immunization schedule.

[0116] In a multiple-dose schedule, various doses can be given by the same route or different routes, e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. The multiple doses are typically administered at least one week apart (e.g., about two weeks, about three weeks, about four weeks, about six weeks, about eight weeks, about ten weeks, about twelve weeks, about sixteen weeks, etc.). In one embodiment, the multiple doses can be administered at approximately 6, 10, and 14 weeks of age, e.g., 6, 10, and 14 weeks of age, as often used in the World Health Organization's Expanded Programme on Immunization ("EPI"). In an alternative embodiment, two primary doses are administered about two months apart, e.g., about 7, 8, or 9 weeks apart, followed by one or more booster doses about six months to one year after the second primary dose, e.g., about 6, 8, 10, or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, for example, about seven, eight, or nine weeks apart, followed by one or more booster doses about six months to one year after the third primary dose.

[0117] In various embodiments, the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include bringing into association the active ingredient(s) with an excipient(s) and / or one or more other accessory ingredients.

[0118] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject, and / or a convenient fraction of such a dosage (including, but not limited to, one-half or one-third of such a dosage).

[0119] The relative amounts of the 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, as well as the route by which the composition is administered. For example, the composition may contain from 0.1% to 99% (w / w) of the active ingredient.

[0120] Pharmaceutical formulations may further comprise pharmaceutically acceptable excipients, which, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, suitable for the particular dosage form desired. Various excipients for formulating 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 any conventional excipient vehicle is contemplated herein except insofar as the conventional excipient vehicle may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition.

[0121] In some embodiments, the size of the lipid particles may be increased and / or decreased. Altering particle size may help combat biological responses, such as, but not limited to, inflammation, or may enhance the biological effectiveness of the NAT delivered to a mammal by altering its biodistribution. Size may also be used to determine target tissues, with larger particles being rapidly cleared and smaller particles reaching various organ systems.

[0122] Pharmaceutically acceptable excipients used in preparing pharmaceutical compositions comprising LNPs include, but are not limited to, inert diluents, surfactants and / or emulsifiers, preservatives, buffers, lubricants, and / or oils. Such excipients can optionally be included in the pharmaceutical formulations of the invention.

[0123] Below is a description of representative lipid particles prepared with nucleic acids (LNPs), how they are produced, evidence of their benefits, and how they can be used to provide therapeutic benefit. [Example]

[0124] General Considerations: All solvents and reagents were commercially available products and were used as received unless otherwise noted. Temperatures are given in degrees Celsius.

[0125] Abbreviation EPO = erythropoietin GFP = green fluorescent protein ug = μg = microgram pg = picograms ng = nanograms g = grams h=time HPLC = High-Performance Liquid Chromatography MFI = Median Fluorescence Intensity min=minutes mL = milliliters mmol = millimolar N / P ratio = ratio of positively chargeable polymeric amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups PBS = phosphate buffer solution wt=mass Deg. C=Celsius temperature

[0126] "Gene of interest" (GOI) refers to one or more genetic elements intended for expression to achieve a therapeutic goal, including immunization. The A5 SARS Cov-2 antigen coding element and epidermal growth factor (EPO) are examples of GOIs for illustrating the present invention, but the GOI is not limited to these illustrative examples.

[0127] IL = ionizable lipid, which is a lipid that is cationic at higher pH and turns uncharged at lower pH. ILs are commonly used in the formulation of nucleic acid cargo.

[0128] Stabilizer = Any stabilizer including polyethylene glycol derivatives including PEG-DMG 2000 and other suitable polymers, which have the purpose of extending circulation life, among other things.

[0129] The components of the lipid mixture include ionizable lipids, phospholipids, cholesterol, and a stabilizer. Low pH buffers (3-6) may be used. For ionizable amino lipids, the pH of the buffer is typically below the pKa of the lipid.

[0130] 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)butanoate found in PCT Publication No. WO20252589 A1 by Jain N, Thomas A, and Brown A.

[0131] 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 No. WO20252589 by Jain et al.

[0132] PNI 127 is an ionizable lipid found in PCT Publication No. WO 021000041 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(octadeca-9,12-dienoate).

[0133] The following is also found in PCT Publication No. WO2100041 by Thomas et al.

[0134] PNI 550: 3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)cyclopentyl 4-(dimethylamino)butanoate, PNI 580: (2R,3S,4S)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)tetrahydrofuran-3,4-diylbis(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-diylbis(2-hexyldecanoate), PNI 722: 2-(((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methoxy)-N,N-dimethylethan-1-amine, PNI 726: (2R,3S,4S)-2-((3-(dimethylamino)propoxy)methyl)tetrahydrofuran-3,4-diylbis(2-hexyldecanoate), PNI 728: ((2R,3R,4S)-3,4-bis((2-hexyldecyl)oxy)tetrahydrofuran-2-yl)methyl(2-(dimethylamino)ethyl)carbamate, and PNI 730: (2R,3S,4S)-2-((2-(dimethylamino)ethoxy)methyl)tetrahydrofuran-3,4-diylbis(2-hexyldecanoate).

[0135] Example 1 Methods for the synthesis of self-amplifying mRNA Restriction enzyme digestion of the circular plasmid encoding the SARS Covid spike protein was performed according to the manufacturer's instructions for BspQI (New England BioLabs, Cat. No. R0712S) or PmeI (New England BioLabs, Cat. No. R0560S) in the vendor's recommended buffer.

[0136] The 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 centrifuged, after which the upper aqueous phase containing the linearized vector was carefully pipetted into a clean RNase / DNase-free tube, precipitated, and then three volumes of 100% ethanol were added, mixed well, carefully removed, and the DNA pellet was air-dried and resuspended in nuclease-free water. The concentration and purity of the linearized vector were determined using a NanoDrop™ spectrophotometer (VWR).

[0137] In vitro transcription was performed using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England BioLabs, catalog number E2040S), followed by digestion of the linear DNA template using TURBO™ DNase (Thermofisher Scientific, catalog number AM2238), and capping of the final in vitro transcribed self-amplifying RNA (saRNA) using the Vaccina Capping System™ (New England BioLabs, catalog number M2080S). All of these processes were carried out according to the manufacturer's protocol to generate self-amplifying RNA using a DNA template obtained from a vector linearization strategy. Purification of the capped saRNA was performed using standard salting out, followed by 70% ethanol washing and resuspension of the RNA pellet in RNA storage solution (Thermofisher). This is described in PCT Publication No. WO23057979 by Geall et al.

[0138] (Example 2) Microfluidic mixing of nucleic acids into lipid nanoparticles (LNPs) A lipid mixture formulation of lipid particles was generated by rapidly mixing a lipid ethanol solution with an aqueous buffer in a microfluidic stirrer designed to induce chaotic advection and provide a controlled mixing environment at intermediate Reynolds numbers (24 < Re < 1000). The microfluidic channels have a herringbone characteristic or are configured in a manner as shown in PCT Publication No. WO2017117647 or U.S. Patent No. 10,835,878.

[0139] The particle size and "polydispersity index" (PDI) of lipid particles were measured by dynamic light scattering (DLS). PDI indicates the width of the particle distribution. It is a parameter calculated from cumulative analysis of the intensity autocorrelation function measured by (DLS), assuming a single particle size mode and a monoexponential fit to the autocorrelation function. From a biophysical perspective, a PDI of less than 0.1 indicates that the sample is monodisperse. Particles produced by mechanical micro-stirrs, such as the NanoAssemblr® Spark™ and NanoAssemblr® Ignite™ (Precision NanoSystems ULC), are substantially uniform in size, assuming all other variables are neutral. A lower PDI indicates a more uniform population of lipid particles. The Spark™ instrument is used in a screening setting to identify lead compositions. Once a composition is selected, the NanoAssemblr® Ignite™ instrument can be used to fine-tune the lipid particles. Once the process parameters flow rate ratio and total flow rate are identified for a particular nanoparticle formulation, the same process parameter values can be used to scale up the nanoparticle technology.

[0140] The above self-amplifying RNA or nucleic acid therapeutic (NAT) was diluted with sodium acetate buffer to the required concentration. Subsequently, lipid nucleic acid particle (LNP) samples were prepared as described by running both fluids using the NanoAssemblr® Ignite instrument. Briefly, 63 μg of nucleic acid in sodium acetate buffer in a total volume of 0.75 mL was mixed with 0.25 mL of 12.5 mM lipid mixture solution, as required for an N / P ratio of 8, and then the LNP was diluted in PBS at a 2:1 ratio by in-line dilution.

[0141] The resulting lipid-nucleic acid particles (LNPs) were immediately diluted with 48 μL of 1× PBS without Ca++ and Mg++ at pH 7.4 in the aqueous output well. These LNPs were immediately collected in a microcentrifuge tube containing 96 μL of the same buffer at pH 7.4. Encapsulation efficiency was measured by a modified Ribogreen™ assay (Quanti-iT RiboGreen™ RNA Assay Kit, Fisher). This information was used to set the desired dosage.

[0142] Lipid particles were also produced using a larger microfluidic mixing device, the NanoAssemblr® Ignite™, for testing. Briefly, 350 μL of mRNA was diluted using 100 mM sodium acetate buffer to the required concentration of 0.2–0.3 mg / mL. Typically, a 12.5 or 25 mM lipid mixture solution was used. LNPs were then prepared by flowing both fluids, i.e., nucleic acid in aqueous solvent and lipid mixture in ethanol, through the microfluidic mixer at a flow ratio of 3:1 and a total flow rate of 12 mL / min. After mixing in the microfluidic device, the post-cartridge lipid-nucleic acid particle sample was diluted in an RNase-free tube containing 3–40 volumes of PBS (pH 7.4). The ethanol was finally removed by either dialysis in PBS (pH 7), using an Amicon™ centrifugal filter (Millipore, USA) at 3000 RPM, or using a TFF system. Once the required concentration was achieved, the lipid-nucleic acid particles were filter-sterilized using a 0.2 μm filter under sterile conditions. The final encapsulation efficiency was measured by the Ribogreen® assay. Quant-iT™ RiboGreen® RNA Reagent and Kit (Invitrogen) was used according to the manufacturer's instructions. The preparation of the self-amplifying mRNA plasmid NAT is described below. Observed particle characteristics generally ranged in size from 50 to 200 nm for mRNA, depending on the lipid composition.

[0143] Table 1 above lists the lipid compositions of the commercially available LNP formulations ONPATTRO™, SPIKEVAX™, and COMIRNATY™. Table 2 lists the vaccine LNP formulation "Vaccmixb-PNI 516" and formulations according to the present invention identified by prefixes beginning with V46. These new vaccine formulations have a significantly different ratio of ionizable lipid to phospholipid than the compositions in Table 1 above, offering a surprising safety advantage since ionizable lipids can be irritating and PEG-lipids are known to cause allergic reactions.

[0144] [Table 2A]

[0145] [Table 2B]

[0146] For Tables 3 and 4, all formulations were made with saRNA expressing the Covid spike protein. Both PNI 516 and MC3 (an ionizable lipid) were formulated at N / P 8, and the RNA concentration for each formulation was kept constant at 84ug / mL.

[0147] [Table 3]

[0148] [Table 4]

[0149] Example 3 Characterization and Encapsulation of Lipid Nucleic Acid Particles or "LNPs" After lipid particles were prepared as described above, LNP particle size (hydrodynamic diameter of the particle) was determined by dynamic light scattering (DLS) using a ZetaSizer™ Nano ZS™ (Malvern Instruments, UK). A He / Ne laser with a wavelength of 633 nm was used as the light source. Data were measured from scattered intensity data performed in backscatter detection mode (measurement angle = 173°). Measurements were the average of 10 runs with two cycles per sample. The Z-average size reported as particle size is defined as the harmonic intensity average particle size. Encapsulation efficiency was measured by a modified Ribogreen™ assay (Quanti-iT RiboGreen™ RNA Assay Kit, Fisher). Encapsulation was good in all formulations (see Table 2 for composition), with polydispersity indices (PDI) less than 0.19.

[0150] Post-encapsulation steps included tangential flow filtration (TFF) concentration for approximately 40 minutes and diafiltration. These steps had minimal impact on LNP properties. Results are not shown. The properties of the lipid mixtures and LNPs of the present invention and the control LNPs are listed in Tables 5 and 6.

[0151] [Table 5]

[0152] [Table 6]

[0153] Example 4 High-throughput LNP potency assay in BHK cells BHK 570 cells were purchased from ATCC and cultured as normal. They were transfected in 96-well plates with LNPs loaded with 1000 to 0.49 ng / mL saRNA A5. LNPs composed of V46-PNI 516 showed significantly higher EC than Vaccmixb-PNI 516. 50The V46 lipid mixture unexpectedly demonstrated a 12.6-fold improvement in EC 50 showed a 12-fold improvement in

[0154] SARsCOV-2 protein expression was measured after transfection of BHK 570 cells with various PNI 516 LNP formulations containing nCoV PNI A5 saRNA as disclosed in PCT Publication No. WO23057979 by Abraham et al.

[0155] [Table 7]

[0156] Visual demonstration of SARsCOV-2 protein expression after transfection in BHK 570 cells was achieved, with green fluorescence representing SARsCOV-2 protein stained with a fluorescently labeled anti-SARsCOV-2 spike protein antibody (FAB105403G) after transfection of BHK 570 cells with Vaccmixb PN1516 or V46-PN1516 at 31.25 ng / well of RNA. BHK 570 cells were plated in 96-well plates (20,000 cells / well) for 48 hours prior to 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 1:50 dilution. Cells were imaged with a Cytation 7™ cell imaging multimode reader (BioTek, Agilent, Santa Clara, CA).

[0157] The dose response of SARsCOV-2 protein expression after transfection with Vaccmixb PN1516 and V46-PN1516 in BHK 570 cells is shown graphically in Figure 1. Dose-response studies were performed by testing decreasing amounts of RNA from 1,000 ng / mL to 0.49 ng / mL. BHK 570 cells were plated in 96-well plates (20,000 cells / well) for 48 hours prior to 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 1:50 dilution. Cells were imaged using a Cytation 7™ (BioTek, Agilent, Santa Clara, CA) multimode reader.

[0158] SARsCOV-2 protein expression was measured after transfection of BHK 570 cells with various PNI516 LNP formulations containing nCoV PNI A5 saRNA. A dose-response study was performed by testing decreasing amounts of RNA from 1,000 ng / mL to 0.49 ng / mL. BHK 570 cells were plated in 96-well plates (20,000 cells / well) for 48 hours prior to 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 1:50 dilution. Cells were imaged using a Cytation 7™. The results are shown in Figure 2.

[0159] For mRNA testing, CLEANCAP™ EGFP mRNA (Trilink Biotechnologies, Cat. No. L-7601) was used with the same testing protocol as above. The various ECs shown in the last columns of Table 8 (N / P 8) and Table 9 (N / P 12) were used. 50A reading is given, with lower levels indicating greater therapeutic efficacy. This finding supports the chemical nature of the ionizable lipids as well as the precise interplay between the optimized ratios of the components. "IL" is the ionizable lipid used. EC 50 "NP" in the column means that the reading was too high to make a meaningful measurement (negative result).

[0160] [Table 8]

[0161] Table 8 shows that there is a large variability in the efficacy of the formulations of the present invention depending on the ionizable lipid used. 50 is a measure of the efficacy of the LNPs to transfect cells and express the GOI. The lower the value, the more effective the formulation.

[0162] [Table 9]

[0163] [Table 10]

[0164] Example 5 In vivo testing The expression of SARS-CoV-2 spike protein-specific IgG in mouse serum was measured after intramuscular LNP injection into BALB / c mice at 1 μg / mouse. Enzyme-linked immunosorbent assay (ELISA) was performed according to established methods to measure SARS-CoV-2 spike protein-specific IgG antibodies in serum samples collected from mice on days 21 and 42 after immunization.

[0165] Reagents for ELISA: Ca2+- and Mg2+-free sterile DPBS 1X (pH 7.4) (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 x-reactivity) antibody, and TMB substrate solution (all BioLegend, San Diego, CA), 70% isopropyl alcohol (IPA) and water (nuclease-free) (VWR), Nunc MaxiSorp™ flat-bottom 96-well plates and stop solution (Thermo Fisher), and SARS-CoV-2 (2019-nCoV) spike S1+S2 ECD-His recombinant protein (Sino Biologicals). Figure 3 shows IgG levels for the various groups on day 21. By day 42, results were beneficially approximately 10-fold higher (not shown), which is a favorable result.

[0166] Example 6 3-month stability test This test was performed on V46 using PNI 516 as the ionizable lipid. Ten aliquots of frozen LNPs (50 μg / mL saRNA) in pH 7.4 buffer were thawed at 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, and then 1 month and 3 months. At each time point, LNP particle size was measured by dynamic light scattering, and saRNA encapsulation was determined using the Quant-it™ RiboGreen RNA Assay Kit™ (Thermo Fisher). The results are shown in Table 11.

[0167] [Table 11]

[0168] Example 7 Cryo-transmission electron microscopy (CryoTEM) structure of LNPs LNPs containing saRNA-encapsulated PNI 516, DSPC, cholesterol, and PEG-DMG were cryopreserved, sectioned, and prepared for cryo-TEM examination. CryoTEM of LNPs containing a standard IL:DSPC ratio of 3.8 yielded mostly dense unilamellar vesicles with a few multicompartmental vesicles, whereas LNPs containing IL:DSPC with a DMG-PEG ratio of 0.58 showed multilamellar vesicles with a greater number of multicompartmental vesicles.

[0169] PEG-free LNPs with an IL:DSPC ratio of 0.58 also exhibited more multilamellar vesicles, but the particles were closer together compared to the PEGylated version of this composition. Figure 4 shows exemplary images of A: Vaccmixb-PNI 516, B: V46-PNI 516, and C: V47-PNI 516.

[0170] Example 8 Stability and activity of LNPs after lyophilization In this experiment, 2 mL glass vials were filled with 500 μL of the LNP formulation described below, each containing PNI 516 as the ionizable lipid. The LNP samples were then equilibrated at 5 °C and 750 Torr pressure. The temperature was then lowered from 5 °C to -50 °C at 400-500 Torr. The samples were held at -50 °C for 3 h. Primary drying was then initiated at -50 °C for 120 min by reducing the pressure to 0.062 Torr. The shelf temperature was then increased to -40 °C at 0.062 Torr pressure, and these conditions were maintained for 12 h (720 min). Finally, secondary drying was performed at 10 °C and 0.062 Torr for 180 min.

[0171] 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, SA RNA encapsulation, and bioactivity as described above.

[0172] EC of the formulation 50The effect of α- and β-actin on the expression of α- and β-actin was determined using an in vitro dose-response assay by decreasing the amount of RNA from 1000 ng / mL to 0.49 ng / mL. BHK 570 cells were plated in 96-well plates (20,000 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 (Sigma-Aldrich) and stained with a 1:50 dilution of FAB105403G antibody (Bio-Techne, Toronto, Ontario). Cells were imaged using a Cytation 7™ (Agilent, Santa Clara, CA) reader.

[0173] The lyophilized versions of V46-PNI 516 LNP and V47-PNI 516 showed no significant change in particle size after rehydration, in contrast to Vaccmixb PNI 516, which showed a significant increase in size. Additionally, Vaccmixb PNI 516 showed a 7-fold decrease in activity compared to V46-PNI 516 LNP and V47-PNI 516 in transfection of BHK 570 cells.

[0174] [Table 12]

[0175] The combination of ionizable lipids in the high phospholipid composition according to the present invention was prepared as described above. LNP particle size (hydrodynamic diameter of the particle) and encapsulation efficiency were measured by the modified Ribogreen™ assay performed as described above. All formulations had good encapsulation size and PDI (see Table 13 for composition), with polydispersity (PDI) less than 0.25 and size less than 117 nm. EC 50 was determined as above in BHK 570 cells using an in vitro dose response by testing decreasing amounts of RNA from 1 μg / well to 0.00049 μg / well.

[0176] Table 13 shows the composition and ratio of various mixtures of ionizable lipids and the effect of LNPs made from them on the GFP saRNA EC50, and the difference in EC50 results from the Vaccmixb (control) EC50. V76 and V68, representing ionizable lipid combinations, showed exceptionally good EC50 (pg / ml) scores. V75, V69, V46, V74, and V76, all representing ionizable lipid mixtures, also performed very well compared to the control.

[0177] [Table 13]

[0178] The mixed ionizable lipid mixtures were also tested for LNP physical properties, and the results are shown in Table 13.

[0179] [Table 14]

[0180] Example 9 This example demonstrates the in vitro transfection ability of saRNA LNPs made with various lipid mixtures in Jurkat cells. Following transfection of V02 Vaccmixb, V46-PNI516, V47-PNI516, and V22-PNI516 at 0.32 μg / mL in Jurkat cells, GFP positivity was assessed using flow cytometry. Jurkat cells (50,000 cells / well) were added to GFP saRNA LNPs to achieve a final saRNA concentration of 3.2 μg / mL. After 24 hours of incubation, cells were resuspended in BSA staining buffer and analyzed using a BECKMAN COULTER™ Cytoflex S flow cytometer. The results are shown in Figure 5.

[0181] Example 10 This example demonstrates the in vitro transfection ability of mRNA LNPs prepared using various lipid mixtures in Jurkat cells. Following transfection of V46-PNI516, V02-PNI516, and V22-PNI516 in Jurkat cells, the GFP positivity rate was assessed in a dose-response experiment. Jurkat cells (50,000 cells / well) were tested for dose response by adding decreasing amounts of GFP mRNA LNPs from 3 μg / well to 0.001465 μg / well. After 24 hours of incubation, the cells were resuspended in BSA staining buffer and analyzed using a Beckman Coulter Cytoflex S flow cytometer. The results are shown in Figure 6.

[0182] Example 11 This example demonstrates the transfection ability of EGFP saRNA LNPs prepared using various lipid mixtures in T cells isolated from PBMCs. Human PBMCs were activated for 3 days in ImmunoCult-XF T cell growth medium supplemented with activator / recombinant human IL-2. After activation, cells were diluted with ApoE to a concentration of 0.25 x 10 cells / ml. LNP formulations containing EGFP saRNA (described in Figure 7) were added to the cell culture medium, and transfection was performed for 24 hours. CD4+ and CD8+ T cells were gated using specific antibodies, and the GFP positivity rate in T cells was assessed using standard flow cytometry analysis. Dead cells were excluded from the analysis using viability staining. Dead cells are susceptible to nonspecific staining by fluorescent antibodies and are generally excluded by gating them out. The results are shown in Figure 7.

[0183] While specific embodiments of the present invention have been described and illustrated, such embodiments should be considered merely as examples of the invention and not as limitations on the invention as interpreted according to the appended claims.

[0184] Example 12 This example demonstrates the efficacy of the LNPs of the present invention in an intravenous protein replacement model.

[0185] Two different LNPs according to embodiments of the present invention were intravenously injected into mice at 0.1 mg / kg. The payload was luciferase mRNA (Trilink-CLEANCAP™ FLuc mRNA (5 moU), catalog number L-7202). The top row of photographs in Figure 8 , showing fluorescence 4 hours after administration, was achieved by intravenous administration of LNPs containing 30% PNI 516, 56% DPSC, 12.5% cholesterol, and 1.5% DMG-PEG. The brighter bottom row in Figure 8 was achieved by LNPs containing 30% PNI 580, 56% DSPC, 12.5% cholesterol, and 1.5% DMG-PEG. This demonstrates that LNPs of the present invention successfully express proteins in vivo after intravenous administration. PNI 580 appears to be slightly more effective than PNI 516 in enabling non-native protein expression in this model.

Claims

1. A lipid formulation comprising an ionizable lipid and a phospholipid, having a ratio of ionizable lipid to phospholipid of 0.1 mol:mol to 2.0 mol:mol, which can form lipid-based nanoparticles suitable for vaccines.

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

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

4. The lipid preparation according to claim 1, further comprising sterols and optionally comprising a stabilizer.

5. The lipid preparation according to any one of claims 1 to 4, wherein the ratio of ionizable lipids to phospholipids is 0.1 mol:mol to 1.3 mol:mol.

6. The lipid preparation according to any one of claims 1 to 4, wherein the ratio of ionizable lipids to phospholipids is 0.3 mol:mol to 1.2 mol:mol.

7. A lipid preparation according to any one of claims 1 to 4, wherein the ratio of ionizable lipids to phospholipids is 0.4 mol:mol to 0.70 mol:mol.

8. A lipid preparation according to any one of claims 1 to 4, wherein the ratio of ionizable lipids to phospholipids is approximately 0.70, approximately 0.69, approximately 0.68, approximately 0.67, approximately 0.66, approximately 0.65, approximately 0.64, approximately 0.63, approximately 0.62, approximately 0.61, approximately 0.60, approximately 0.59, approximately 0.58, approximately 0.57, approximately 0.56, approximately 0.55, approximately 0.54, approximately 0.53, approximately 0.52, approximately 0.51, approximately 0.50, approximately 0.49, approximately 0.48, approximately 0.47, approximately 0.46, approximately 0.45, approximately 0.44, approximately 0.43, approximately 0.42, approximately 0.41, or approximately 0.

40.

9. A lipid preparation according to any one of claims 1 to 4, wherein ionizable lipids are present in an amount of 10 to 42 mol% of the lipid preparation.

10. A lipid preparation according to any one of claims 1 to 4, wherein the ionizable lipid comprises a mixture of ionizable lipids.

11. A lipid preparation according to any one of claims 1 to 4, wherein the ionizable lipid comprises one or more of PNI 127, PNI 516, PNI 550, PNI 560, PNI 580, PNI 659, PNI 721, PNI 722, PNI 726, PNI 728, and PNI 730.

12. The lipid preparation according to any one of claims 1 to 4, wherein phospholipids are present in an amount of 20 to 75 mol of the lipid preparation.

13. A lipid preparation according to any one of claims 1 to 4, wherein phospholipids are present in an amount of 40 to 60 mol of the lipid preparation.

14. A lipid preparation according to any one of claims 1 to 4, wherein the phospholipid is DSPC or DOPE.

15. The lipid preparation according to claim 4, wherein sterols are present in an amount of 12 to 25 mol% of the lipid preparation.

16. The lipid preparation according to claim 4, wherein the sterol is cholesterol or cholesteryl hemysuccinate.

17. A lipid preparation according to any one of claims 1 to 4, which does not contain a stabilizer.

18. The lipid preparation according to claim 4, wherein the stabilizer is present in an amount of 0.0 to 15 mol% of the lipid preparation.

19. The lipid preparation according to claim 4, wherein the stabilizer is present in an amount of 0.0 to 2.5 mol% of the lipid preparation.

20. A lipid formulation according to claim 18 or 19, wherein the stabilizer comprises polyethylene glycol (PEG), polysorbate (Tween), TPGS (vitamin E polyethylene glycol succinate), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™ 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij™ S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), Myrj™ 52 (polyoxyethylene (40) stearate), or polyethylene glycol-bound lipids.

21. The lipid preparation according to claim 4, comprising 10 to 42 mol% of an ionizable lipid, 20 to 75 mol% of a phospholipid, 12 to 25 mol% of a sterol, and 0 to 15 mol% of a stabilizer.

22. A lipid formulation for encapsulating a nucleic acid payload in nanoparticles, comprising an ionizable lipid, a sterol, and a phospholipid, wherein the phospholipid content is 25 to 60%, and the molar ratio of the ionizable lipid to the phospholipid is 0.33 to 1.

2.

23. A lipid preparation comprising an ionizable lipid and a phospholipid, having a ratio of ionizable lipid to phospholipid of 0.1 mol:mol to 2.0 mol:mol, and Nucleic acid vaccine elements encapsulated in lipid formulations A vaccine containing [this ingredient].

24. The vaccine according to claim 23, wherein the nucleic acid vaccine element encodes an antigen selected from coronavirus spike protein and influenza hemagglutinin protein.

25. The vaccine according to claim 23, wherein the nucleic acid vaccine element is derived from an influenza virus or coronavirus.

26. The vaccine according to any one of claims 23 to 25, wherein the lipid preparation comprises 10 to 42 mol% of ionizable lipids and 20 to 75 mol% of phospholipids.

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