Lipid nanoparticles for inducing an immunological response

Abstract

The present disclosure provides lipid nanoparticles that are particularly beneficial in inducing an immune response in a subject, particularly by stimulating production of antibodies by the subject. The lipid nanoparticles can include one or more of a low conjugated lipid concentration, a neutral phospholipid, and a concentration of an ionizable lipid and / or sterol that satisfy certain thresholds dependent on the conjugated lipid concentration. The disclosure also provides vaccines, other pharmaceutical compositions, and methods including the provided lipid nanoparticles.

Description

LIPID NANOPARTICLES FOR INDUCING AN IMMUNOLOGICAL RESPONSECROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 574,476 filed April 4, 2024, the full disclosure of which is incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Lipid nanoparticles (LNPs) have emerged as a critical technology in the field of drug delivery, particularly for the encapsulation and systemic delivery of nucleic acids such as mRNA and siRNA. Their importance lies in their ability to protect these therapeutic agents from degradation, enhance cellular uptake, and control the release of the encapsulated agent. For example lipid nanoparticles have emerged as a promising technology for the delivery of vaccines. The particles can, for example, protect immunogenic nucleic acids such as mRNA molecules from degradation, aid in their intracellular delivery, and facilitate endosomal escape, thus enabling the mRNA to be translated into a target protein, such as an antigenic protein.

[0003] Despite their success, however, there is a pressing need for improved LNPs, including those used for delivery of prophylactic vaccines. Current LNPs can have limitations including suboptimal biodistribution and / or clearance rates, induction of undesired immune responses, potential for off-target effects, and low expression efficiencies of delivered payloads such as mRNA. For example, inflammation-related side effects, such as pain, swelling, fever, and sleepiness, can be associated with some current RNA-LNP-based vaccines. Additionally, some LNP designs can inadvertently result in substantial mRNA expression in off-target tissues and organs, thereby reducing prophylactic efficiency and increasing the likelihood of unwanted side effects. Therefore, the development of next-generation LNPs with, for example, enhanced stability, targeted delivery, and minimized side effects is important for fully realizing the potential of LNPs in the field of nucleic acid delivery for prophylactic and therapeutic care. The present disclosure addresses these needs and provides associated and other advantages.BRIEF SUMMARY

[0004] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Covered embodiments of the disclosure are defined by the claims, not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0005] In one aspect, the disclosure provides a vaccine that includes a lipid nanoparticle. The lipid nanoparticle includes a conjugated lipid. The conjugated lipid includes no more than X mol% of the total lipid of the lipid nanoparticle, where X has a value that is no more than 1. The lipid nanoparticle further includes a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net neutral charge at physiological pH. The lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof. The lipid nanoparticle further includes cholesterol or a derivative thereof. The lipid nanoparticle further includes a nucleic acid encoding a protein of interest, e.g., an antigen, or a fragment thereof. The lipid nanoparticle has a composition satisfying at least one of the following conditions: (a) the ionizable lipid or the pharmaceutically acceptable salt thereof includes at least Y mol% of the total lipid of the lipid nanoparticle, where Y has a value defined by the formula: 51.1 - 5.7X + 8. OX2; and (b) the cholesterol or the derivative thereof includes no more than Z mol% of the total lipid of the nanoparticle, where Z has a value defined by the formula: 39.3 + 2.3X - 5.6X2.

[0006] In another aspect, the disclosure provides a method of producing a lipid nanoparticle. The method includes providing an aqueous solution that includes a nucleic acid encoding a protein of interest, e.g., an antigen, or a fragment thereof. The method further includes providing an alcoholic solution comprising an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, cholesterol or a derivative thereof, and a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net neutral charge at physiological pH. The method further includes combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle. The conjugated lipid includes no more than X mol% of the total lipid of the alcoholic solution, where X has a value that is no more than 1. The alcoholic solution has acomposition satisfying at least one of the following conditions: (a) the ionizable lipid or the pharmaceutically acceptable salt thereof includes at least Y mol% of the total lipid of the alcoholic solution, where Y has a value defined by the formula: 51.1 - 5.7X + 8. OX2; and (b) the cholesterol or the derivative thereof includes no more than Z mol% of the total lipid of the alcoholic solution, where Z has a value defined by the formula: 39.3 + 2.3X - 5.6X2.

[0007] In another aspect, the disclosure provides a method of inducing an immune response against the protein of interest, e.g., antigen, of a vaccine as disclosed herein in a subject. The method includes administering to the subject a therapeutically effective amount of the vaccine.

[0008] In another aspect, the disclosure provides a method of preventing or treating a disease in a subject. The method includes administering to the subject a therapeutically effective amount of a vaccine as disclosed herein.BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG. 1 presents a graph plotting anti-HA IgG levels 28 days after immunization with 10 pg HA LNP containing various levels of PEG-lipid and ionizable lipid. N=4 BALB / C mice, SEM. LNP compositions are described in Table 1.

[0010] FIG. 2 presents a graph plotting anti-OVA IgG levels 35 days after prime / boost immunization with 1 pg HA LNP containing various levels of PEG-lipid and ionizable lipid on Day 1 and Day 21. N=4 BALB / C mice, SEM. LNP compositions are described in Table 2.

[0011] FIG. 3 presents a graph plotting anti-HA IgG levels 28 days after immunization with 1 pg HA mRNA-LNP containing alternative mol% of PEG-lipid and differing cationic lipid. N=4 BALB / C mice, SEM. LNP compositions are described in Table 3.

[0012] FIG. 4 presents a graph plotting anti-HA IgG levels 28 days after immunization with 10 pg HA LNP containing various mol% of conjugated lipids. N=4 BALB / C mice, SEM. LNP compositions are described in Table 4.DETAILED DESCRIPTIONA. INTRODUCTION

[0013] The present disclosure generally relates to lipid nanoparticles that, when employed for example in vaccines, provide advantageous improvements in inducing an immune response, e.g., an antibody response. For example, it is beneficial for vaccinations to stimulate a high, i.e., strong, production of antibodies. Strong antibody production indicates a robust immuneresponse, which is essential for effectively neutralizing pathogens. Adequate antibody levels confer heightened protection against the targeted infectious agent over an extended period, offering a sustained defense that can reduce the likelihood of infection and subsequent illness. Strong antibody responses can play a crucial role in preventing outbreaks of existing or novel infectious diseases. By minimizing the pool of susceptible individuals, high levels of immunity within a community act as a barrier to the transmission of the pathogen, thereby reducing the likelihood of outbreaks and epidemics.

[0014] It has proven difficult, however, for vaccines using lipid nanoparticles as antigen carriers to elicit high antibody responses in vaccinated individuals. In some cases, lipid nanoparticles may trigger immune tolerance, wherein the immune system becomes desensitized to the vaccine antigens. This can hinder the generation of robust antibody responses as the immune system fails to mount an adequate reaction to the vaccine components. Further, the size and structure of lipid nanoparticles can impact their ability to deliver vaccine antigens to antigen-presenting cells, limiting the generation of antibody-producing immune cells. Also, lipid nanoparticles can potentially modulate the immune response in ways that may not favor the generation of high antibody levels. For example, certain lipid components or adjuvants used in lipid nanoparticle formulations may skew the immune response towards a less antibody-centric profde, leading to diminished antibody production.

[0015] The materials and methods provided by the present disclosure address these difficulties by using particular lipid nanoparticle compositions that are demonstrated herein as being surprisingly effective in inducing high levels of antibody production. These provided lipid nanoparticle formulations include selected materials that are combined in relative amounts having specific relationships to one another. Notably, the importance of these relative amounts and the specific relationships between them had not previously been appreciated, particularly with regard to their beneficial effects on stimulation of an enhanced immune response.B. DEFINITIONS

[0016] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0017] The abbreviations used herein have their conventional meaning within the chemical and biological arts. Description of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds that are not inherently unstable and / or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, or physiological conditions. Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0018] Unless otherwise stated, the compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labeled with isotopes, such as for example deuterium (2H), tritium (3H), iodine- 125 (125I), carbon- 13 (13C), or carbon- 14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0019] As used herein, the term “substitution,” when used in relation to a chemical substance, refers to replacement of a hydrogen atom with a non-hydrogen atom or covalently bonded group of atoms. The atom or group of atoms replacing the hydrogen atom is referred to as a “substituent.”

[0020] As used herein, the term “member,” when used in relation to a chemical substance, refers to a non-hydrogen atom of a covalently bonded group of atoms, e.g., a compound or substituent thereof.

[0021] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a phospholipid” optionally includes a combination of two or more phospholipids, and the like.

[0022] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0023] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0024] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of’ is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of’ limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0025] The term “optional” or “optionally” means that the subsequently described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0026] The terms “first,” “second,” “third,” and the like when used herein with reference to elements or properties, are simply to more clearly distinguish or identify multiple elements or properties, and are not intended to indicate an order or other serial or numerical limitation, or to require that each of the multiple elements or properties are present.

[0027] As used herein, the terms “lipid particle,” “lipid nanoparticle,” and “LNP” refer to a particle comprising a phospholipid and an ionizable lipid. A lipid particle may comprise additional lipid components, such as a sterol and / or a conjugated lipid, and may further comprise a nucleic acid, wherein the nucleic acid may be encapsulated within the particle . Lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Application Publication Nos. 2004 / 0142025 and 2007 / 0042031, and International Patent Application Publication No. WO 2012 / 000104.

[0028] As used herein, the term “phospholipid” refers to a lipid species having a phosphate- containing hydrophilic “head group” and a hydrophobic moiety. The hydrophobic moiety can comprise one or more hydrophobic groups, most typically two hydrophobic groups. The hydrophobic groups are also referred to as hydrophobic “tails,” and can be derived from fatty acids and joined by an alcohol residue, e.g., glycerol. Exemplary structures of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine .

[0029] As used herein, the term “ionizable lipid” refers to a lipid species that carries a net positive charge at a selected pH, such as an acidic pH or physiological pH. In some cases, an ionizable lipid includes an ionizable primary, secondary, or tertiary amine (e.g., pH titratable) head group. In some instances, ionizable lipids promote encapsulation of a negatively charged nucleic acid (e.g., mRNA or siRNA) payload during particle formation. In some instances, ionizable lipids promote endosomal fusion and cytoplasmic release of a payload following cellular uptake of a lipid nanoparticle.

[0030] As used herein, the terms “permanently charged lipid,” “permanently charged cationic lipid,” and “permanently charged ionic lipid” generally refer to a lipid species that carries a net positive charge regardless of the pH of its biological environment.

[0031] As used herein, the term “conjugated lipid” refers to a polymer-conjugated lipid, e.g., a polymer-conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), polysarcosine-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the polymer to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.

[0032] As used herein, the term “salt” refers to acid or base salts of the compounds of the present disclosure. A “pharmaceutically acceptable salt” is one that is compatible with other ingredients of a formulation composition containing the compound, and that is not deleterious to a recipient thereof, i.e., a subject. It is thus understood that the pharmaceutically acceptable salts do not cause a significant adverse toxicological effect on the subject.

[0033] As used herein, the term “nucleic acid” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, that have similar binding properties as the reference nucleotide and are metabolized in a manner similar to reference nucleotides. Non-limiting examples of nucleotideanalogs are described in, e.g., International Patent Application No. WO 2007 / 024708. Examples of nucleic acids including such nucleotide analogs, modified backbone residues, or linkages include, without limitation, those containing phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine (Batzer et al., (191) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605; and Rossolini et al., (1994) Mol. Cell. Probes 8:91).

[0034] Non-limiting examples of polynucleotides or nucleic acids include DNA, RNA, coding or noncoding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), small nucleolar RNA(snoRNA), ribozymes, deoxynucleotides (dNTPs), or dideoxynucleotides (ddNTPs). Polynucleotides can also include complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification. Polynucleotides can also include DNA molecules produced synthetically or by amplification, genomic DNA (gDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, or primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides, nucleotide analogs, and / or nucleosides suitable for reducing the immunogenicity of RNA, such as those described in International Patent Application No. WO 2007 / 024708. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Polynucleotide sequences, when provided, are listed in the 5' to 3' direction, unless stated otherwise.

[0035] Nucleic acids or polynucleotides can be double- or triple -stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acidstrands need not be coextensive, for example, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands.

[0036] Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications include base modifications such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of S-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0037] Nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, in vitro transcription such as described in, e.g., International Patent Application Publication No. WO 2007 / 024708, or from a combination of those processes.

[0038] As used herein in the context of lipid nanoparticles and nucleic acids, the term “fully encapsulated” indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Fully encapsulated also suggests that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[0039] As used herein, the term “vaccine” refers to a composition comprising at least one antigen or immunogen, or comprising a nucleic acid molecule encoding at least one antigen or immunogen, optionally in a pharmaceutically acceptable carrier or lipid particle, where the composition is useful for inducing an immune response against the antigen or immunogen in a subject for the purpose of improving immunity against a disease and / or infection in the subject.

[0040] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, thesubject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0041] As used herein, the terms “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like. One of skill in the art will recognize that other pharmaceutically acceptable excipients and carriers are useful in the present disclosure.

[0042] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, intrathecal, intracerebroventricular, intraparenchymal, subretinal, or intravitreal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0043] As used herein, the term “therapeutically effective amount” refers to an amount or dose of a compound, composition, or formulation that produces therapeutic effects for which it is administered. The exact amount or dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 23rd Edition (2020), Gennaro, Ed., Lippincott, Williams & Wilkins).

[0044] As used here, the terms “treat”, “treating” and “treatment” refers to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology orcondition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of one or more symptoms. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination or laboratory test.C. LIPID NANOPARTICLES AND VACCINES

[0045] In one aspect, the present disclosure provides various lipid nanoparticles, as well as vaccines that include the provided lipid nanoparticles. The lipid nanoparticles generally include a conjugated lipid, a phospholipid or a pharmaceutically acceptable salt thereof, an ionizable lipid or a pharmaceutically acceptable salt thereof, a cholesterol or a derivative thereof, and a nucleic acid encoding a protein of interest, e .g . , an antigen, or a fragment thereof. The particular selection, composition, and amounts (e.g., relative amounts) of these components provide surprising improvements in the ability of the lipid nanoparticles and vaccines to induce immune responses, e.g., immune responses characterized by high levels of antibody production. For example, the lipid nanoparticles and vaccines are demonstrated herein as inducing strong immune responses when (1) the phospholipid is one having a net neutral charge (e.g., a net neutral charge at a physiological pH), (2) the concentration of the conjugated lipid in the lipid nanoparticle is below a certain threshold (e.g., less than 1 mol% of the total lipid of the lipid nanoparticle), (3) the concentration of the ionizable lipid or the pharmaceutically acceptable salt thereof is above a certain threshold that is a function of the conjugated lipid concentration (e.g., greater than 51.1 - 5 ,7X + 8. OX2mol% of the total lipid of the lipid nanoparticle, where X is the conjugated lipid mol% of the total lipid of the lipid nanoparticle), and / or (4) the concentration of the cholesterol or the derivate thereof in the lipid nanoparticle is below a certain threshold that is a function of the conjugated lipid concentration (e.g., less than 39.3 + 2.3X - 5 ,6X2mol% of the total lipid of the lipid nanoparticle, where X is the conjugated lipid mol% of the total lipid of the lipid nanoparticle).

[0046] In some examples of the provided lipid nanoparticles and vaccines, the conjugated lipid of the lipid nanoparticle comprises between about 0.6 mol% and about 1 mol% of the total lipid of the lipid nanoparticle (e.g., between about 0.6 mol% and about 0.9 mol%, between about 0.6 mol% and about 0.8 mol%, between about 0.6 mol% and 0.7 mol%, between about 0.7 mol% and about 1 mol%, between about 0.7 mol% and about 0.9 mol%, between about 0.7 mol% and about 0.8 mol%, between about 0.8 mol% and about 1 mol%, between about 0.8 mol% and about 0.9 mol%, or between about 0.9 mol% and about 1 mol%), the phospholipidor the pharmaceutically acceptable salt thereof comprises between about 8.5 mol% and about11.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 8.5 mol% and about10.75 mol%, between about 8.5 mol% and about 10 mol%, between about 8.5 mol% and about 9.25 mol%, between about 9.25 mol% and about 11.5 mol%, between about 9.25 mol% and about 10.75 mol%, between about 9.25 mol% and about 10 mol%, between about 10 mol% and about 11.5 mol%, between about 10 mol% and about 10.75 mol%, or between about 10.75 mol% and about 11 mol%), the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 50.5 mol% and 58.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 50.5 mol% and about 56.5 mol%, between about 50.5 mol% and about 54.5 mol%, between about 50.5 mol% and about 52.5 mol%, between about 52.5 mol% and about58.5 mol%, between about 52.5 mol% and about 56.5 mol%, between about 52.5 mol% and about 54.5 mol%, between about 54.5 mol% and about 58.5 mol%, between about 54.5 mol% and about 56.5 mol%, or between about 56.5 mol% and 58.5 mol%), and the cholesterol or the derivative thereof comprises between 29 mol% and 36 mol% of the total lipid of the lipid nanoparticle (e.g., between about 29 mol% and about 34.25 mol%, between about 29 mol% and about 32.5 mol%, between about 29 mol% and about 30.75 mol%, between about 30.75 mol% and about 36 mol%, between about 30.75 mol% and about 34.25 mol%, between about30.75 mol% and 32.5 mol%, between about 32.5 mol% and about 36 mol%, between about32.5 mol% and about 34.25 mol%, or between about 34.25 mol% and about 36 mol%).

[0047] In other examples of the provided lipid nanoparticles and vaccines, the conjugated lipid of the lipid nanoparticle comprises between about 0.1 mol% and about 0.6 mol% of the total lipid of the lipid nanoparticle (e.g., between about 0.1 mol% and about 0.475 mol%, between about 0.1 mol% and about 0.35 mol%, between about 0.1 mol% and 0.225 mol%, between about 0.225 mol% and about 6 mol%, between about 0.225 mol% and about 0.475 mol%, between about 0.225 mol% and about 0.35 mol%, between about 0.35 mol% and about 0.6 mol%, between about 0.35 mol% and about 0.475 mol%, or between about 0.475 mol% and about 0.6 mol%), the phospholipid or the pharmaceutically acceptable salt thereof comprises between about 8.5 mol% and about 11.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 8.5 mol% and about 10.75 mol%, between about 8.5 mol% and about 10 mol%, between about 8.5 mol% and about 9.25 mol%, between about 9.25 mol% and about 11.5 mol%, between about 9.25 mol% and about 10.75 mol%, between about 9.25 mol% and about 10 mol%, between about 10 mol% and about 11.5 mol%, between about 10 mol% and about 10.75 mol%, or between about 10.75 mol% and about 11 mol%), the ionizablelipid or the pharmaceutically acceptable salt thereof comprises between 53.5 mol% and 61.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 53.5 mol% and about 59.5 mol%, between about 53.5 mol% and about 57.5 mol%, between about 53.5 mol% and about 55.5 mol%, between about 55.5 mol% and about 61.5 mol%, between about 55.5 mol% and about 59.5 mol%, between about 55.5 mol% and about 57.5 mol%, between about 57.5 mol% and about 61.5 mol%, between about 57.5 mol% and about 59.5 mol%, or between about 59.5 mol% and 61.5 mol%), and the cholesterol or the derivative thereof comprises between 32 mol% and 39 mol% of the total lipid of the lipid nanoparticle (e.g., between about 32 mol% and about 37.25 mol%, between about 32 mol% and about 35.5 mol%, between about 32 mol% and about 33.75 mol%, between about 33.75 mol% and about 39 mol%, between about 33.75 mol% and about 37.25 mol%, between about 33.75 mol% and 35.5 mol%, between about 35.5 mol% and about 39 mol%, between about 35.5 mol% and about 37.25 mol%, or between about 37.25 mol% and about 39 mol%).

[0048] In certain examples, the provided lipid nanoparticle can further include a permanently charged cationic lipid. The lipid nanoparticle can include a permanently charged cationic lipid that comprises, for example, between about 1 mol% and 10 mol% of the total lipid of the lipid nanoparticle, e.g., between about 1 mol% and about 5.6 mol%, between about 1 mol% and about 3.2 mol%, between about 1 mol% and about 1.8 mol%, between about 1.8 mol% and about 10 mol%, between about 1.8 mol% and about 5.6 mol%, between about 1.8 mol% and about 3.2 mol%, between about 3.2 mol% and about 10 mol%, between about 3.2 mol% and5.6 mol%, or between about 5.6 mol% and about 10 mol%. In terms of upper limits, the permanently charged cationic lipid can comprise, for example, no more than about 10 mol% of the total lipid of the lipid nanoparticle, e.g., no more than about 6.8 mol%, no more than about4.6 mol%, no more than about 3.2 mol%, no more than about 2.2 mol%, no more than about 1.5 mol%, or no more than about 1 mol%. In terms of lower limits, the permanently charged cationic lipid can comprise, for example, no less than about 1 mol% of the total lipid of the lipid nanoparticle, e.g., no less than about 1.5 mol%, no less than about 2.2 mol%, no less than about 3.2 mol%, no less than about 4.6 mol%, or no less than about 6.8 mol%. In some examples, the provided lipid nanoparticle does not include a permanently charged cationic lipid.

[0049] The identity and relative amounts of the lipid nanoparticle components can also be selected to provide the lipid nanoparticle with a pKa that is advantageous for use in a vaccine. For example, in some embodiments, the lipid nanoparticle has a pKa from about 5.8 to about6.9, e.g., from about 5.8 to about 6.8, from about 5.8 to about 6.7, from about 5.8 to about 6.6, from about 5.8 to about 6.5, from about 5.8 to about 6.4, from about 5.8 to about 6.3, from about 5.8 to about 6.2, from about 5.8 to about 6.1, from about 5.8 to about 6.0, from about 5.8 to about 5.9, from about 5.9 to about 6.9, from about 5.9 to about 6.8, from about 5.9 to about 6.7, from about 5.9 to about 6.6, from about 5.9 to about 6.5, from about 5.9 to about 6.4, from about 5.9 to about 6.3 from about 5.9 to about 6.2, from about 5.9 to about 6.1, from about 5.9 to about 6.0, from about 6.0 to about 6.9, from about 6.0 to about 6.8, from about 6.0 to about 6.7, from about 6.0 to about 6.6, from about 6.0 to about 6.5, from about 6.0 to about 6.4, from about 6.0 to about 6.3, from about 6.0 to about 6.2, from about 6.0 to about 6.1, from about 6.1 to about 6.9, from about 6.1 to about 6.8, from about 6. 1 to about 6.7, from about 6. 1 to about 6.6, from about 6. 1 to about 6.5, from about 6.1 to about 6.4, from about 6. 1 to about 6.3, from about 6.1 to about 6.2, from about 6.2 to about 6.9, from about 6.2 to about 6.8, from about 6.2 to about 6.7, from about 6.2 to about 6.6, from about 6.2 to about 6.5, from about 6.2 to about 6.4, from about 6.2 to about 6.3, from about 6.3 to about 6.9, from about 6.3 to about 6.8, from about 6.3 to about 6.7, from about 6.3 to about 6.6, from about 6.3 to about 6.5, from about 6.3 to about 6.4, from about 6.4 to about 6.9, from about 6.4 to about 6.4 to about 6.8, from about 6.4 to about 6.7, from about 6.4 to about 6.6, from about 6.4 to about 6.5, from about 6.5 to about 6.9, from about 6.5 to about 6.8, from about 6.5 to about 6.6, from about 6.6 to about 6.9, from about 6.6 to about 6.8, from about 6.6 to about 6.7, from about 6.7 to about 6.8, or from about 6.8 to about 6.9.

[0050] The identity and relative amounts of the lipid nanoparticle components can also be selected to provide the lipid nanoparticle with a size that is beneficial for use in a vaccine. The lipid nanoparticles can have a mean diameter that is, for example, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 60 to about 90 nm, or from about 70 to about 90 nm.

[0051] The provided lipid nanoparticles can further be configured, e.g., through the selection of their components, to be substantially non-toxic. When the lipid nanoparticles are used in a vaccine, the vaccine can additionally include a pharmaceutically acceptable excipient.1. Conjugated Lipid

[0052] The lipid nanoparticles disclosed herein generally include at least one conjugated lipid. The conjugated lipid can be selected or designed to provide the lipid nanoparticle, or avaccine that includes the lipid nanoparticle, with improved performance in inducing a desired immune response, such as an increase production of antibodies associated with a target antigen. Also, the amount of the conjugated lipid present in the lipid nanoparticle is shown in the present disclosure to influence the ability of the lipid nanoparticle to induce a strong immune response. For example, conjugated lipid amounts below certain thresholds, and optionally also above certain other thresholds, are demonstrated as beneficially resulting in lipid nanoparticles that stimulate high antibody production levels.

[0053] In some examples, the provided lipid nanoparticles and vaccines induce strong immune responses when the amount of conjugated lipid in the lipid nanoparticle is less than about 1 mol% of the total lipid of the lipid nanoparticle. The conjugated lipid of the provided lipid nanoparticle can comprise, for example, between about 0.1 mol% and about 1 mol% of the total lipid of the lipid nanoparticle, e.g., between about 0.1 mol% and about 0.56 mol%, between about 0.1 mol% and about 0.32 mol%, between about 0.1 mol% and about 0.18 mol%, between about 0.18 mol% and about 1 mol%, between about 0.18 mol% and about 0.56 mol%, between about 0.18 mol% and about 0.32 mol%, between about 0.32 mol% and about 1 mol%, between about 0.32 mol% and about 0.56 mol%, or between about 0.56 mol% and about 1 mol%.

[0054] In certain examples, the provided lipid nanoparticles and vaccines induce strong immune responses when the amount of conjugated lipid in the lipid nanoparticle is also greater than about 0.5 mol% of the total lipid of the lipid nanoparticle. The conjugated lipid can comprise, for example, between about 0.5 mol% and about 1 mol% of the total lipid of the lipid nanoparticle, e.g., between about 0.5 mol% and about 0.875 mol%, between about 0.5 mol% and about 0.75 mol%, between about 0.5 mol% and about 0.625 mol%, between about 0.625 mol% and about 1 mol%, between about 0.625 mol% and about 0.875 mol%, between about 0.625 mol% and about 0.75 mol%, between about 0.75 mol% and about 1 mol%, between about 0.75 mol% and about 0.875 mol%, or between about 0.875 mol% and about 1 mol%.

[0055] In terms of upper limits, the conjugated lipid of the provided lipid nanoparticle can comprise, for example, no more than about 1 mol% of the total lipid of the lipid nanoparticle, e.g., no more than about 0.95 mol%, no more than about 0.9 mol%, no more than about 0.85 mol%, no more than about 0.8 mol%, no more than about 0.75 mol%, no more than about 0.7 mol%, no more than about 0.65 mol%, no more than about 0.6 mol%, no more than about 0.55 mol%, no more than about 0.5 mol%, no more than about 0.45 mol%, no more than about 0.4mol%, no more than about 0.35 mol%, no more than about 0.3 mol%, no more than about 0.25 mol%, no more than about 0.2 mol%, no more than about 0.15 mol%, or no more than about 0.1 mol%. In terms of lower limits, the conjugated lipid can comprise, for example, no less than about 0.1 mol% of the total lipid of the lipid nanoparticle, e.g., no less than about 0.15 mol%, no less than about 0.2 mol%, no less than about 0.25 mol%, no less than about 0.3 mol%, no less than about 0.35 mol%, no less than about 0.4 mol%, no less than about 0.45 mol%, no less than about 0.5 mol%, no less than about 0.55 mol%, no less than about 0.6 mol% no less than about 0.65 mol%, no less than about 0.7 mol%, no less than about 0.75 mol%, no less than about 0.8 mol%, no less than about 0.85 mol%, no less than about 0.9 mol%, or no less than about 0.95 mol%.

[0056] In some embodiments, the lipid nanoparticle includes one species of conjugated lipid. In other embodiments, the lipid nanoparticle includes two or more species of conjugated lipids, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of conjugated lipids. The one or more conjugated lipids can each independently be selected or configured to control the size of the particle during formation, and / or to prevent particle aggregation by sterically stabilizing the lipid nanoparticle. The conjugated lipid of the lipid nanoparticle can be situated at the surface of the particle, with the hydrophilic polymer of the conjugated lipid oriented outwardly and interfacing with the aqueous environment, and the lipid component of the conjugated lipid buried in the particle to anchor it in place.

[0057] Conjugated lipids suitable for use with the provided lipid nanoparticle include, but are not limited to, polyethylene glycol (PEG)-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), polyoxazoline (POZ)-lipids, polysarcosine (pSAR)- lipids and mixtures thereof. In certain embodiments, the lipid nanoparticle comprises either a PEG-lipid conjugate or an ATTA-lipid conjugate optionally together with a CPL.

[0058] In a preferred embodiment, the conjugated lipid includes or consists of a PEG-lipid. Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., International Patent Application Publication No. WO 05 / 026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Application Publication Nos. 2003 / 0077829 and 2005 / 0008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613, PEG conjugated to cholesterol or a derivative thereof,mPEG(2000)-N,N-ditetradecylacetamide (ALC-0159), mPEG(2000)-stearate, 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), R-3-[(co- methoxy-poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3 -amine (PEG-C- DOMG), any of the PEG lipids described in International Patent Application Publication No. WO 2022 / 133344, and mixtures thereof. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. The provided lipid nanoparticle can include one or more methyl capped PEG-lipids, one or more uncapped PEG- lipids, or a combination thereof.

[0059] PEG is a linear, water-soluble polymer of ethylene glycol repeating units. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. Such molecular weights are average molecular weights due to polydispersity. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG- NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (Me PEG-IM). Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present disclosure. The disclosures of these patents are herein incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol acetic acid (MePEG-CEECOOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.

[0060] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0061] In certain instances, the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid viaa linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester containing linker moiety. As used herein, the term “non-ester containing linker moiety” refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-). Suitable non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (NHC(O)O-), urea (-NHC(O)NH-), disulphide (-S-S-), ether (-O-), succinyl ((O)CCH2CH2C(O)-), succinimidyl (-NHC(O)CH2CH2C(O)NH-), as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.

[0062] In other embodiments, an ester containing linker moiety is used to couple the PEG to the lipid. Suitable ester containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.

[0063] In addition to the foregoing, it will be readily apparent to those of skill in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, ATTA, cationic-polymers, polyoxazoline (POZ), polysarcosine, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and poly dimethylacrylamide, polylactic acid, polyglycolic acid, poly (ethyl ethylene phosphate) (PEEP), and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0064] The conjugated lipid of the provided lipid nanoparticles can include or consist of, e.g., one or more of the following: a polyethylene glycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, or mixtures thereof. In one preferred embodiment, the nucleic acid- lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. The conjugated lipids may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (Cl 2), a PEG- dimyristyloxypropyl (Cl 4), a PEG-dipalmityloxypropyl (Cl 6), a PEG-distearyloxypropyl (Cl 8), or mixtures thereof.

[0065] Additional PEG-lipid conjugates suitable for use in the provided lipid nanoparticle include, but are not limited to, mPEG2000-l,2-di-0-alkyl-sn3-carbomoylglyceride (PEG-C- DOMG). The synthesis of PEG-C-DOMG is described in PCT Application No.PCI7US08 / 88676, filed Dec. 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, l-[8'-(l,2-dimyristoyl-3-propanoxy)-carboxamido- 3',6'-dioxaoctanyl]carbamoyl-w-methylpoly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S. Pat. No. 7,404,969, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0066] In some examples, the provided lipid nanoparticles and vaccines including the lipid nanoparticles exhibit an ability to induce a strong immune response in part because the conjugated lipid of the lipid nanoparticles is one having two or more terminal hydrocarbon chains (e.g., terminal alkyl chains or terminal alkenyl chains) that each have five or more members, and that each are not directly bonded to an ester. Lipid nanoparticles including conjugated lipids having such hydrophobic tails are demonstrated herein as stimulating advantageously high productions of antibodies. The conjugated lipid can include or consist of, for example, N- [(methoxypoly (ethylene glycol))carbamoyl]-l,2-dimyristyloxy-propylamine (PEG-c-DMA).

[0067] The terminal hydrocarbon chains, e.g., alkyl chains, of the conjugated lipid in the lipid nanoparticle are not particularly limited in terms of length. Each terminal hydrocarbon chain of the conjugated lipid can independently have a length that is, for example C12, C14, Cl 6, Cl 8, C20, C22, or C24. In some examples, the conjugated lipid includes one or two terminal hydrocarbon chains that are each independently Cl 2, Cl 4, C16, or Cl 8.

[0068] By controlling the composition and concentration of the conjugated lipid in the provided lipid nanoparticle, one can control the rate at which the conjugated lipid exchanges out of the lipid nanoparticle and, in turn, the rate at which the lipid nanoparticle becomes fusogenic. For instance, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as the conjugated lipid, the rate at which the lipid nanoparticle becomes fusogenic can be varied, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide. In addition, other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and / or control the rate at which the lipid nanoparticle becomes fusogenic. Other methods which can be used to control the rate at which the lipid nanoparticle becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.2. Ionizable Lipid

[0069] The lipid nanoparticles disclosed herein generally include at least one ionizable lipid, or a pharmaceutically acceptable salt thereof. The ionizable lipid can be selected or designed to provide the lipid nanoparticle, or a vaccine that includes the lipid nanoparticle, with improved performance in inducing a desired immune response, such as an increase production of antibodies associated with a target antigen. Also, the amount of the ionizable lipid present in the lipid nanoparticle is shown in the present disclosure to influence the ability of the lipid nanoparticle to induce a strong immune response. For example, ionizable lipid amounts greater than certain thresholds are demonstrated as beneficially resulting in lipid nanoparticles that stimulate high antibody production levels.

[0070] Further, the present disclosure reveals that not only the absolute amount of ionizable lipid in a lipid nanoparticle, but also the relative amount of the ionizable lipid relative to that of the conjugated lipid of the lipid nanoparticle, can enhance the immune response induced by the lipid nanoparticle. This particular relationship between the ionizable lipid concentration and the conjugated lipid concentration, which had not been previously recognized, can be expressed in terms of a function of these two concentrations. The function can define a threshold criterion, such that lipid nanoparticles including a molar concentration of the ionizable lipid greater than a threshold value induce stronger immune responses, where the threshold value is calculated as a function of the conjugated lipid concentration of the lipid nanoparticle. The function can be defined, for example, by the formula Y > 51.1 - 5.7X + 8. OX2, where X represents the mol% ofthe conjugated lipid among the total lipids of the lipid nanoparticle, and the Y calculated as a function of X is a threshold mol% of the ionizable lipid or pharmaceutically acceptable salt thereof among the total lipids of the nanoparticle.

[0071] In some examples, the conjugated lipid comprises 1 mol% of the total lipids of the lipid nanoparticle, and the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least 53.4 mol% of the total lipid of the lipid nanoparticle. The lipid nanoparticle can include, for example, 0.9 mol% of the conjugated lipid and at least 52.45 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.8 mol% of the conjugated lipid and at least 51.66 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.7 mol% of the conjugated lipid and at least 51.03 mol% of the ionizable lipid or thepharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.6 mol% of the conjugated lipid and at least 50.56 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.5 mol% of the conjugated lipid and at least 50.25 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.4 mol% of the conjugated lipid and at least 50.1 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.35 mol% of the conjugated lipid and at least 50.09 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.3 mol% of the conjugated lipid and at least 50.11 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.25 mol% of the conjugated lipid and at least 50.18 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.2 mol% of the conjugated lipid and at least 50.28 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.15 mol% of the conjugated lipid and at least 52.43 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The lipid nanoparticle can include, for example, 0.1 mol% of the conjugated lipid and at least 50.6 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof.

[0072] In some embodiments, the lipid nanoparticle includes one species of ionizable lipid. In other embodiments, the lipid nanoparticle includes two or more species of ionizable lipids, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of ionizable lipids.

[0073] Ionizable lipids which can be used with the provided lipid include, for example, N,N- dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), l,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl- N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)- carbamoyljcholesterol (DC-Chol), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), l,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3.beta.-oxy)-3'-oxapentoxy)-3- dimethy-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOB A), 1 ,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane(DOcarbDAP), l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- dilinoleoylcarbamyl-3 -dimethylaminopropane (DLinCDAP), 2.2-dilinoleyl-4-(2- dimethylaminoethyl)-[ 1 ,3] -dioxolane (DLin-K-C2-DMA), 2.2-dilinoleyl-4-(3 - dimethylaminopropyl) -[1,3] -dioxolane (DLin-K-C3-DMA), 2.2-dilinoleyl-4-(4- dimethylaminobutyl)-[ 1,3] -dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5- dimethylaminomethyl-[l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpiperazino- [ 1 ,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[ 1 ,3]-dioxolane (DLin-K-DMA), l,2-dilinoleylcarbamoyloxy-3 -dimethylaminopropane (DLin-C-DAP), 1,2- dilinoley oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoley oxy-3 - morpholinopropane (DLin-MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3 - dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin- TAP.C1), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N- dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and mixtures thereof.

[0074] In some examples, the provided lipid nanoparticles and vaccines including the lipid nanoparticles exhibit an ability to induce a strong immune response in part because the ionizable lipid of the lipid nanoparticles is one having three or more terminal hydrocarbon chains (e.g., terminal alkyl chains or terminal alkenyl chains) that each have five or more members. For instance, the lipid nanoparticles can include a trialkyl cationic lipid as described in International Patent Application Publication WO 2013 / 126803, an ionizable lipid as described in International Patent Application Publication WO 2017 / 049245, or an ionizable lipid as described in International Patent Application Publication WO 2017 / 075531. The ionizable lipid can include or consist of, for example, (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa- 6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (3D-P-DMA). In other examples, the ionizable lipid is one having two terminal hydrocarbon chains. The ionizable lipid can include or consistof, for example, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (MC3).3. Cholesterol

[0075] The lipid nanoparticles disclosed herein generally include a sterol, e.g., cholesterol, or one or more derivatives thereof. The sterol can be selected or designed to provide the lipid nanoparticle, or a vaccine that includes the lipid nanoparticle, with improved performance in inducing a desired immune response, such as an increase production of antibodies associated with a target antigen. Also, the amount of the sterol present in the lipid nanoparticle is shown in the present disclosure to influence the ability of the lipid nanoparticle to induce a strong immune response. For example, sterol amounts less than certain thresholds are demonstrated as beneficially resulting in lipid nanoparticles that stimulate high antibody production levels.

[0076] Further, the present disclosure reveals that not only the absolute amount of cholesterol in a lipid nanoparticle, but also the relative amount of cholesterol to conjugated lipid of the lipid nanoparticle, can enhance the immune response induced by the lipid nanoparticle. This particular relationship between the cholesterol concentration and the conjugated lipid concentration, which had not been previously recognized, can be expressed in terms of a function of these two concentrations. The function can define a threshold criterion, such that lipid nanoparticles including a molar concentration of cholesterol less than a threshold value induce stronger immune responses, where the threshold value is calculated as a function of the conjugated lipid concentration of the lipid nanoparticle. The function can be defined, for example, by the formula Z < 39.3 + 2.3X - 5.6X2, where X represents the mol% of the conjugated lipid among the total lipids of the lipid nanoparticle, and the Z calculated as a function of X is a threshold mol% of the cholesterol or derivative thereof among the total lipids of the nanoparticle.

[0077] In some examples, the conjugated lipid comprises 1 mol% of the total lipids of the lipid nanoparticle, and cholesterol or the derivative thereof comprises no more than 36 mol% of the total lipid of the lipid nanoparticle. The lipid nanoparticle can include, for example, 0.9 mol% of the conjugated lipid and no more than 36.83 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.8 mol% of the conjugated lipid and no more than 37.56 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.7 mol% of the conjugated lipid and no more than 38.17 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.6 mol%of the conjugated lipid and no more than 38.67 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.5 mol% of the conjugated lipid and no more than 39.05 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.4 mol% of the conjugated lipid and no more than 39.32 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.35 mol% of the conjugated lipid and no more than 39.42 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.3 mol% of the conjugated lipid and no more than 39.49 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.25 mol% of the conjugated lipid and no more than 39.53 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.2 mol% of the conjugated lipid and no more than 39.54 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.15 mol% of the conjugated lipid and no more than 39.52 mol% of cholesterol or the derivative thereof. The lipid nanoparticle can include, for example, 0.1 mol% of the conjugated lipid and no more than 39.47 mol% of cholesterol or the derivative thereof.

[0078] In some embodiments, the lipid nanoparticle includes one species of sterols. In other embodiments, the lipid nanoparticle includes two or more species of sterols, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of ionizable lipids. The lipid nanoparticle can include, for example, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, cholesterol hydroxyethyl ether, cholesterol hydroxyhexyl ether, cholesterol stearate, cholesterol oleate, 7-betahydroxycholesterol, 7-alphahydroxycholesterol, 4-betahydroxycholesterol, cholesterol PEG, beta sitosterol, or any combination thereof. In certain examples, the lipid nanoparticle includes cholesterol, but substantially no derivative of cholesterol.4. Phospholipid

[0079] The lipid nanoparticles disclosed herein generally include at least one phospholipid or pharmaceutically acceptable salt thereof. The phospholipid can be selected or designed to provide the lipid nanoparticle, or a vaccine that includes the lipid nanoparticle, with improved performance in inducing a desired immune response, such as an increase production of antibodies associated with a target antigen. In some embodiments, the lipid nanoparticle includes one species of phospholipid. In other embodiments, the lipid nanoparticle includestwo or more species of phospholipids, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of phospholipids.

[0080] In some examples, the provided lipid nanoparticles and vaccines including the lipid nanoparticles exhibit an ability to induce a strong immune response in part because the phospholipid of the lipid nanoparticles is one having a net neutral charge at physiological pH. Lipid nanoparticles including such neutral phospholipids are demonstrated herein as stimulating advantageously high productions of antibodies. The phospholipid can include or consist of, for example, a phosphatidylcholine, a phosphatidylethanolamine, or a combination thereof.

[0081] Phosphatidylcholine lipids suitable for use with the provided lipid nanoparticles include, for example, l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2-didecanoyl-sn- glycero-3 -phosphocholine, l,2-diundecanoyl-sn-glycero-3-phosphocholine, 1,3 -dipalmitoyl - rac-glycero-2 -phosphocholine, l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- ditridecanoyl-sn-glycero-3 -phosphocholine, l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), l,2-dipentadecanoyl-sn-glycero-3 -phosphocholine, l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-diheptadecanoyl-sn-glycero-3-phosphocholine, 1,2- dinonadecanoyl-sn-glycero-3-phosphocholine, l,2-diarachidoyl-sn-glycero-3- phosphocholine, 1,2-divaccenoyl -sn-glycero-3 -phosphocholine, l,2-di[(8Z)octadecenoyl]-sn- glycero-3 -phosphocholine, 1 ,2-dimyristoleoyl-sn-glycero-3 -phosphocholine, 1,2- dimyristelaidoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dipalmitelaidoyl-sn-glycero-3 -phosphocholine, 1,2-dipetroselenoyl-sn- glycero-3 -phosphocholine, l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dielaidoyl-sn-glycero-3 -phosphocholine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine, 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dieicosenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l-pentadecanoyl-2-oleoyl-sn-glycero-3- phosphocholine, 1 -myristoyl -2 -palmitoyl-sn-glycero-3 -phosphocholine (MPPC), 1 -myristoyl - 2-stearoyl-sn-glycero-3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, l-palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 -palmitoyl -2-oleoyl- glycero-3 -phosphocholine (POPC), l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1- palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), 1 -stearoyl -2-myristoyl-sn- glycero-3 -phosphocholine (SMPC), l-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1- stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-arachidonoyl-sn-glycero-3 -phosphocholine, 1 -oleoyl -2-myristoyl-sn-glycero-3-phosphocholine (OMPC), 1 -oleoyl -2 -palmitoyl-sn-glycero-3- phosphocholine, l-oleoyl-2-stearoyl-sn-glycero-3 -phosphocholine, l-(8Z-octadecenoyl)-2- palmitoyl-sn-glycero-3-phosphocholine, or a combination thereof.

[0082] Phosphatidylethanolamine lipids suitable for use with the provided lipid nanoparticles include, for example, l,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, l,2-didecanoyl-sn-glycero-3 -phosphoethanolamine, l,2-dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE), l,2-dipentadecanoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), . l,2-distearoyl-sn-glycero-3- phosphoethanolamine, l,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), 1,2- dipalmitoleoyl-sn-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE), 1 -pentadecanoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine, 1 -palmitoyl -2 -linoleoyl-sn-glycero-3 -phosphoethanolamine, 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 -stearoyl -2 -oleoyl-sn- glycero-3-phosphoethanolamine, l-palmitoyl-2-arachidonoyl-sn-glycero-3- phosphoethanolamine, l-pahnitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3-phosphoethanolamine, 1 -stearoyl -2 -arachidonoyl-sn- glycero-3 -phosphoethanolamine, 1 -stearoyl -2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine, or a combination thereof.

[0083] In addition to the species of phospholipid, the amount of phospholipid included in the provided lipid nanoparticle has also been shown to provide the particle with its advantageous characteristics. The phospholipid present in the provided lipid nanoparticle can comprise, for example, from about 3 mol % to about 20 mol % of the total lipid present in the particle, e.g., from about 5 mol % to about 20 mol %, from about 8 mol % to about 20 mol %, from about 10 mol % to about 20 mol %, from about 3 mol % to about 15 mol %, from about 5 mol % to about 15 mol %, from about 8 mol %to about 15 mol %, from about 10 mol %to about 15 mol %, or from about 8 mol % to about 12 mol % of the total lipid present in the particle. In some embodiments, the phospholipid present in the lipid nanoparticle comprises about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, or about 15 mol % of the total lipid present in the particle.5. Nucleic Acid

[0084] The lipid nanoparticles disclosed herein generally include a nucleic acid. The composition of the lipid nanoparticle can be selected or configured such the nucleic acids present in lipid nanoparticles are resistant in aqueous solution to degradation with a nuclease. In some embodiments, the nucleic acid is at least 50% encapsulated within the lipid nanoparticle; in one embodiment, the nucleic acid is at least 75% encapsulated within the lipid nanoparticle; in one embodiment, the nucleic acid is at least 90% encapsulated within the lipid nanoparticle; and in one embodiment, the nucleic acid is fully encapsulated within the lipid nanoparticle.

[0085] In certain embodiments, the present disclosure provides a lipid nanoparticle formulation comprising a plurality or population of lipid nanoparticles. In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid nanoparticles such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range therein) of the lipid nanoparticles in the plurality or population of lipid nanoparticles have the nucleic acid encapsulated therein.

[0086] The nucleic acid that is present in the lipid nanoparticles described herein can include or consist of any form of nucleic acid that is known. The nucleic acids used herein can be single-stranded DNA or RNA (e.g., ssDNA or ssRNA), or double -stranded DNA or RNA (e.g., dsDNA or dsRNA), or DNA-RNA hybrids. Single -stranded nucleic acids include, e.g., mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA).

[0087] Nucleic acids may be of various lengths, generally dependent upon the particular form of nucleic acid. For example, in particular embodiments, mRNA, plasmids, or genes may be from about 500 to about 100,000 nucleotides in length. In particular embodiments, oligonucleotides may range from about 10 to about 100 nucleotides in length. In various relatedembodiments, oligonucleotides, which may be single-stranded, double-stranded, or triplestranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length.

[0088] In some embodiments, the nucleic acid of a provided lipid nanoparticle comprises or consists of a modified or substituted polynucleotide or oligonucleotide. Modified or substituted polynucleotides and oligonucleotides can be preferred over native forms in some instances because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. a) Protein of Interest

[0089] The nucleic acid of the provided lipid nanoparticles generally encode a protein of interest, or a fragment thereof. The protein of interest can be, for example, any protein that can be expressed by a target cell or target organism, i.e., a cell or organism to which the lipid nanoparticles are administered. In some embodiments, the protein of interest is a protein that can be secreted by a target cell, e.g., a target cell of a target organism. In some embodiments, the protein of interest is a protein that can be displayed on the surface of a target cell.

[0090] In preferred embodiments, the protein of interest is an antigen. The presence of the antigen in the lipid nanoparticles, and the provided vaccines that include the lipid nanoparticles, allow the lipid nanoparticles to induce an immune response in subjects to which the lipid nanoparticles or vaccines are administered. For example, following administration of a provided lipid nanoparticle or vaccine to a subject, and as a result of this administration, the subject can produce antibodies associated with the antigen encoded by the nucleic acid of the lipid nanoparticles.

[0091] The nucleic acid of the provided lipid nanoparticles can encode an antigen associated with an infection. In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is derived from a pathogen. The pathogen can be a bacterial pathogen. For example, the pathogen can be Achromobacter xylosoxidans, Acinetobacter baumannii, Actinomyces such as Actinomyces israelii, Aeromonas species, Bacillus species, Bacteroides fragilis, Bacteroides melaninogenicus , Bartonella species, Bordetella pertussis, Borrelia species, Brucella species, Burkholderia species, Campylobacter, Capnocytophaga species, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter species, Clostridiumspecies, Corynebacterium species, Coxiella burnetiid, Ehrlichia species, Eikenella corrodens, Enterobacter species, Enterococcus faecalis, Enterococcus fitecium. Escherichia coli, Francisella tularensis, Fusobacterium necrophorum. Gardnerella vaginalis, Haemophilus species, Helicobacter pylori, Klebsiella species, Lactobacillus species, Legionella species, Leptospira species, Listeria monocytogenes, Moraxella catarrhalis, Morganella species, Mycoplasma pneumonia, Neisseria species, Nocardia species, Pasteurella multocida, Peptostreptococcus species. Porphyromonas gingivalis, Propionibacterium acnes, Proteus species, Providencia species, Pseudomonas aeruginosa, Salmonella species, Serratia marcescens, Shigella species, Staph epidermidis, Staph hominis, Staph, haemolyticus , Staphylococcus aureus, Staphylococcus saprophyticus , Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus pneumoniae, Streptococcus pyogenes (Groups A, B, C, G, and / or F), Treponema pallidum, Vibrio species, or a combination thereof.

[0092] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is derived from a fungal pathogen. For example the pathogen can be Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces glabrata, (Candida glabrata), Histoplasma species, a Eumycetoma causative agent, Mucorales, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Cryptococcus gattii, Lomentospora prolificans, Talaromyces marneffei, Coccidioides species, Pneumocystis jirovecii, Pichia kudriavzeveii (Candida krusei), Paracoccidioides species, or a combination thereof.

[0093] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is derived from a protozoan pathogen. For example the pathogen can be Cryptosporidium species; Toxoplasma gondii,' Plasmodium species such as Plasmodium malaria, Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale,' Trypanosoma cruzi, Trypanosoma brucei,' Leishmania species; Giardia duodenalis,' Entamoeba histolytica,' Naegleria fowleri,' Babesia microti, Balamuthia mandrillaris, Blastocystis hominis, Trichomoniasis vaginalis, Schistosoma species, or a combination thereof.

[0094] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is derived from a helminthic pathogen. For example the pathogen can be Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Strongyloides stercoralis, Ancylostoma duodenale, Hymenolepis nana, Taenia saginata, Enterobius species, Fasciolahepatica, Schistosoma mansoni. Toxocara cants, Toxocara cati, Wuchereria bancroft i. Brugia malayi. Onchocerca volvulus, Trichostrongylus species, Dracunculus medinensis, Baylisascaris species, Echinococcus species, Taenia multiceps, Taenia serialis, Taenia glomerata, Taenia brauna, species, Clonorchis sinensis, Fasciolopsis buski, Opisthorchis species, Paragonimus species, Schistosoma species, Moniliformis species, or a combination thereof.

[0095] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is derived from a viral pathogen. For example the antigen can be from a coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), influenza virus (e.g., influenza A, B, and C viruses), filovirus (e.g., Ebola virus, Marburg virus), arenavirus (e.g., Lassa virus, Junin virus, Machupo virus, Guanarito virus, Sabia virus), Zika virus, rabies virus, rhinovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), hepatitis viruses (e.g., hepatitis C virus), herpes simplex virus, human papilloma virus (HPV), or Epstein-Barr virus. In particular embodiments, the infectious disease antigen is a SARS-CoV-2 protein selected from the group consisting of S (spike) protein, E (envelope) protein, M (membrane) protein, N (nucleocapsid) protein, and an antigenic fragment thereof. In other embodiments, the infectious disease antigen is a hemagglutinin (HA) protein of a virus of the Paramyxoviridae or Orthomyxoviridae families, e.g., an influenza virus. Example 2, Example 4, and Example 5 demonstrate use of provided lipid nanoparticles to encapsulate a nucleic acid, i.e., mRNA, encoding hemagglutinin.

[0096] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is derived from a pathogenic toxin. The antigen can be associated with any non- enzymatic product of any pathogen (e.g., any of the bacterial, fungal, protozoan, helminthic, or viral pathogens described above), where the pathogen product is harmful to the subject infected with the pathogen. In some examples, the antigen is derived from a pathogenic adhesion element, i.e., any product of any pathogen (e.g., any of the bacterial, fungal, protozoan, helminthic, or viral pathogens described above), where the pathogen product participates in adhesion of the pathogen to cells of the subject infected with the pathogen. b) mRNA

[0097] The nucleic acid encoding the protein of interest of the provided lipid nanoparticles can include or consist of an RNA molecule that encodes the protein of interest or a fragment thereof. In some examples, the nucleic acid of the provided lipid nanoparticles includes orconsists of one or more messenger RNA (mRNA) molecules, e.g., a cocktail of mRNA molecules. The mRNA molecules can be selected or designed for transfer to a cell within a subject (e.g., a mammal, such as a human) to which a provided lipid nanoparticle or vaccine is administered. The mRNA molecules can encode one or more antigens that is / are expressed within the cell to elicit immunity against a disease. The compositions and methods described herein are therefore useful, for example, in preventing an infectious disease caused by a pathogen such as a virus (e.g., a coronavirus such as SARS-CoV-2) by expressing antigenic polypeptides (e.g., from mRNA molecules encoding viral proteins such as S (spike), E (envelope), M (membrane), or N (nucleocapsid) proteins or antigenic fragments thereof) to produce an immune response within an organism (e.g., a mammal, such as a human) by stimulating the adaptive immune system to create antibodies that target the pathogen.

[0098] In some embodiments, the mRNA molecules are fully encapsulated in lipid particles. In some examples, the RNA of a provided lipid nanoparticle or a provided population of nanoparticles includes a cocktail of one or more types of mRNA together with one or more other types of RNA, e.g., gRNA, siRNA, miRNA, etc. With respect to formulations comprising an mRNA cocktail, the different types of mRNA species present in the cocktail (e.g., mRNA having different sequences) may be co-encapsulated in the same particle, or each type of mRNA species present in the cocktail may be encapsulated in a separate particle. The mRNA cocktail may be formulated in the particles described herein using a mixture of two or more individual mRNAs (each having a unique sequence) at identical, similar, or different concentrations or molar ratios. In one embodiment, a cocktail of mRNAs (corresponding to a plurality of mRNAs with different sequences) is formulated using identical, similar, or different concentrations or molar ratios of each mRNA species, and the different types of mRNAs are co-encapsulated in the same particle. In another embodiment, each type of mRNA species present in the cocktail is encapsulated in different particles at identical, similar, or different mRNA concentrations or molar ratios, and the particles thus formed (each containing a different mRNA payload) are administered separately (e.g., at different times in accordance with a prophylactic or therapeutic regimen), or are combined and administered together as a single unit dose (e.g., with a pharmaceutically acceptable carrier). In particular embodiments, the lipid particles are serum-stable, are resistant to nuclease degradation, and are substantially non-toxic to mammals such as humans.(1) Modifications to mRNA

[0099] The mRNA molecules present in the provided lipid nanoparticles can include one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in a cell into which the mRNA is introduced, relative to a corresponding unmodified mRNA.

[0100] In some embodiments, modified nucleosides include or consist of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5 -hydroxyuridine, 3 -methyluridine, 5 -carboxymethyl -uridine, 1- carboxymethyl-pseudouridine, 5 -propynyl -uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl -pseudouridine, 5 -taurinomethyl -2 -thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl -uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, or any combination thereof.

[0101] In some embodiments, modified nucleosides include or consist of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5 -formylcytidine, N4- methylcytidine, 5 -hydroxymethylcytidine, 1 -methyl -pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5 -methyl -cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5 -methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl - pseudoisocytidine, or any combination thereof.

[0102] In other embodiments, modified nucleosides include or consist of 2-aminopurine, 2,6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2 -aminopurine, 7-deaza-8- aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cA- hydroxyisopentenyl)adenosine, 2-mcthylthio-N6-(c / .s-hydroxyisopcntcnyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2 -methoxy-adenine .

[0103] In certain embodiments, the modified nucleosides include or consist of 5'-O-(l- thiophosphate)-adenosine, 5 '-O-( 1 -thiophosphate)-cytidine, 5 '-O-( 1 -thiophosphate)- guanosine, 5'-O-(l-thiophosphate)-uridine, 5'-O-(l-thiophosphate)-pseudouridine, or a combination thereof. The a-thio substituted phosphate moiety is provided to confer stability to RNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate RNA polymers have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate-linked nucleic acids are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0104] In certain embodiments, it is desirable to intracellularly degrade a modified nucleic acid introduced into the cell, for example, if precise timing of protein production is desired. Thus, the present disclosure provides a modified nucleic acid containing a degradation domain, which is capable of being acted on in a directed manner within a cell.

[0105] In other embodiments, modified nucleosides include or consist of inosine, 1 -methylinosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7- methyl -guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, or a combination thereof.(2) Optional mRNA Components

[0106] In further embodiments, the mRNA molecules present in the provided lipid nanoparticles may include other optional components. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intronic nucleotide sequences, internal ribosome entry site (IRES), 5' caps, and poly-A tails. For example, a 5' untranslated region (UTR) and / or a 3' UTR may be included, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Also provided are mRNA molecules containing a Kozak sequence. Additionally, provided herein are mRNA molecules containing one or more intronic nucleotide sequences capable of being excised from the mRNA sequence.(a) Untranslated Regions (UTRs)

[0107] Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is a growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the mRNA molecule and translation. The regulatory features of a UTR can be incorporated into the mRNA used in the lipid particles described herein to increase the stability of the molecule. The specific features can also be incorporated to ensure controlled downregulation of the transcript in case they are misdirected to undesired tissue or organ sites.(b) 5' Capping

[0108] The 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability, and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns during mRNA splicing.

[0109] The mRNA molecules may be 5 '-end capped, generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. This 5 '-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anti-terminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O-methylated. 5'- decapping through hydrolysis and cleavage of the guanylate cap structure may target an mRNA molecule for degradation.(c) IRES Sequences

[0110] mRNA containing an internal ribosome entry site (IRES) are also useful in the lipid nanoparticles described herein. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic mRNA”). When mRNA are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences include, without limitation, those from picomaviruses (e.g., FMDV), pest viruses (e.g., CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immune deficiency viruses (S1V), and cricket paralysis viruses (CrPV).(d) Poly-A Tails

[0111] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3 ' end of the transcript may be cleaved to free a 3 ' hydroxyl . Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between 100 and 250 residues long.

[0112] Generally, the length of a poly-A tail is greater than 30 nucleotides in length. In some embodiments, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater in length than the mRNA. In other embodiments, the poly-A tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the total length of the mRNA.(3) Generation of mRNA Molecules

[0113] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., Molecular Cloning, A Laboratory Manual (2nded. 1989)) as are PCR methods (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those of skill in the art. Additional basic texts disclosing the general methods of use in the present disclosure include Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). In some embodiments, mRNA molecules are generated by in vitro transcription, followed by purification. Standard techniques for purifying mRNA, e.g., mRNA produced via in vitro transcription, are well known in the art and include, for example, oligo(dT) affinity purification, cellulose based purification, and other chromatography based methods Examples of available mRNA purification techniques are provided by Zhang, J. et al. Pharmaceutics, 15:2182 (2023).

[0114] In some embodiments, the provided lipid nanoparticle contains mRNA that is synthesized from a DNA template. Processes for synthesizing mRNA from a DNA template are well-known, and generally involve the use of enzymes (e.g., T7 RNA polymerase and / or derivatives thereof, such as optimized alternative enzymes), nucleotides (optionally including modified nucleotides as described herein), and optional mRNA components (e.g., 5' caps). In some embodiments, the lipid nanoparticle contains mRNA that is generated via in vitro transcription using a commercially available kit. Commercially available kits suitable for generating mRNA contained by the provided lipid nanoparticles include, for example, the MEGASCRIPT™ T7 Transcription Kit available from Thermo Fisher Scientific. Alternatively or additionally, mRNA for use with the provided lipid nanoparticles can be generated using chemical synthesis processes, such as, for example, those described by Abe et al., ACS Chem. Biol. 2022, 17, 6, 1308-1314. In further embodiments, the lipid nanoparticles include mRNA generated with mRNA printers, for example, any of those reviewed by Sheridan, 2022 Nature Biotechnol. 2022, 40, 1160-1162.

[0115] In some embodiments, the mRNA generated by any one or more of the methods described herein is purified before being used to form one or more provided lipid nanoparticles. mRNA purification techniques suitable for use with the mRNA of the provided lipid nanoparticles include, but are not limited to, HPLC purification, cellulose purification, tangential flow filtration, bead based purification (e.g., with purification with Dynabeads), silica based purification, precipitation, and combinations thereof. Non-limiting examples of mRNA purification are described in, for example, International Patent Application Publication No. WO 2018 / 006052 Al. c) circRNA

[0116] In some examples, the nucleic acid of the provided lipid nanoparticles includes or consists of one or more circular RNA (circRNA) molecules, e.g., a cocktail of circRNA molecules. The circRNA molecules can be selected or designed for transfer to a cell within a subject (e.g., a mammal, such as a human) to which a provided lipid nanoparticle or vaccine is administered. The circRNA molecules can encode one or more antigens that is / are expressed within the cell to elicit immunity against a disease. The compositions and methods described herein are therefore useful, for example, in preventing an infectious disease caused by a pathogen such as a virus (e.g., a coronavirus such as SARS-CoV-2) by expressing antigenic polypeptides (e.g., from circRNA molecules encoding viral proteins such as S (spike), E(envelope), M (membrane), or N (nucleocapsid) proteins or antigenic fragments thereof) to produce an immune response within an organism (e.g., a mammal, such as a human) by stimulating the adaptive immune system to create antibodies that target the pathogen.

[0117] In some embodiments, the circRNA molecules are fully encapsulated in lipid particles. In some examples, the RNA of a provided lipid nanoparticle or a provided population of nanoparticles includes a cocktail of one or more types of circRNA together with one or more other types of RNA, e.g., gRNA, siRNA, miRNA, etc. With respect to formulations comprising a circRNA cocktail, the different types of circRNA species present in the cocktail (e.g., circRNA having different sequences) may be co-encapsulated in the same particle, or each type of circRNA species present in the cocktail may be encapsulated in a separate particle. The circRNA cocktail may be formulated in the particles described herein using a mixture of two or more individual circRNAs (each having a unique sequence) at identical, similar, or different concentrations or molar ratios. In one embodiment, a cocktail of circRNAs (corresponding to a plurality of circRNAs with different sequences) is formulated using identical, similar, or different concentrations or molar ratios of each circRNA species, and the different types of circRNAs are co-encapsulated in the same particle. In another embodiment, each type of circRNA species present in the cocktail is encapsulated in different particles at identical, similar, or different circRNA concentrations or molar ratios, and the particles thus formed (each containing a different circRNA payload) are administered separately (e.g., at different times in accordance with a prophylactic or therapeutic regimen), or are combined and administered together as a single unit dose (e.g., with a pharmaceutically acceptable carrier). In particular embodiments, the lipid particles are serum-stable, are resistant to nuclease degradation, and are substantially non-toxic to mammals such as humans.

[0118] In further embodiments, the circRNA molecules present in the provided lipid nanoparticles may include other optional components. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intronic nucleotide sequences, internal ribosome entry site (IRES), in-frame 2A peptide coding sequences, and m6A modification motif sequences. For example, a 5' untranslated region (UTR) and / or a 3' UTR may be included, wherein either or both may independently contain one or more different nucleoside modifications, e.g., any of the nucleoside modifications described in Sections C.5.b and C.5.b(l). In such embodiments, nucleoside modifications may also be present in the translatable region. Also provided are circRNA molecules containing a Kozak sequence.Additionally, provided herein are circRNA molecules containing one or more intronic nucleotide sequences capable of being excised from the circRNA sequence.

[0119] The production of circRNAs can be performed using various methods provided in the art. For example, U.S. Patent No. 6,210,931 teaches a method of synthesizing circRNAs by inserting DNA fragments into a plasmid containing sequences having the capability of spontaneous cleavage and self-circularization. U.S. Patent No. 5,773,244 teaches producing circRNAs by making a DNA construct encoding an RNA cyclase ribozyme, expressing the DNA construct as RNA, and then allowing the RNA to self-splice, which produces a circRNA free from intron in vitro. International Patent Application Publication WO 1992 / 001813 teaches a process of making single strand circular nucleic acids by synthesizing a linear polynucleotide, combining the linear nucleotide with a complementary linking oligonucleotide under hybridization conditions, and ligating the linear polynucleotide. Other methods for producing circRNA are provided in, for example, International Patent Application Publications WO 2015 / 034925 and WO 2016 / 011222. d) saRNA

[0120] In some examples, the nucleic acid of the provided lipid nanoparticles includes or consists of one or more self-amplifying RNA (saRNA) molecules, e.g., a cocktail of saRNA molecules. The saRNA molecules can be selected or designed for transfer to a cell within a subject (e.g., a mammal, such as a human) to which a provided lipid nanoparticle or vaccine is administered. The saRNA molecules can encode one or more antigens that is / are expressed within the cell to elicit immunity against a disease. The compositions and methods described herein are therefore useful, for example, in preventing an infectious disease caused by a pathogen such as a virus (e.g., a coronavirus such as SARS-CoV-2) by expressing antigenic polypeptides (e.g., from saRNA molecules encoding viral proteins such as S (spike), E (envelope), M (membrane), or N (nucleocapsid) proteins or antigenic fragments thereof) to produce an immune response within an organism (e.g., a mammal, such as a human) by stimulating the adaptive immune system to create antibodies that target the pathogen.

[0121] In some embodiments, the saRNA molecules are fully encapsulated in lipid particles. In some examples, the RNA of a provided lipid nanoparticle or a provided population of nanoparticles includes a cocktail of one or more types of saRNA together with one or more other types of RNA, e.g., gRNA, siRNA, miRNA, etc. With respect to formulations comprising a saRNA cocktail, the different types of saRNA species present in the cocktail (e.g., saRNAhaving different sequences) may be co-encapsulated in the same particle, or each type of saRNA species present in the cocktail may be encapsulated in a separate particle. The saRNA cocktail may be formulated in the particles described herein using a mixture of two or more individual saRNAs (each having a unique sequence) at identical, similar, or different concentrations or molar ratios. In one embodiment, a cocktail of saRNAs (corresponding to a plurality of saRNAs with different sequences) is formulated using identical, similar, or different concentrations or molar ratios of each saRNA species, and the different types of saRNAs are co-encapsulated in the same particle. In another embodiment, each type of saRNA species present in the cocktail is encapsulated in different particles at identical, similar, or different saRNA concentrations or molar ratios, and the particles thus formed (each containing a different saRNA payload) are administered separately (e.g., at different times in accordance with a prophylactic or therapeutic regimen), or are combined and administered together as a single unit dose (e.g., with a pharmaceutically acceptable carrier). In particular embodiments, the lipid particles are serum-stable, are resistant to nuclease degradation, and are substantially non-toxic to mammals such as humans.

[0122] In further embodiments, the saRNA molecules present in the provided lipid nanoparticles may include other optional components. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intronic nucleotide sequences, internal ribosome entry site (IRES), caps, and poly-A tails. For example, a 5' untranslated region (UTR) and / or a 3' UTR may be included, wherein either or both may independently contain one or more different nucleoside modifications, e.g., any of the nucleoside modifications described in Sections C.5.b and C.5.b(l). In such embodiments, nucleoside modifications may also be present in the translatable region. Also provided are saRNA molecules containing a Kozak sequence. Additionally, provided herein are saRNA molecules containing one or more intronic nucleotide sequences capable of being excised from the saRNA sequence.D. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0123] In another aspect, the disclosure provides a pharmaceutical composition that includes one or more of any of the lipid nanoparticles or vaccines described herein in Section C. In some embodiments, the pharmaceutical composition further includes a therapeutically effective amount of a pharmaceutically acceptable excipient. Additionally or alternatively, the pharmaceutical composition can include a pharmaceutically acceptable carrier.

[0124] The pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) can be selected in accordance with the route of administration and standard pharmaceutical practice. Generally, normal buffered saline (e.g., 135-150 mM NaCl) is used as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Additional suitable carriers are described in, e.g., Remington: The Science and Practice of Pharmacy, 23rd Edition (2020). The pharmaceutically acceptable carrier is generally added following particle formation. Thus, after the particle is formed, the particle can be diluted into pharmaceutically acceptable carriers such as normal buffered saline.

[0125] Examples of suitable excipients for use with the provided pharmaceutical compositions include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.

[0126] The provided pharmaceutical compositions can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; coloring agents; and / or flavoring agents. The compositions can also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0127] Pharmaceutical compositions that are liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution (preferably physiological saline), Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates or phosphates; detergents, such as nonionic surfactants, polyols; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The liquid compositions can include aqueous or oilsuspensions, or emulsions, with sesame oil, com oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0128] The provided pharmaceutical compositions for administration are preferably sterile. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and thimerosal. Compositions for administration can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic, or other material.

[0129] For oral administration, the provided pharmaceutical compositions can be in the form of tablets, lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays, powders, and / or sustained-release formulations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

[0130] For topical administration, the provided pharmaceutical compositions can be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments, and / or transdermal patches. For delivery by inhalation, the composition can be delivered as a dry powder or in liquid form via a nebulizer. For parenteral administration, the compositions can be in the form of sterile injectable solutions and / or sterile packaged powders. Preferably, injectable solutions are formulated at a pH of about 4.5 to about 7.5.

[0131] The concentration of lipid nanoparticles in the provided pharmaceutical formulations can vary widely, e.g., from less than about 0.05%, usually at or at least about 2 to about 5%, to as much as about 10 to about 90% by weight, and can be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, lipid nanoparticles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.

[0132] The provided pharmaceutical compositions can be sterilized by conventional, well- known sterilization techniques. Aqueous solutions can be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and / or calcium chloride. Additionally, the lipid nanoparticle suspension can include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free- radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.E. KITS

[0133] In another aspect, the disclosure provides kits to facilitate and / or standardize the use of the materials described herein (i.e., the lipid nanoparticles and vaccines described in Section C and / or the pharmaceutical compositions described in Section D), as well as to facilitate the methods described herein (i.e., the methods described in Section F). Materials and reagents to carry out these various methods can be provided in kits to facilitate execution of the methods.

[0134] The provided kits can contain chemical reagents as well as other components. In addition, the kits containing the provided lipid nanoparticles, ionizable lipids, and / or pharmaceutical compositions can include, without limitation, instructions to the kit user. Kits can also be packaged for convenient storage and safe shipping, for example, as ampules or other vials packaged in a box having a lid.

[0135] The provided kits may comprise one or more containers, each of which is compartmentalized for holding the various elements of the lipid nanoparticles (e.g., the nucleic acids and the individual lipid components of the nanoparticles). In some embodiments, the kit may further comprise an endosomal membrane destabilizer (e.g., calcium ions). The kit typically contains the lipid nanoparticle compositions of the present disclosure, preferably in dehydrated form, with instructions for their rehydration and administration.F. METHODS

[0136] In other aspects, the disclosure provides several methods for making and / or using the lipid nanoparticles and vaccines described in Section C, the pharmaceutical compositionsdescribed in Section D, and / or the kits described in Section E. The methods disclosed herein benefit from the improved properties of these provided materials, e.g., properties advantageous for inducing an immune response in a subject. These improved properties include, for example, stimulation of antibody production in the subject.1. Inducing an Immune Response

[0137] Some methods provided by the disclosure are useful for inducing an immune response in a subject. These methods generally include administering one or more lipid nanoparticles, one or more vaccines, or one or more pharmaceutical compositions to the subject, wherein the lipid nanoparticles and vaccines include those described in Section C and the pharmaceutical compositions include those described in Section D. In some embodiments, the inducing of the immune response includes increasing production of antibodies in the subject. The provided methods can be used to induce an immune response against any of the pathogens described in Section C.5.a.

[0138] The disclosed lipid nanoparticles, vaccines, and pharmaceutical compositions can be administered using the provided method as a single dose or as multiple doses, for example, two doses administered at an interval of about one week, about two weeks, about three weeks, about one month, about two months, about three months, about six months, or about 12 months. Other suitable dosage schedules can be determined by a medical practitioner. In some embodiments, additional compounds or medications can be co-administered to the subject. Such compounds or medications can be co-administered to, for example, alleviate signs or symptoms of a disease caused by a pathogenic infection being treated, or to reduce side effects caused by induction of the immune response.

[0139] The administration of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions can be carried out via any of the generally accepted modes of administration. Thus, administration can be, for example, intramuscular, intradermal, intravenous, topical, subcutaneous, transcutaneous, transdermal, oral, intra-joint, parenteral, intra-arteriole, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation. In preferred embodiments , the lipid nanoparticle, vaccine, and / or pharmaceutical composition is administered intramuscularly or intradermally. In particularly preferred embodiments, the lipid nanoparticle, vaccine, and / or pharmaceutical composition is administered intramuscularly.

[0140] In some embodiments, the immune response is assessed by detecting antibodies obtained from the subject, for example using an antigen binding assay such as an enzyme- linked immunosorbent assay (ELISA). In some embodiments, the immune response includes generation of antibodies that recognize an antigen, where the antigen, or a fragment thereof, is encoded by the nucleic acid of the lipid nanoparticle or vaccine. The antigen can be any of those described herein, e.g., any of the antigens described in Section C.5.a.

[0141] In some embodiments, the immune response induced by the provided method is greater than an immune response induced using a corresponding composition that does include a lipid nanoparticle having the formulation described in Section C.2. Preventing or Treating a Disease or Disorder

[0142] Some methods provided by the disclosure are useful for preventing or treating a disease or a disorder of a subject. These methods generally include administering one or more lipid nanoparticles, one or more vaccines, or one or more pharmaceutical compositions to the subject, wherein the lipid nanoparticles and vaccines include those described in Section C and the pharmaceutical compositions include those described in Section D. In some embodiments, the treating of the disease in the subject includes decreasing or eliminating one or more signs or symptoms of the disease.

[0143] In general, the provided methods are useful for preventing or treating an infection or infectious disease caused by a pathogen, e.g., a virus, bacterium, fungus, parasite, or any other infectious agent. Non-limiting examples of infectious diseases suitable for treatment using the provided methods include acquired immunodeficiency syndrome (AIDS / HIV) or HIV-related disorders, Alpers syndrome, anthrax, bovine spongiform encephalopathy (mad cow disease), chicken pox, cholera, conjunctivitis, Creutzfeldt-Jakob disease (CJD), dengue fever, Ebola, elephantiasis, encephalitis, fatal familial insomnia, Fifth’s disease, Gerstmann-Straussler- Scheinker syndrome, hantavirus, helicobacter pylori, hepatitis (hepatitis A, hepatitis B, hepatitis C), herpes, influenza (e.g., avian influenza A (bird flu)), Kuru, leprosy, Lyme disease, malaria, hemorrhagic fever (e.g., Rift Valley fever, Crimean-Congo hemorrhagic fever, Lassa fever, Marburg virus disease, and Ebola hemorrhagic fever), measles, meningitis (viral, bacterial), mononucleosis, nosocomial infections, otitis media, pelvic inflammatory disease (PID), plague, pneumonia, polio, prion disease, rabies, rheumatic fever, roseola, Ross River virus infection, rubella, salmonellosis, septic arthritis, sexually transmitted diseases (STDs), shingles, smallpox, strep throat, tetanus, toxic shock syndrome, toxoplasmosis, trachoma,tuberculosis, tularemia, typhoid fever, valley fever, whooping cough, and yellow fever. The provided methods can be used to prevent or treat infections by any of the pathogens described in Section C.5.a.

[0144] In some embodiments, the provided method further includes obtaining a test sample from the subject. The test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof. In some embodiments, the provided method further includes determining the level of one or more biomarkers in the obtained test sample. Determining the presence or level of biomarkers(s) can be used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease.

[0145] In examples where the provided lipid nanoparticle, vaccine, or pharmaceutical composition includes a nucleic acid encoding a protein of interest or a fragment thereof, the measured biomarker can be an indicator of an expression level of the protein or fragment by a target cell or target organism, e.g., the cell or organism to which the lipid nanoparticle, vaccine, or pharmaceutical composition is administered. For example, the biomarker can be the protein or fragment itself, such that a measured level of the protein or fragment indicates the level of its expression. When the protein of interest is an antigen, the measured biomarker can include or consist of one or more components of the immune system of the target cell or target organism. For example, the biomarker can include or consist of one or more antibodies or other immunoglobulins. In these ways, the determination of a level of a biomarker in a test sample can provide information related to the efficiency and / or effectiveness of the administration of the lipid nanoparticle in preventing or treating a disease or disorder. Example 3 demonstrates quantification of IgG antibodies as a biomarker following administration of lipid nanoparticles encapsulating nucleic acid (i.e., mRNA) encoding an antigen (i.e., hemagglutinin).

[0146] In some embodiments, the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample. The reference sample can be obtained, for example, from the subject, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided materials. In this way, the reference sample can provide information about baseline levels of the biomarkers in the sample before the treatment, and the test sample can provide information about levels of the biomarkers after the treatment.

[0147] Alternatively, the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the treatment is not provided according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment. The reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the treatment is not provided according to the provided method. In this way, the reference sample can provide population-averaged information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.

[0148] The reference sample can also be obtained from an individual or a population of individuals after treatment is provided according to the provided methods, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.

[0149] Depending on the biomarker, an increase or decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease. In some embodiments, an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., an infectious disease or cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1.1 -fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11- fold, at least 12-fold, at least 13 -fold, at least 14-fold, at least 15 -fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold higher in comparison to a control. In other embodiments, a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower in comparison to a control.

[0150] The biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers. Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads. In some instances, nucleic acid sequencing is employed.

[0151] In certain embodiments, the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, e.g., quantitative PCR. This detectable signal can be compared to the signal from a reference sample or to a threshold value.

[0152] In some embodiments, the results of the biomarker level determinations are recorded in a tangible medium. For example, the results of diagnostic assays, e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., an infectious disease or cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive.

[0153] In some embodiments, the provided method further includes the step of providing to the subject a diagnosis and / or the results of treatment.3. Preparing Lipid Nanoparticles

[0154] Some methods provided by the disclosure are useful for preparing one or more lipid nanoparticles, wherein the lipid nanoparticles include those described in Section C. The lipid nanoparticles can be prepared via, for example, a continuous mixing method and / or a direct dilution process. In some embodiments, the methods include selecting, designing, and / or preparing one or more components of the lipid nanoparticle being prepared. For example, the methods can include selecting, designing, and / or preparing a phospholipid, an ionizable lipid, a cholesterol or derivative thereof, a conjugated lipid, and / or a nucleic acid.

[0155] In some embodiments, the lipid nanoparticles described herein are produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid (e.g., any of the nucleic acids described in Section C.5) in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with theaqueous solution so as to substantially instantaneously produce a lipid particle encapsulating the nucleic acid. Examples of this process and the apparatus for carrying out this process are described in U.S. Patent Application Publication No. 2004 / 0142025.

[0156] In some examples, the organic lipid solution is an alcoholic solution that includes an alcoholic organic solvent and the lipids that will form the lipid nanoparticles. These lipids can include one or more conjugated lipids, one or more ionizable lipids or pharmaceutically acceptable salts thereof, one or more sterols or derivatives thereof, and one or more phospholipids or pharmaceutically acceptable salts thereof. For example, the conjugated lipid can be any of those described in Section C.l, the ionizable lipid can be any of those described in Section C.2, the sterol can be cholesterol or any of its derivatives described in Section C.3, and the phospholipid can be any of those described in Section C.4. In some embodiments, the alcoholic organic solvent of the alcoholic solution includes or consists of ethanol. In some examples, the alcoholic solution is the product of combining the alcoholic organic solvent and a phospholipid solution, where the phospholipid solution includes or consists of a nonalcoholic organic solvent and the one or more phospholipids or pharmaceutically acceptable salts thereof. In some embodiments, the nonalcoholic organic solvent includes or consists of tetrahydrofuran.

[0157] The action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a lipid particle substantially instantaneously upon mixing. As used herein, the phrase “continuously diluting a lipid solution with a buffer solution” (and variations) generally means that the lipid solution is diluted sufficiently rapidly in a hydration process with sufficient force to effectuate vesicle generation. By mixing the aqueous solution comprising a nucleic acid with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a lipid particle.

[0158] The lipid nanoparticles formed using the continuous mixing method typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 60 to about 90 nm, or from about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0159] In some embodiments, the lipid nanoparticles described herein are produced via a direct dilution process that includes forming a lipid particle solution and immediately and directly introducing the lipid particle solution into a collection vessel containing a controlled amount of dilution buffer. In certain embodiments, the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In one embodiment, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid particle solution introduced thereto. As a non-limiting example, a lipid particle solution in 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.

[0160] In some embodiments, the lipid nanoparticles described herein are produced via a direct dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region. In this embodiment, the lipid particle solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region. In certain embodiments, the second mixing region includes a T-connector arranged so that the lipid particle solution and the dilution buffer flows meet as opposing 1800flows; however, connectors providing shallower angles can be used, e.g., from about 270to about 180 °. A pump mechanism delivers a controllable flow of buffer to the second mixing region. In one embodiment, the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of lipid particle solution introduced thereto from the first mixing region. This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid particle solution in the second mixing region, and therefore also the concentration of lipid particle solution in buffer throughout the second mixing process. Such control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations. Examples of these processes and the apparatuses for carrying out these direct dilution processes are described in U.S. Patent No. 9,005,654.

[0161] The lipid particles formed using the direct dilution process typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 60 to about 90 nm, or from about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0162] If needed, the lipid particles described herein can be sized by any of the methods available to one of skill in the art. The sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes. Several techniques are available for sizing the particles to a desired size, e.g. such as described in Lam et al. 2023, Adv. Mat., 35:26, 2211420. One sizing method, used for liposomes and equally applicable to the present particles, is described in U.S. Patent No. 4,737,323. Sonicating a particle suspension either by bath or probe sonication produces a progressive size reduction down to particles of less than about 50 nm in size. Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones. In a typical homogenization procedure, particles are recirculated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 nm and about 80 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.

[0163] Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.

[0164] In some embodiments, the nucleic acid to lipid ratios (mass / mass ratios) in a formed lipid particle ranges from about 0.01 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0. 1, or from about 0.01 to about 0.08. The ratio of the starting materials can also fall within this range. In other embodiments, the lipid particle preparation uses about 400 pg nucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratio of about 0.01 to about 0.08, e.g., about 0.04, which corresponds to 1.25 mg of total lipid per 50 pg of nucleic acid. In certain embodiments, the particle has a nucleic acid:lipid mass ratio of about 0.08.

[0165] In other embodiments, the lipid to nucleic acid ratios (mass / mass ratios) in a formed lipid particle ranges from about 1 (1: 1) to about 100 (100: 1), from about 5 (5: 1) to about 100 (100: 1), from about 1 (1 : 1) to about 50 (50: 1), from about 2 (2: 1) to about 50 (50: 1), from about 3 (3: 1) to about 50 (50: 1), from about 4 (4: 1) to about 50 (50: 1), from about 5 (5: 1) to about 50 (50: 1), from about 1 (1: 1) to about 25 (25: 1), from about 2 (2: 1) to about 25 (25: 1), from about 3 (3: 1) to about 25 (25: 1), from about 4 (4: 1) to about 25 (25: 1), from about 5 (5: 1) to about 25(25: 1), from about 5 (5: 1) to about 20 (20: 1), from about 5 (5: 1) to about 15 (15: 1), from about 5 (5: 1) to about 10 (10: 1), about 5 (5: 1), 6 (6: 1), 7 (7: 1), 8 (8: 1), 9 (9: 1), 10 (10: 1), 11 (11: 1), 12 (12: 1), 13 (13: 1), 14 (14: 1), or 15 (15: 1). The ratio of the starting materials can also fall within this range.G. EXEMPLARY EMBODIMENTS

[0166] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0167] Embodiment 1: A lipid nanoparticle comprising: a conjugated lipid, the conjugated lipid comprising no more than X mol% of the total lipid of the lipid nanoparticle, X having a value that is no more than 1 ; a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net neutral charge at physiological pH; an ionizable lipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; and a nucleic acid encoding a protein of interest or a fragment thereof; wherein the lipid nanoparticle has a composition satisfying at least one of the following conditions: (a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the lipid nanoparticle, Y having a value defined by the formula: 51.1 - 5.7X + 8.0X2; and (b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the nanoparticle, Z having a value defined by the formula: 39.3 + 2.3X - 5.6X2.

[0168] Embodiment 2: An embodiment of embodiment 1, wherein: (a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the nanoparticle; and (b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the nanoparticle.

[0169] Embodiment 3 : An embodiment of embodiment 1 or 2, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

[0170] Embodiment 4: An embodiment of embodiment 3, wherein the ionizable lipid comprises (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (3D-P-DMA).

[0171] Embodiment 5 : An embodiment of embodiment 1 or 2, wherein the ionizable lipid comprises (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (MC3).

[0172] Embodiment 6: An embodiment of any one of embodiments 1-5, wherein X has a value that is at least 0.5.

[0173] Embodiment 7: An embodiment of any one of embodiments 1-6, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains that each have five or more members and that each are not directly bonded to an ester.

[0174] Embodiment 8: An embodiment of any one of embodiments 1-7, wherein the conjugated lipid comprises a carbamate linkage.

[0175] Embodiment 9: An embodiment of any one of embodiments 1-8, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

[0176] Embodiment 10: An embodiment of any one of embodiments 1-9, wherein the conjugated lipid comprises N- [(methoxypoly (ethylene glycol))carbamoyl]-l,2-dimyristyloxy- propylamine (PEG-c-DMA).

[0177] Embodiment 11: An embodiment of any one of embodiments 1-10, wherein the phospholipid comprises a phosphatidylcholine.

[0178] Embodiment 12: An embodiment of embodiment 11, wherein the phosphatidylcholine comprises l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1,2- didecanoyl-sn-glycero-3-phosphocholine, l,2-diundecanoyl-sn-glycero-3 -phosphocholine, 1, 3 -dipalmitoyl-rac-glycero-2 -phosphocholine, l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1 ,2-ditridecanoyl-sn-glycero-3 -phosphocholine, 1 ,2-dimyristoyl-sn-glycero-3 - phosphocholine (DMPC), l,2-dipentadecanoyl-sn-glycero-3 -phosphocholine, 1,2-dipalmitoyl- sn-glycero-3 -phosphocholine (DPPC), 1 ,2-diheptadecanoyl-sn-glycero-3 -phosphocholine,1.2-dinonadecanoyl-sn-glycero-3 -phosphocholine, l,2-diarachidoyl-sn-glycero-3- phosphocholine, 1,2-divaccenoyl -sn-glycero-3 -phosphocholine, l,2-di[(8Z)octadecenoyl]-sn- glycero-3 -phosphocholine, 1 ,2-dimyristoleoyl-sn-glycero-3 -phosphocholine, 1,2- dimyristelaidoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dipalmitelaidoyl-sn-glycero-3 -phosphocholine, 1,2-dipetroselenoyl-sn- glycero-3 -phosphocholine, l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dielaidoyl-sn-glycero-3 -phosphocholine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine, 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dieicosenoyl-sn-glycero-3 -phosphocholine,1.2-diarachidonoyl-sn-glycero-3 -phosphocholine, l-pentadecanoyl-2-oleoyl-sn-glycero-3- phosphocholine, 1 -myristoyl -2 -palmitoyl-sn-glycero-3 -phosphocholine (MPPC), 1 -myristoyl -2-stearoyl-sn-glycero-3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, l-palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 -palmitoyl -2-oleoyl- glycero-3 -phosphocholine (POPC), l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1- palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), 1 -stearoyl -2-myristoyl-sn- glycero-3 -phosphocholine (SMPC), l-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1- stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1 -oleoyl -2- myristoyl-sn-glycero-3-phosphocholine (OMPC), 1 -oleoyl -2 -palmitoyl-sn-glycero-3- phosphocholine, l-oleoyl-2-stearoyl-sn-glycero-3 -phosphocholine, l-(8Z-octadecenoyl)-2- palmitoyl-sn-glycero-3-phosphocholine, or a combination thereof.

[0179] Embodiment 13: An embodiment of any one of embodiments 1-12, wherein the phospholipid comprises a phosphatidylethanolamine.

[0180] Embodiment 14: An embodiment of embodiment 13, wherein the phosphatidylethanolamine comprises l,2-diheptadecanoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-didecanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilauroyl-sn- glycero-3 -phosphoethanolamine (DLPE), l,2-dipentadecanoyl-sn-glycero-3- phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), .1,2- distearoyl-sn-glycero-3 -phosphoethanolamine, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine (DAPE), 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-linoleoyl-sn- glycero-3-phosphoethanolamine, 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine, l-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl- 2-arachidonoyl-sn-glycero-3-phosphoethanolamine, l-stearoyl-2-docosahexaenoyl-sn- glycero-3 -phosphoethanolamine, or a combination thereof.

[0181] Embodiment 15: An embodiment of any one of embodiments 1-14, wherein the lipid nanoparticle further comprises a permanently charged cationic lipid, the permanently charged cationic lipid comprising no more than 10 mol% of the total lipid of the lipid nanoparticle.

[0182] Embodiment 16: An embodiment of any one of embodiments 1-14, wherein the lipid nanoparticle does not comprise a permanently charged cationic lipid.

[0183] Embodiment 17: An embodiment of any one of embodiments 1-16, wherein: the conjugated lipid comprises between 0.6 mol% and 1 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 50.5 mol% and 58.5 mol% of the total lipid of the nanoparticle; and the cholesterol or the derivative thereof comprises between 29 mol% and 36 mol% of the total lipid of the nanoparticle.

[0184] Embodiment 18: An embodiment of any one of embodiments 1-16, wherein: the conjugated lipid comprises between 0.1 mol% and 0.6 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 53.5 mol% and 61.5 mol% of the total lipid in the lipid nanoparticle; and the cholesterol or the derivative thereof comprises between 32 mol% and 39 mol% of the total lipid of the nanoparticle.

[0185] Embodiment 19: An embodiment of any one of embodiments 1-18, wherein: the ionizable lipid comprises 3D-P-DMA; the conjugated lipid comprises PEG-c-DMA; and the phospholipid comprises DSPC, MC3, or a combination thereof.

[0186] Embodiment 20: An embodiment of any one of embodiments 1-19, wherein the nucleic acid comprises RNA.

[0187] Embodiment 21: An embodiment of embodiment 20, wherein the RNA comprises mRNA, circRNA, saRNA, or a combination thereof.

[0188] Embodiment 22: An embodiment of any one of embodiments 1-19, wherein the nucleic acid comprises DNA.

[0189] Embodiment 23: An embodiment of any one of embodiments 1-22, wherein the protein of interest comprises an antigen.

[0190] Embodiment 24: An embodiment of embodiment 23, wherein the antigen is derived from a pathogen, a pathogenic toxin, or a pathogenic adhesion element.

[0191] Embodiment 25: An embodiment of any one of embodiments 1-24, wherein the lipid nanoparticle further comprises a pharmaceutically acceptable excipient.

[0192] Embodiment 26: A method of producing a lipid nanoparticle, the method comprising: providing an aqueous solution comprising a nucleic acid encoding a protein of interest or a fragment thereof; providing an alcoholic solution comprising an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, cholesterol or a derivative thereof, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net neutral charge at physiological pH; and combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle; wherein the conjugated lipid comprises no more than X mol% of the total lipid of the alcoholic solution, X having a value that is no more than 1, and the alcoholic solution has a composition satisfying at least one of the following conditions: (a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the alcoholic solution, Y having a value defined by the formula: 51.1 - 5.7X + 8. OX2; and (b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the alcoholic solution, Z having a value defined by the formula: 39.3 + 2.3X - 5.6X2.

[0193] Embodiment 27: An embodiment of embodiment 26, wherein the method further comprises removing at least a portion of the alcoholic organic solvent from the lipid nanoparticle solution.

[0194] Embodiment 28: An embodiment of embodiment 27, wherein the removing of at least a portion of the alcoholic organic solvent comprises dialyzing the lipid nanoparticle solution.

[0195] Embodiment 29: An embodiment of any one of embodiments 26-28, wherein the method further comprises concentrating the lipid nanoparticle solution.

[0196] Embodiment 30: An embodiment of embodiment 29, wherein the concentrating of the lipid nanoparticle precursor solution comprises centrifuging the lipid nanoparticle solution.

[0197] Embodiment 31: An embodiment of any one of embodiments 26-30, wherein the method further comprises filtering the lipid nanoparticle solution.

[0198] Embodiment 32: An embodiment of any one of embodiments 26-31, wherein the alcoholic organic solvent comprises ethanol.

[0199] Embodiment 33: An embodiment of any one of embodiments 26-32, wherein the providing of the alcoholic solution comprises: forming a phospholipid solution comprising a nonalcoholic organic solvent and the phospholipid or a pharmaceutically acceptable salt thereof; and combining the phospholipid solution and the alcoholic organic solvent.

[0200] Embodiment 34: An embodiment of embodiment 33, wherein the nonalcoholic organic solvent comprises tetrahydrofuran.

[0201] Embodiment 35: An embodiment of any one of embodiments 26-34, wherein: (a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the alcoholic solution; and (b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the alcoholic solution.

[0202] Embodiment 36: An embodiment of any one of embodiments 26-35, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

[0203] Embodiment 37: An embodiment of embodiment 36, wherein the ionizable lipid comprises 3D-P-DMA.

[0204] Embodiment 38: An embodiment of any one of embodiments 26-35, wherein the ionizable lipid comprises MC3.

[0205] Embodiment 39: An embodiment of any one of embodiments 26-38, wherein X has a value that is at least 0.5.

[0206] Embodiment 40: An embodiment of any one of embodiments 26-39, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains that each have five or more members and that each are not bonded directly to an ester.

[0207] Embodiment 41: An embodiment of any one of embodiments 26-40, wherein the conjugated lipid comprises a carbamate linkage.

[0208] Embodiment 42: An embodiment of any one of embodiments 26-41, wherein the conjugated lipid comprises a PEG-lipid conjugate.

[0209] Embodiment 43: An embodiment of any one of embodiments 26-42, wherein the conjugated lipid comprises PEG-c-DMA.

[0210] Embodiment 44: An embodiment of any one of embodiments 26-43, wherein the phospholipid comprises a phosphatidylcholine.

[0211] Embodiment 45: An embodiment of embodiment 44, wherein the phosphatidylcholine comprises DSPC, l,2-didecanoyl-sn-glycero-3 -phosphocholine, 1,2- diundecanoyl-sn-glycero-3-phosphocholine, l,3-dipalmitoyl-rac-glycero-2-phosphocholine, DLPC, l,2-ditridecanoyl-sn-glycero-3-phosphocholine, DMPC, 1,2-dipentadecanoyl-sn- glycero-3 -phosphocholine, DPPC, l,2-diheptadecanoyl-sn-glycero-3-phosphocholine, 1,2- dinonadecanoyl-sn-glycero-3-phosphocholine, l,2-diarachidoyl-sn-glycero-3- phosphocholine, 1,2-divaccenoyl -sn-glycero-3 -phosphocholine, l,2-di[(8Z)octadecenoyl]-sn- glycero-3 -phosphocholine, 1 ,2-dimyristoleoyl-sn-glycero-3 -phosphocholine, 1,2- dimyristelaidoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dipalmitelaidoyl-sn-glycero-3 -phosphocholine, 1,2-dipetroselenoyl-sn- glycero-3 -phosphocholine, DOPC, l,2-dielaidoyl-sn-glycero-3-phosphocholine, 1,2- dilinoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- dieicosenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, l-pentadecanoyl-2-oleoyl-sn-glycero-3-phosphocholine, MPPC, MSPC, 1 -palmitoyl -2- myristoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-stearoyl-sn-glycero-3- phosphocholine, POPC, 1 -palmitoyl -2 -linoleoyl-sn-glycero-3 -phosphocholine, PAPC, SMPC, l-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1 -stearoyl -2 -oleoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, l-stearoyl-2- arachidonoyl-sn-glycero-3-phosphocholine, OMPC, 1 -oleoyl -2 -palmitoyl-sn-glycero-3- phosphocholine, l-oleoyl-2-stearoyl-sn-glycero-3 -phosphocholine, l-(8Z-octadecenoyl)-2- palmitoyl-sn-glycero-3-phosphocholine, or a combination thereof.

[0212] Embodiment 46: An embodiment of any one of embodiments 26-45, wherein the phospholipid comprises a phosphatidylethanolamine.

[0213] Embodiment 47: An embodiment of embodiment 46, wherein the phosphatidylethanolamine comprises l,2-diheptadecanoyl-sn-glycero-3- phosphoethanolamine, l,2-didecanoyl-sn-glycero-3 -phosphoethanolamine, DLPE, 1,2- dipentadecanoyl-sn-glycero-3 -phosphoethanolamine, DPPE, . l,2-distearoyl-sn-glycero-3- phosphoethanolamine, DMPE, l,2-dipahnitoleoyl-sn-glycero-3-phosphoethanolamine,DOPE, l,2-dielaidoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, DAPE, l-pentadecanoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine, 1 -palmitoyl -2 -linoleoyl-sn-glycero-3 -phosphoethanolamine, POPE, 1 -stearoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine, 1 -palmitoyl -2-arachidonoyl- sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine, l-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl- 2-arachidonoyl-sn-glycero-3-phosphoethanolamine, l-stearoyl-2-docosahexaenoyl-sn- glycero-3 -phosphoethanolamine, or a combination thereof.

[0214] Embodiment 48: An embodiment of any one of embodiments 26-47, wherein the alcoholic solution further comprises a permanently charged cationic lipid, and the permanently charged cationic lipid comprises no more than 10 mol% of the total lipid of the alcoholic solution.

[0215] Embodiment 49: An embodiment of any one of embodiments 26-47, wherein the alcoholic solution does not comprise a permanently charged cationic lipid.

[0216] Embodiment 50: An embodiment of any one of embodiments 26-49, wherein: the conjugated lipid comprises between 0.6 mol% and 1 mol% of the total lipid of the alcoholic solution; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 50.5 mol% and 58.5 mol% of the total lipid of the alcoholic solution; and the cholesterol or the derivative thereof comprises between 29 mol% and 36 mol% of the total lipid of the alcoholic solution.

[0217] Embodiment 51: An embodiment of any one of embodiments 26-49, wherein: the conjugated lipid comprises between 0. 1 mol% and 0.6 mol% of the total lipid of the alcoholic solution; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 53.5 mol% and 61.5 mol% of the total lipid of the alcoholic solution; and the cholesterol or the derivative thereof comprises between 32 mol% and 39 mol% of the total lipid of the alcoholic solution.

[0218] Embodiment 52: An embodiment of any one of embodiments 26-51, wherein: the ionizable lipid comprises 3D-P-DMA; the conjugated lipid comprises PEG-c-DMA; and the phospholipid comprises DSPC, MC3, or a combination thereof.

[0219] Embodiment 53: An embodiment of any one of embodiments 26-52, wherein the nucleic acid comprises RNA.

[0220] Embodiment 54: An embodiment of embodiment 53, wherein the RNA comprises mRNA, circRNA, saRNA, or a combination thereof.

[0221] Embodiment 55: An embodiment of any one of embodiments 26-52, wherein the nucleic acid comprises DNA.

[0222] Embodiment 56: An embodiment of any one of embodiments 26-55, wherein the protein of interest comprises an antigen.

[0223] Embodiment 57: An embodiment of embodiment 56, wherein the antigen is derived from a pathogen, a pathogenic toxin, or a pathogenic adhesion element.

[0224] Embodiment 58: A method of inducing an immune response against the protein of interest of the lipid nanoparticle of any one of embodiments 1-25 in a subject, the method comprising administering to the subject a therapeutically effective amount of the lipid nanoparticle.

[0225] Embodiment 59: An embodiment of embodiment 58, wherein the immune response comprises increased production of antibodies.

[0226] Embodiment 60: An embodiment of embodiment 58 or 59, wherein the administering is performed intradermally or intramuscularly.

[0227] Embodiment 61 : An embodiment of any one of embodiments 1-25 for use in inducing an immune response against the protein of interest of the lipid nanoparticle in a subject.

[0228] Embodiment 62: An embodiment of embodiment 61, wherein the immune response comprises increased production of antibodies.

[0229] Embodiment 63: A use of the lipid nanoparticle of any one of embodiments 1-25 to prepare a medicament for inducing an immune response against the protein of interest of the lipid nanoparticle in a subject.

[0230] Embodiment 64: An embodiment of embodiment 63, wherein the immune response comprises increased production of antibodies.

[0231] Embodiment 65 : A method of preventing or treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the lipid nanoparticle of any one of embodiments 1-25.

[0232] Embodiment 66: An embodiment of embodiment 65, wherein the disease comprises an infection.

[0233] Embodiment 67: An embodiment of embodiment 65, wherein the administering is performed intradermally or intramuscularly.

[0234] Embodiment 68: An embodiment of any one of embodiments 1-25 for use in preventing or treating a disease in a subject.

[0235] Embodiment 69: An embodiment of embodiment 68, wherein the disease comprises an infection.

[0236] Embodiment 70: A use of the lipid nanoparticle of any one of embodiments 1-25 to prepare a medicament for preventing or treating a disease in a subject.

[0237] Embodiment 71: An embodiment of embodiment 70, wherein the disease comprises an infectionEXAMPLES

[0238] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner.Example 1. Preparation of lipid nanoparticles.

[0239] Nucleic acid was encapsulated in LNPs using a controlled mixing process (US 9,005,654) in which an aqueous solution of mRNA in 100 mM acetate buffer at pH 5.0 was combined with an ethanolic solution of lipids, containing a PEG-conjugated lipid, an ionizable lipid (3D-P-DMA: (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate; WO 2013 / 126803), cholesterol, and l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) at various molar ratios as outlined in Table 1 (Example 2), Table 2 (Example 3), Table 3 (Example 4), and Table 4 (Example 5). The ethanol was then removed, and the external buffer was replaced with Tris / NaCl buffer by dialysis. Following dialysis, the formulations were concentrated using VivaSpin concentrator units (MWCO 100,000) andfiltered through a 0.2-pm pore sterile filter. Aliquots were subsequently stored frozen at -80 °C in Tris-sucrose buffer, pH 8.0. Nucleic acid concentration was determined by the RiboGreen assay. Particle size and polydispersity were determined using a Malvern Nano Series Zetasizer.Example 2. Comparison of mRNA-LNP formulations used as a vaccine against Hemagglutinin.

[0240] Codon-optimized A / Puerto Rico / 8 / 1934(HlNl) Hemagglutinin (HA) (SEQ ID NO. 1) was synthesized (GenScript) and cloned into an mRNA production plasmid. HA mRNA was produced using T7 RNA polymerase (MEGASCRIPT, Ambion) on linearized plasmids. Post transcription the RNA was enzymatically polyadenylated (NEB). Pseudouridine-5'- triphosphate (TriLink) was used instead of UTP (uridine 5 ’-triphosphate) to generate HA with modified nucleoside-containing mRNA. Capping of the in vv / ro-transcribcd mRNAs was performed co-transcriptionally using the trinucleotide capl analog, CleanCap (TriLink). mRNA was purified by cellulose purification, as previously described (Baiersdorfer et al., Mol. Ther. Nucleic Acids 15, (2019): 26).

[0241] Lipid nanoparticles containing HA mRNA were prepared according to Table A. 10 pg of the each LNP formulation (or PBS control) was injected intramuscularly to female Balb / c mice (7-8 weeks old) to immunize against HA protein. Serum was collected 28 days after the immunization and the potency of each formulation was determined via an ELISA to quantify the amount of IgG antibodies developed against the antigen encoded by the mRNA (HA). Briefly, for Anti-HA ELISA CORNING® 96-well Clear Flat Bottom Polystyrene High Bind Microplates (Coming #9018) were coated with 1 pg / ml purified HA (SinoBiological #11684- V08H) in PBS (100 pL / well) overnight at 4 °C. The following day, the coated plates were washed three times with wash buffer (0.05% Tween-20 in PBS). The plates were then blocked with 10% FBS in PBS for 1 h at room temperature and washed three times with wash buffer. Mouse sera and an anti -HA Ab (SinoBiological #11684-MM03) used to generate a standard curve were diluted in blocking buffer and incubated for 2 h at room temperature, followed by three washes. HRP-conjugated anti-mouse secondary antibody (Jackson Immunoresearch (#115-036-071) was diluted 1: 10,000 in blocking buffer and incubated for 1 h, followed by three washes. BD TMB Substrate Reagent Set (#BDB555214) was applied to the plate and the reaction was stopped with 2 N sulfuric acid. The absorbance was measured at 450 nm using a Tecan Safire 2 microplate reader.Table 1aZ-average and PDI were measured on a Malvern Zetasizer with dynamic light scattering.bPDI = polydispersity index.cEE =Encapsulation Efficiency.Example 3. Comparison of mRNA-LNP formulations used as a vaccine against Ovalbumin.

[0242] Commercially available modified Ovalbumin (OVA) mRNA was purchased from Trilink (L-7210, 5moU). Lipid nanoparticles containing OVA mRNA were prepared according to Table 2. For each test case, 1 pg of the LNP formulation (or PBS control) was injected intramuscularly to female Balb / c mice (7-8 weeks old) on Day 0 and Day 21 to immunize against OVA protein. Serum was collected 14 days after the second immunization and the potency of each formulation was determined via an ELISA to quantify the amount of IgG antibodies developed against the antigen encoded by the mRNA (OVA). Serum Anti-OVA IgG antibody titres were determined using a commercially available kit (Chondrex #3011) and by following the manufacturers instructions.Table 2Example 4. Applicability of low conjugated lipid formulation to alternative cationic lipid identity.

[0243] HA mRNA was produced as described in Example 2. Lipid nanoparticles containing HA mRNA were prepared according to Table 3. For this example, two different cationic lipids were compared in formulations with varying percentages of PEG lipid. Either 1 pg or 5 pg of the tested LNP formulation (or PBS control) was injected intramuscularly to female Balb / c mice (7-8 weeks old) to immunize against HA protein. Serum was collected 28 days after the immunization and the potency of each formulation was determined via an ELISA to quantify the amount of IgG antibodies developed against the antigen encoded by the mRNA (HA). The ELISA was generally conducted as described in Example 2.Table 3Example 5. Applicability of low mol% conjugated lipid formulation to alternative PEG lipids and PEOZ lipid.

[0244] HA mRNA was produced as described in Example 2. Lipid nanoparticles containingHA mRNA were prepared according to Table 4. For this example, different PEG lipids orsubstitution with a PEOZ lipid were compared with varying mol %. For each test case, 10 pg of LNP formulation (or PBS control) was injected intramuscularly to female Balb / c mice (7-8 weeks old) to immunize against HA protein. Serum was collected 28 days after the immunization and the potency of each formulation was determined via an ELISA to quantify the amount of IgG antibodies developed against the antigen encoded by the mRNA (HA). The ELISA was conducted as described in Example 2.Table 4

[0245] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference.SEQUENCE LISTING

Claims

WHAT IS CLAIMED IS:

1. A lipid nanoparticle comprising: a conjugated lipid, the conjugated lipid comprising no more than X mol% of the total lipid of the lipid nanoparticle, X having a value that is no more than 1 ; a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net neutral charge at physiological pH; an ionizable lipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; and a nucleic acid encoding a protein of interest or a fragment thereof; wherein the lipid nanoparticle has a composition satisfying at least one of the following conditions:(a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the lipid nanoparticle, Y having a value defined by the formula: 51.1 - 5.7X + 8. OX2; and(b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the nanoparticle, Z having a value defined by the formula: 39.3 + 2.3X - 5.6X2.

2. The lipid nanoparticle of claim 1, wherein:(a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the nanoparticle; and(b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the nanoparticle.

3. The lipid nanoparticle of claim 1 or 2, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

4. The lipid nanoparticle of claim 3, wherein the ionizable lipid comprises (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (3D-P- DMA).

5. The lipid nanoparticle of claim 1 or 2, wherein the ionizable lipid comprises (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (MC3).

6. The lipid nanoparticle of any one of claims 1-5, wherein X has a value that is at least 0.5.

7. The lipid nanoparticle of any one of claims 1-6, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains that each have five or more members and that each are not directly bonded to an ester.

8. The lipid nanoparticle of any one of claims 1-7, wherein the conjugated lipid comprises a carbamate linkage.

9. The lipid nanoparticle of any one of claims 1-8, wherein the conjugated lipid comprises a polyethylene glycol (PEG) -lipid conjugate.

10. The lipid nanoparticle of any one of claims 1-9, wherein the conjugated lipid comprises N- [(methoxypoly (ethylene glycol))carbamoyl]-l,2-dimyristyloxy- propylamine (PEG-c-DMA).

11. The lipid nanoparticle of any one of claims 1-10, wherein the phospholipid comprises a phosphatidylcholine.

12. The lipid nanoparticle of claim 11, wherein the phosphatidylcholine comprises l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-didecanoyl-sn-glycero-3- phosphocholine, 1 ,2-diundecanoyl-sn-glycero-3 -phosphocholine, 1 ,3 -dipalmitoyl -rac- glycero-2-phosphocholine, l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- ditridecanoyl-sn-glycero-3 -phosphocholine, l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), l,2-dipentadecanoyl-sn-glycero-3 -phosphocholine, l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-diheptadecanoyl-sn-glycero-3-phosphocholine, 1,2- dinonadecanoyl-sn-glycero-3-phosphocholine, l,2-diarachidoyl-sn-glycero-3- phosphocholine, 1,2-divaccenoyl -sn-glycero-3 -phosphocholine, l,2-di[(8Z)octadecenoyl]-sn- glycero-3 -phosphocholine, 1 ,2-dimyristoleoyl-sn-glycero-3 -phosphocholine, 1,2- dimyristelaidoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoleoyl-sn-glycero-3- phosphocholine, l,2-dipalmitelaidoyl-sn-glycero-3 -phosphocholine, 1,2-dipetroselenoyl-sn- glycero-3 -phosphocholine, l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dielaidoyl-sn-glycero-3 -phosphocholine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine, 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dieicosenoyl-sn-glycero-3 -phosphocholine,l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l-pentadecanoyl-2-oleoyl-sn-glycero-3- phosphocholine, 1 -myristoyl -2 -palmitoyl-sn-glycero-3 -phosphocholine (MPPC), 1 -myristoyl - 2-stearoyl-sn-glycero-3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, l-palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 -palmitoyl -2-oleoyl- glycero-3 -phosphocholine (POPC), l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1- palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), 1 -stearoyl -2-myristoyl-sn- glycero-3 -phosphocholine (SMPC), l-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1- stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, 1 -oleoyl -2- myristoyl-sn-glycero-3-phosphocholine (OMPC), 1 -oleoyl -2 -palmitoyl-sn-glycero-3- phosphocholine, l-oleoyl-2-stearoyl-sn-glycero-3 -phosphocholine, l-(8Z-octadecenoyl)-2- palmitoyl-sn-glycero-3-phosphocholine, or a combination thereof.

13. The lipid nanoparticle of any one of claims 1-12, wherein the phospholipid comprises a phosphatidylethanolamine.

14. The lipid nanoparticle of claim 13, wherein the phosphatidylethanolamine comprises l,2-diheptadecanoyl-sn-glycero-3- phosphoethanolamine, l,2-didecanoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilauroyl-sn- glycero-3 -phosphoethanolamine (DLPE), l,2-dipentadecanoyl-sn-glycero-3- phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), .1,2- distearoyl-sn-glycero-3 -phosphoethanolamine, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), l,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine (DAPE), 1- pentadecanoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-linoleoyl-sn- glycero-3-phosphoethanolamine, 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphoethanolamine, l-palmitoyl-2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine, l-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl- 2-arachidonoyl-sn-glycero-3-phosphoethanolamine, l-stearoyl-2-docosahexaenoyl-sn- glycero-3 -phosphoethanolamine, or a combination thereof.

15. The lipid nanoparticle of any one of claims 1-14, wherein the lipid nanoparticle further comprises a permanently charged cationic lipid, the permanently charged cationic lipid comprising no more than 10 mol% of the total lipid of the lipid nanoparticle.

16. The lipid nanoparticle of any one of claims 1-14, wherein the lipid nanoparticle does not comprise a permanently charged cationic lipid.

17. The lipid nanoparticle of any one of claims 1-16, wherein: the conjugated lipid comprises between 0.6 mol% and 1 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 50.5 mol% and 58.5 mol% of the total lipid of the nanoparticle; and the cholesterol or the derivative thereof comprises between 29 mol% and 36 mol% of the total lipid of the nanoparticle.

18. The lipid nanoparticle of any one of claims 1-16, wherein: the conjugated lipid comprises between 0.1 mol% and 0.6 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 53.5 mol% and 61.5 mol% of the total lipid in the lipid nanoparticle; and the cholesterol or the derivative thereof comprises between 32 mol% and 39 mol% of the total lipid of the nanoparticle.

19. The lipid nanoparticle of any one of claims 1-18, wherein: the ionizable lipid comprises 3D-P-DMA; the conjugated lipid comprises PEG-c-DMA; and the phospholipid comprises DSPC, MC3, or a combination thereof.

20. The lipid nanoparticle of any one of claims 1-19, wherein the nucleic acid comprises RNA.

21. The lipid nanoparticle of claim 20, wherein the RNA comprises mRNA, circRNA, saRNA, or a combination thereof.

22. The lipid nanoparticle of any one of claims 1-19, wherein the nucleic acid comprises DNA.

23. The lipid nanoparticle of any one of claims 1-22, wherein the protein of interest comprises an antigen.

24. The lipid nanoparticle of claim 23, wherein the antigen is derived from a pathogen, a pathogenic toxin, or a pathogenic adhesion element.

25. The lipid nanoparticle of any one of claims 1-24, wherein the lipid nanoparticle further comprises a pharmaceutically acceptable excipient.

26. A method of producing a lipid nanoparticle, the method comprising: providing an aqueous solution comprising a nucleic acid encoding a protein of interest or a fragment thereof; providing an alcoholic solution comprising an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, cholesterol or a derivative thereof, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net neutral charge at physiological pH; and combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle; wherein the conjugated lipid comprises no more than X mol% of the total lipid of the alcoholic solution, X having a value that is no more than 1, and the alcoholic solution has a composition satisfying at least one of the following conditions:(a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the alcoholic solution, Y having a value defined by the formula: 51.1 - 5.7X + 8. OX2; and(b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the alcoholic solution, Z having a value defined by the formula: 39.3 + 2.3X - 5.6X2.

27. The method claim 26, wherein the method further comprises removing at least a portion of the alcoholic organic solvent from the lipid nanoparticle solution.

28. The method of claim 27, wherein the removing of at least a portion of the alcoholic organic solvent comprises dialyzing the lipid nanoparticle solution.

29. The method of any one of claims 26-28, wherein the method further comprises concentrating the lipid nanoparticle solution.

30. The method of claim 29, wherein the concentrating of the lipid nanoparticle precursor solution comprises centrifuging the lipid nanoparticle solution.

31. The method of any one of claims 26-30, wherein the method further comprises filtering the lipid nanoparticle solution.

32. The method of any one of claims 26-31, wherein the alcoholic organic solvent comprises ethanol.

33. The method of any one of claims 26-32, wherein the providing of the alcoholic solution comprises: forming a phospholipid solution comprising a nonalcoholic organic solvent and the phospholipid or a pharmaceutically acceptable salt thereof; and combining the phospholipid solution and the alcoholic organic solvent.

34. The method of claim 33, wherein the nonalcoholic organic solvent comprises tetrahydrofuran.

35. The method of any one of claims 26-34, wherein:(a) the ionizable lipid or the pharmaceutically acceptable salt thereof comprises at least Y mol% of the total lipid of the alcoholic solution; and(b) the cholesterol or the derivative thereof comprises no more than Z mol% of the total lipid of the alcoholic solution.

36. The method of any one of claims 26-35, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members .

37. The method of claim 36, wherein the ionizable lipid comprises 3D-P- DMA.

38. The method of any one of claims 26-35, wherein the ionizable lipid comprises MC3.

39. The method of any one of claims 26-38, wherein X has a value that is at least 0.5.

40. The method of any one of claims 26-39, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains that each have five or more members and that each are not bonded directly to an ester.

41. The method of any one of claims 26-40, wherein the conjugated lipid comprises a carbamate linkage.

42. The method of any one of claims 26-41, wherein the conjugated lipid comprises a PEG-lipid conjugate.

43. The method of any one of claims 26-42, wherein the conjugated lipid comprises PEG-c-DMA.

44. The method of any one of claims 26-43, wherein the phospholipid comprises a phosphatidylcholine.

45. The method of claim 44, wherein the phosphatidylcholine comprisesDSPC, l,2-didecanoyl-sn-glycero-3-phosphocholine, l,2-diundecanoyl-sn-glycero-3- phosphocholine, l,3-dipalmitoyl-rac-glycero-2-phosphocholine, DLPC, 1,2-ditridecanoyl-sn- glycero-3-phosphocholine, DMPC, l,2-dipentadecanoyl-sn-glycero-3 -phosphocholine, DPPC, 1 ,2-diheptadecanoyl-sn-glycero-3 -phosphocholine, 1 ,2-dinonadecanoyl-sn-glycero-3 - phosphocholine, l,2-diarachidoyl-sn-glycero-3 -phosphocholine, 1,2-divaccenoyl -sn-glycero- 3 -phosphocholine, 1 ,2-di [(8Z)octadecenoyl] -sn-glycero-3 -phosphocholine, 1,2- dimyristoleoyl-sn-glycero-3 -phosphocholine, l,2-dimyristelaidoyl-sn-glycero-3- phosphocholine, 1 ,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine, 1 ,2-dipalmitelaidoyl-sn- glycero-3 -phosphocholine, l,2-dipetroselenoyl-sn-glycero-3 -phosphocholine, DOPC, 1,2- dielaidoyl-sn-glycero-3 -phosphocholine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine, 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dieicosenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l-pentadecanoyl-2-oleoyl-sn-glycero-3- phosphocholine, MPPC, MSPC, l-palmitoyl-2-myristoyl-sn-glycero-3 -phosphocholine, 1- palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, POPC, 1 -palmitoyl -2-linoleoyl-sn- glycero-3 -phosphocholine, PAPC, SMPC, 1 -stearoyl -2 -palmitoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, l-stearoyl-2-linoleoyl-sn- glycero-3 -phosphocholine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, OMPC, 1 -oleoyl -2 -palmitoyl-sn-glycero-3-phosphocholine, 1 -oleoyl -2-stearoyl-sn-glycero-3- phosphocholine, l-(8Z-octadecenoyl)-2-palmitoyl-sn-glycero-3-phosphocholine, or a combination thereof.

46. The method of any one of claims 26-45, wherein the phospholipid comprises a phosphatidylethanolamine.

47. The method of claim 46, wherein the phosphatidylethanolamine comprises l,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-didecanoyl-sn- glycero-3 -phosphoethanolamine, DLPE, 1 ,2-dipentadecanoyl-sn-glycero-3 - phosphoethanolamine, DPPE, . l,2-distearoyl-sn-glycero-3 -phosphoethanolamine, DMPE, 1,2- dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DOPE, l,2-dielaidoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, DAPE, 1 -pentadecanoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine, l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, POPE, 1 -stearoyl -2 -oleoyl-sn- glycero-3-phosphoethanolamine, l-palmitoyl-2-arachidonoyl-sn-glycero-3- phosphoethanolamine, l-pahnitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3-phosphoethanolamine, 1 -stearoyl -2 -arachidonoyl-sn- glycero-3 -phosphoethanolamine, 1 -stearoyl -2-docosahexaenoyl-sn-glycero-3- phosphoethanolamine, or a combination thereof.

48. The method of any one of claims 26-47, wherein the alcoholic solution further comprises a permanently charged cationic lipid, and the permanently charged cationic lipid comprises no more than 10 mol% of the total lipid of the alcoholic solution.

49. The method of any one of claims 26-47, wherein the alcoholic solution does not comprise a permanently charged cationic lipid.

50. The method of any one of claims 26-49, wherein: the conjugated lipid comprises between 0.6 mol% and 1 mol% of the total lipid of the alcoholic solution; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 50.5 mol% and 58.5 mol% of the total lipid of the alcoholic solution; and the cholesterol or the derivative thereof comprises between 29 mol% and 36 mol% of the total lipid of the alcoholic solution.

51. The method of any one of claims 26-49, wherein: the conjugated lipid comprises between 0.1 mol% and 0.6 mol% of the total lipid of the alcoholic solution; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8.5 mol% and 11.5 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 53.5 mol% and 61.5 mol% of the total lipid of the alcoholic solution; and the cholesterol or the derivative thereof comprises between 32 mol% and 39 mol% of the total lipid of the alcoholic solution.

52. The method of any one of claims 26-51, wherein: the ionizable lipid comprises 3D-P-DMA; the conjugated lipid comprises PEG-c-DMA; and the phospholipid comprises DSPC, MC3, or a combination thereof.

53. The method of any one of claims 26-52, wherein the nucleic acid comprises RNA.

54. The method of claim 53, wherein the RNA comprises mRNA, circRNA, saRNA, or a combination thereof.

55. The method of any one of claims 26-52, wherein the nucleic acid comprises DNA.

56. The method of any one of claims 26-55, wherein the protein of interest comprises an antigen.

57. The method of any one of claim 56, wherein the antigen is derived from a pathogen, a pathogenic toxin, or a pathogenic adhesion element.

58. A method of inducing an immune response against the protein of interest of the lipid nanoparticle of any one of claims 1-25 in a subject, the method comprising administering to the subject a therapeutically effective amount of the lipid nanoparticle.

59. The method of claim 58, wherein the immune response comprises increased production of antibodies.

60. The method of claim 58 or 59, wherein the administering is performed intradermally or intramuscularly.

61. The lipid nanoparticle of any one of claims 1-25 for use in inducing an immune response against the protein of interest of the lipid nanoparticle in a subject.

62. The lipid nanoparticle of claim 61, wherein the immune response comprises increased production of antibodies.

63. A use of the lipid nanoparticle of any one of claims 1-25 to prepare a medicament for inducing an immune response against the protein of interest of the lipid nanoparticle in a subject.

64. The use of claim 63, wherein the immune response comprises increased production of antibodies.

65. A method of preventing or treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the lipid nanoparticle of any one of claims 1-25.

66. The method of claim 65, wherein the disease comprises an infection.

67. The method of claim 65, wherein the administering is performed intradermally or intramuscularly.

68. The lipid nanoparticle of any one of claims 1-25 for use in preventing or treating a disease in a subject.

69. The lipid nanoparticle of claim 68, wherein the disease comprises an infection.

70. A use of the lipid nanoparticle of any one of claims 1-25 to prepare a medicament for preventing or treating a disease in a subject.

71. The use of claim 70, wherein the disease comprises an infection.

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