Lipid nanoparticles for stimulating t cells

Abstract

The present disclosure provides lipid nanoparticles that are particularly beneficial in inducing an immune response in a subject, particularly by stimulating CD8+ T cells associated with protein or protein fragments of interest. The lipid nanoparticles can have a negative zeta potential and / or particular configurations and / or relative amounts of a phospholipid, an ionizable lipid, and a conjugated lipid. The disclosure also provides vaccines, other pharmaceutical compositions, and methods including the provided lipid nanoparticles.

Description

LIPID NANOPARTICLES FOR STIMULATING T CELLSCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 574,479 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 that can be used to stimulate a high CD8+T cell response in a subject to whom the LNPs are administered. Preventative and curative therapies that can benefit from such elevated CD8+T cell stimulation include, for example, those targeting cancers, infectious diseases, and autoimmune diseases. Current LNPs, however, can suffer from limitations reducing their efficacy in these therapies. These limitations can include 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 and / or therapeutic 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 inKILPATRICK TOWNSEND & STOCKTON LLPthe 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 lipid nanoparticle. The lipid nanoparticle includes a phospholipid or a pharmaceutically acceptable salt thereof. The lipid nanoparticle further includes a conjugated lipid. The total lipid of the lipid nanoparticle includes between 0.1 mol% and 5 mol% of the conjugated lipid. The lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof. The ionizable lipid includes three or more terminal hydrocarbon chains that each have five or more members. The lipid nanoparticle further includes a nucleic acid. The nucleic acid encodes a protein of interest or a fragment thereof. The lipid nanoparticle is configured to have a negative zeta potential at physiological pH.

[0006] In another aspect, the disclosure provides another lipid nanoparticle. The lipid nanoparticle includes a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net negative charge at physiological pH. The lipid nanoparticle further includes a conjugated lipid. The total lipid of the lipid nanoparticle includes less than 1 mol% of the conjugated lipid. The lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof. The lipid nanoparticle further includes a nucleic acid encoding a protein of interest or a fragment thereof.

[0007] In another aspect, the disclosure provides another lipid nanoparticle. The lipid nanoparticle includes a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net negative charge at physiological pH. The total lipid of the lipid nanoparticle includes at least 8 mol% of the phospholipid or the pharmaceutically acceptablesalt thereof. The lipid nanoparticle further includes a conjugated lipid. The total lipid of the lipid nanoparticle includes between 0.1 mol % and 5 mol % of the conjugated lipid. The lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof. The lipid nanoparticle further includes a nucleic acid. The nucleic acid encodes a protein of interest or a fragment thereof.

[0008] In another aspect, the disclosure provides another lipid nanoparticle. The lipid nanoparticle includes a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net negative charge at physiological pH. The lipid nanoparticle further includes a conjugated lipid. The total lipid of the lipid nanoparticle includes between 0. 1 mol% and 5 mol% of the conjugated lipid. The lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof. The total lipid of the lipid nanoparticle includes more than 1 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The ionizable lipid does not include sulfur. The lipid nanoparticle further includes a nucleic acid encoding a protein of interest or a fragment thereof.

[0009] In another aspect, the disclosure provides a method of producing a lipid nanoparticle. The method includes providing an aqueous solution. The aqueous solution includes a nucleic acid encoding a protein of interest or a fragment thereof. The method further includes providing an alcoholic solution. The alcoholic solution includes an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, and a phospholipid or a pharmaceutically acceptable salt thereof. The total lipid of the alcoholic solution includes between 0.1 mol% and 5 mol% of the conjugated lipid. The method further includes combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle. The composition of the alcoholic solution is configured such that the lipid nanoparticle has a negative zeta potential at physiological pH.

[0010] In another aspect, the disclosure provides another method of producing a lipid nanoparticle. The method includes providing an aqueous solution. The aqueous solution includes a nucleic acid encoding a protein of interest or a fragment thereof. The method further includes providing an alcoholic solution. The alcoholic solution includes an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, and a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net negative charge at physiological pH. The total lipid of the alcoholic solution includes less than 1 mol% of the conjugated lipid. The method further includes combining the aqueous solutionand the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle.

[0011] In another aspect, the disclosure provides another method of producing a lipid nanoparticle. The method includes providing an aqueous solution. The aqueous solution includes a nucleic acid encoding a protein of interest or a fragment thereof. The method further includes providing an alcoholic solution. The alcoholic solution includes an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, and a phospholipid or a pharmaceutically acceptable salt thereof. The phospholipid has a net negative charge at physiological pH. The total lipid of the alcoholic solution includes between 0.1 mol% and 5 mol% of the conjugated lipid. The total lipid of the alcoholic solution further includes at least 8 mol% of the phospholipid or the pharmaceutically acceptable salt thereof. The method further includes combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle.

[0012] In another aspect, the disclosure provides another method of producing a lipid nanoparticle. The method includes providing an aqueous solution. The aqueous solution includes a nucleic acid encoding a protein of interest or a fragment thereof. The method further includes providing an alcoholic solution. The alcoholic solution includes an alcoholic organic solvent, a conjugated lipid; an ionizable lipid or a pharmaceutically acceptable salt thereof, and a phospholipid or a pharmaceutically acceptable salt thereof. The ionizable lipid does not include sulfur. The total lipid of alcoholic solution includes between 0.1 mol% and 5 mol% of the conjugated lipid. The total lipid of the alcoholic solution further includes more than 1 mol% of the ionizable lipid or the pharmaceutically acceptable salt thereof. The phospholipid has a net negative 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.

[0013] In another aspect, the disclosure provides another lipid nanoparticle. The lipid nanoparticle is obtainable by any of the methods disclosed herein.

[0014] In another aspect, the disclosure provides a vaccine. The vaccine includes any of the lipid nanoparticles disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 presents a graph plotting the variations of Zeta Potential measurements across a range of pH values for four lipid nanoparticle formulations provided in Table 1.

[0016] FIG. 2 presents a graph comparing relative measured amounts of CD8+IFN-y+T cells following administration of two different lipid nanoparticle formulations containing HA mRNA to mice.

[0017] FIG. 3 presents a graph comparing relative measured amounts of CD8+TNF-a+T cells following administration of two different lipid nanoparticle formulations containing HA mRNA to mice.

[0018] FIG. 4 presents a graph comparing relative measured amounts of CD8+IL-2+T cells following administration of two different lipid nanoparticle formulations containing HA mRNA to mice.DETAILED DESCRIPTIONA. INTRODUCTION

[0019] The present disclosure generally relates to lipid nanoparticles that, when employed in preventative or curative therapies, provide advantageous improvements in inducing an immune response, e.g., an immune response characterized by an enhanced stimulation of particular T cells. For example, the elicitation of antigen-specific CD8+T cell responses can be a critical aspect of targeted immunotherapies, given the unique role these cells play in adaptive immunity. CD8+T cells, often referred to as cytotoxic T lymphocytes (CTLs), are adept at recognizing and binding to specific antigens presented by MHC class I molecules on the surface of infected or malignant cells. Upon antigen recognition, these CTLs can directly exert cytotoxic effects, leading to the lysis of the aberrant cells. Therapies that stimulate the production of antigen-specific CD8+T cells can therefore enhance the immune system’s precision in eradicating cells expressing the antigen of interest, while minimizing collateral damage to healthy cells. This specificity not only bolsters therapeutic efficacy but also mitigates off-target cytotoxicity, thereby improving the safety profile of the treatment. Consequently, the capacity to stimulate antigen-specific CD8+T cell production can be pivotal in advancing efficacious immunotherapies. Moreover, certain disorders such as cancers,autoimmune diseases, and some infections can be more effectively prevented or mitigated with immunotherapies that involve CD8+T cell stimulation.

[0020] It has proven difficult, however, to use lipid nanoparticles as antigen carriers for the purpose of inducing strong CD8+T cell responses in vaccinated individuals. This difficulty results from several challenges associated with the complex nature of immune activation and the physicochemical properties of LNPs. For example, to trigger the desired CD8+cell response, the antigen encapsulated by an LNP must be effectively delivered to targeted cells or organs, where it can be properly processed and presented to CD8+T cells. However, many LNPs are preferentially taken up by endocytic pathways, which often leads to lysosomal degradation of the encapsulated antigen and limits the efficiency of targeted delivery. Additionally, the charge and lipid composition of LNPs can significantly influence their biodistribution, cellular uptake, and endosomal escape, each of which can significantly influence the effectiveness of antigen delivery and presentation.

[0021] 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 stimulating production of CD8+T cells associated with proteins or protein fragments of interest, e.g., antigens of interest. These provided lipid nanoparticle formulations include selected materials that are combined in certain relative amounts to one another. Notably, the importance of these formulations and the resulting configurations and characteristics of lipid nanoparticles they produce had not been previously appreciated, particularly with regard to their beneficial effects on generating an enhanced immune response.B. DEFINITIONS

[0022] 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.

[0023] 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 notinherently 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-.

[0024] 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.

[0025] 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.”

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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”).

[0030] 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.

[0031] As used herein, 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.

[0032] As used herein, 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.

[0033] 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.

[0034] 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 .

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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 nucleotide analogs 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 alsoimplicitly 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).

[0040] 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.

[0041] 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 acid strands need not be coextensive, for example, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands.

[0042] 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 includebase 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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, the subject 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 than70 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.

[0047] 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.

[0048] 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.

[0049] 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).

[0050] 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 or condition 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

[0051] 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, and a nucleic acid encoding a protein of interest 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 T cell (e.g., CD8+T cell) stimulation. For example, the lipid nanoparticles are demonstrated herein as inducing strong immune responses characterized by T cell stimulation when (1) the lipid nanoparticle has a negative zeta potential (e.g., a negative zeta potential at physiological pH), (2) the ionizable lipid includes particular configurations of tail groups (e.g., three or more terminal hydrocarbon chains that each have five or more members), (3) the phospholipid has a net negative charge (e.g., a net negative charge at physiological pH), (4) 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), (5) the concentration of the phospholipid or the pharmaceutically acceptable salt thereof is above a certain threshold (e.g., at least 8 mol% of the total lipid of the lipid nanoparticle), and / or (6) the ionizable lipid does not include a sulfur.

[0052] In some cases, and as demonstrated by data provided by Example 2 and Example 3, the provided lipid nanoparticles capable of eliciting strong T cell responses exhibit a negative zeta potential. In some embodiments, the lipid nanoparticles have a negative zeta potential at physiological pH, i.e., at a pH value of about 7.4. In preferred embodiments, the lipid nanoparticles have a negative zeta potential at all pH values that are greater than about 7.3, greater than about 7.2, greater than about 7.1, greater than about 7.0, greater than about 6.9, greater than about 6.8, greater than about 6.7, greater than about 6.6, greater than about 6.5, greater than about 6.4, greater than about 6.3, greater than about 6.2, or greater than about 6. 1. The negative zeta potential of the lipid nanoparticles when measured at a pH value (e.g., physiological pH) within this range can be, for example, between about - 1 mV and about -20 mV, e.g., between about -1 mV and about -6 mV, between about -1.3 mV and about -8.1 mV, between about -1.8 mV and about -11 mV, between about -2.5 mV and about -15 mV, or between -3.3 mV and about -20 mV. In terms of lower limits, the lipid nanoparticle negative zeta potential can be, for example, greater than about -20 mV, e.g., greater than -15 mV, greaterthan about -11 mV, greater than about -8.1 mV, greater than about -6 mV, greater than about - 4.5 mV, greater than about -3.3 mV, greater than about -2.5 mV, greater than about -1.8 mV, or greater than about -1.3 mV. Lower zeta potentials, e.g., less than about -20 mV, are also contemplated.

[0053] In a preferred embodiment, the zeta potentials of the lipid nanoparticles described herein are determined using an assay measuring particle zeta potential at a temperature between about 20 °C and about 30 °C. In some embodiments, the lipid nanoparticle has a negative zeta potential, as determined using an assay performed at a temperature between about 20 °C and about 30 °C, when measured at physiological pH or within any of the pH ranges listed above. In particularly preferred embodiments, the zeta potential of the lipid nanoparticle described herein is determined using an assay measuring zeta potential at a temperature of about 25 °C. In preferred embodiments, the zeta potential of the lipid nanoparticle is determined using the assay described in Example 2.

[0054] In some examples of the provided lipid nanoparticles, the phospholipid or the pharmaceutically acceptable salt thereof comprises between about 8.5 mol% and about 10.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 8.5 mol% and about 10 mol%, between about 8.5 mol% and about 9.5 mol%, between about 8.5 mol% and about 9 mol%, between about 9 mol% and about 10.5 mol%, between about 9 mol% and about 10 mol%, between about 9 mol% and about 9.5 mol%, between about 9.5 mol% and about 10.5 mol%, between about 9.5 mol% and about 10 mol%, or between about 10 mol% and about 10.5 mol%), the conjugated lipid comprises between about 0.25 mol% and 0.9 mol% of the total lipid of the lipid nanoparticle (between about 0.25 mol% and about 0.74 mol%, between about 0.25 mol% and about 0.58 mol%, between about 0.25 mol% and about 0.41 mol%, between about 0.41 mol% and about 0.9 mol%, between about 0.41 mol% and about 0.74 mol%, between about 0.41 mol% and about 0.58 mol%, between about 0.58 mol% and about 0.9 mol%, between about 0.58 mol% and about 0.74 mol%, or between about 0.74 mol% and about 0.9 mol%), and the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between about 48 mol% and about 58 mol% of the total lipid of the lipid nanoparticle (e.g., between about 48 mol% and about 55.5 mol%, between about 48 mol% and about 53 mol%, between about 48 mol% and about 50.5 mol%, between about 50.5 mol% and about 58 mol%, between about 50.5 mol% and about 55.5 mol%, between about 50.5 mol% and about 53 mol%, between about 53 mol% and about 58 mol%, between about 53 mol% and about 55.5 mol%, or between about 55.5 mol% and about 58 mol%). In some embodiments, the lipidnanoparticle further includes cholesterol or a derivative thereof, where the cholesterol or the derivative thereof comprises between about 35 mol% and 39 mol% of the total lipid of the lipid nanoparticle (e.g., between about 35 mol% and about 38 mol%, between about 35 mol% and about 37 mol%, between about 35 mol% and about 36 mol%, between about 36 mol% and about 39 mol%, between about 36 mol% and about 38 mol%, between about 36 mol% and about 37 mol%, between about 37 mol% and about 39 mol%, between about 37 mol% and about 38 mol%, or between about 38 mol% and about 39 mol%).

[0055] In some examples of the provided lipid nanoparticles, the phospholipid or the pharmaceutically acceptable salt thereof comprises between about 9 mol% and about 11 mol% of the total lipid of the lipid nanoparticle (e.g., between about 9 mol% and about 10.5 mol%, between about 9 mol% and about 10 mol%, between about 9 mol% and about 9.5 mol%, between about 9.5 mol% and about 11 mol%, between about 9.5 mol% and about 10.5 mol%, between about 9.5 mol% and about 10 mol%, between about 10 mol% and about 11 mol%, between about 10 mol% and about 10.5 mol%, or between about 10.5 mol% and about 11 mol%), the conjugated lipid comprises between about 0.85 mol% and 2.6 mol% of the total lipid of the lipid nanoparticle (between about 0.85 mol% and about 2.16 mol%, between about 0.85 mol% and about 1.73 mol%, between about 0.85 mol% and about 1.29 mol%, between about 1.29 mol% and about 2.6 mol%, between about 1.29 mol% and about 2.16 mol%, between about 1.29 mol% and about 1.73 mol%, between about 1.73 mol% and about 2.6 mol%, between about 1.73 mol% and about 2.16 mol%, or between about 2.16 mol% and about 2.6 mol%), and the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between about 45 mol% and about 55 mol% of the total lipid of the lipid nanoparticle (e.g., between about 45 mol% and about 52.5 mol%, between about 45 mol% and about 50 mol%, between about 45 mol% and about 47.5 mol%, between about 47.5 mol% and about 55 mol%, between about 47.5 mol% and about 52.5 mol%, between about 47.5 mol% and about 50 mol%, between about 50 mol% and about 55 mol%, between about 50 mol% and about 52.5 mol%, or between about 52.5 mol% and about 55 mol%). In some embodiments, the lipid nanoparticle further includes cholesterol or a derivative thereof, where the cholesterol or the derivative thereof comprises between about 36.5 mol% and 40.5 mol% of the total lipid of the lipid nanoparticle (e.g., between about 36.5 mol% and about 39.5 mol%, between about 36.5 mol% and about 38.5 mol%, between about 36.5 mol% and about 37.5 mol%, between about 37.5 mol% and about 40.5 mol%, between about 37.5 mol% and about 39.5 mol%, between about37.5 mol% and about 38.5 mol%, between about 38.5 mol% and about 40.5 mol%, between about 38.5 mol% and about 39.5 mol%, or between about 39.5 mol% and about 40.5 mol%).

[0056] In certain examples, the provided lipid nanoparticle further includes 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 about1.5 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.

[0057] 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 stimulating an immune response, e.g., stimulating T cell production. For example, in some embodiments, the lipid nanoparticle has a pKa from about 5.8 to about 6.9, 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, fromabout 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, or from about 6.3 to about 6.4.

[0058] 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 stimulating an immune response characterized by enhanced T cell production. 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.

[0059] 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. Phospholipid

[0060] 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 with improved performance in inducing a desired immune response, such as an increased stimulation of T cells. In some embodiments, the lipid nanoparticle includes one species of phospholipid. In other embodiments, the lipid nanoparticle includes two 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.

[0061] In some examples, the provided lipid nanoparticles exhibit an ability to elicit a strong T cell response in part because the phospholipid of the lipid nanoparticles is one having a net negative charge at physiological pH. Lipid nanoparticles including such negative phospholipids are demonstrated herein as, for example, eliciting advantageously high levels of CD8+T cells specific for a protein of interest or a fragment thereof. The phospholipid can include or consist of, for example, a phosphatidylserine, a phosphatidylglycerol, or a combination thereof. In some examples, at least one of the phospholipid species, e.g., negatively charged phospholipidspecies, of the lipid nanoparticle is a naturally occurring phospholipid. In some embodiments each phospholipid of the lipid nanoparticle is a naturally occurring phospholipid.

[0062] Phosphatidylserine lipids suitable for use with the provided lipid nanoparticles include, for example, l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2- diheptadecanoyl-sn-glycero-3-phospho-L-serine, l,2-distearoyl-sn-glycero-3-phospho-L- serine (DSPS), l,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-didecanoyl-sn- glycero-3-phospho-L-serine, l,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2- dilauroyl-sn-glycero-3-phospho-L-serine (DLPS), l,2-dilinoleoyl-sn-glycero-3-phospho-L- serine, l,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine, l-pentadecanoyl-2-oleoyl-sn- glycero-3-phospho-L-serine, l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (SOPS), l-palmitoyl-2-linoleoyl-sn-glycero- 3-phospho-L-serine, 1 -palmitoyl -2 -arachidonoyl-sn-glycero-3-phospho-L-serine, 1 -palmitoyl - 2-docosahexaenoyl-sn-glycero-3-phospho-L-serine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3- phospho-L-serine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, l-stearoyl-2- docosahexaenoyl-sn-glycero-3-phospho-L-serine, and combinations thereof.

[0063] Phosphatidylglycerol lipids suitable for use with the provided lipid nanoparticles include, for example, l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG), 1,2- diheptadecanoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l,2-didecanoyl-sn-glycero-3- phospho-(l'-rac-glycerol), l,2-dilauroyl-sn-glycero-3-phospho-(l'-rac -glycerol) (DLPG), 1,2- dimyristoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol) (DMPG), 1 ,2-dipentadecanoyl-sn- glycero-3 -phospho-( 1 '-rac-glycerol), 1 ,2-dipalmitoyl-sn-glycero-3 -phospho-( 1' -rac-glycerol) (DPPG), l,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DSPG), 1,2-dielaidoyl-sn- glycero-3-phospho-(l'-rac-glycerol), l,2-dilinoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l,2-dilinolenoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l,2-diarachidonoyl-sn-glycero-3- [phospho-rac-( 1 -glycerol)], l,2-didocosahexaenoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol) (POPG), 1 -palmitoyl-2- linoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), 1 -palmitoyl -2 -arachidonoyl-sn-glycero-3- phospho-( 1 '-rac-glycerol) (PAPG), 1 -palmitoyl-2-docosahexaenoyl-sn-glycero-3 -phospho-( 1 '- rac-glycerol), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (SOPG), 1-stearoyl- 2 -linoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l-stearoyl-2-arachidonoyl-sn-glycero-3- phospho-( 1 '-rac-glycerol) (S APG), 1 -stearoyl -2 -docosahexaenoyl-sn-glycero-3 -phospho-( 1 '- rac-glycerol), and combinations thereof

[0064] 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. In some examples, the provided lipid nanoparticles and vaccines induce strong immune responses when the amount of phospholipid or the pharmaceutically acceptable salt thereof in the lipid nanoparticle is between about 1 mol% and about 20 mol% of the total lipid of the lipid nanoparticle. The molar fraction of the phospholipid or the pharmaceutically acceptable salt thereof in the total lipid of the provided lipid nanoparticle can be, for example, between about 1 mol% and about 16.2 mol%, between about 1 mol% and about 12.4 mol%, between about 1 mol% and about 8.6 mol%, between about 1 mol% and about 4.8 mol%, between about 4.8 mol% and about 20 mol%, between about 4.8 mol% and about 16.2 mol%, between about 4.8 mol% and about 12.4 mol%, between about 4.8 mol% and about 8.6 mol%, between about 8.6 mol% and about 20 mol%, between about 8.6 mol% and about 16.2 mol%, or between about 8.6 mol% and about 12.4 mol%.

[0065] In certain examples, the provided lipid nanoparticles and vaccines induce strong immune responses when the amount of the phospholipid or the pharmaceutically acceptable salt thereof in the lipid nanoparticle is additionally or alternatively at least about 8 mol% of the total lipid of the lipid nanoparticle. The molar fraction of the phospholipid or the pharmaceutically acceptable salt thereof in the total lipid of the provided lipid nanoparticle can be, for example, between about between about 8 mol% and about 20 mol% of the total lipid of the lipid nanoparticle, e.g., between about 8 mol% and about 17.6 mol%, between about 8 mol% and about 15.2 mol%, between about 8 mol% and about 12.8 mol%, between about 8 mol% and about 10.4 mol%, between about 10.4 mol% and about 20 mol%, between about 10.4 mol% and about 17.6 mol%, between about 10.4 mol% and about 15.2 mol%, between about 10.4 mol% and about 12.8 mol%, between about 12.8 mol% and about 20 mol%, between about 12.8 mol% and about 17.6 mol%, between about 12.8 mol% and about 15.2 mol%, between about 15.2 mol% and about 20 mol%, between about 15.2 mol% and about 17.6 mol%, or between about 17.6 mol% and about 20 mol%.2. Conjugated Lipid

[0066] 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 a vaccine that includes the lipid nanoparticle, with improved performance in inducing a desired immune response, such as an increase production of T cells, e.g. CD8+T cells specific for aprotein of interest or a fragment thereof. For example, conjugate lipids having two or more hydrophobic tail groups that are not directly bonded to an ester are shown herein as eliciting high T cell production. Further, some lipid nanoparticles providing advantageously high immune responses include conjugated lipids that include a carbamate linkage. 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 T cells, e.g., CD8+T cells specific for a protein of interest or a fragment thereof.

[0067] 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.

[0068] In some examples, at least one of the conjugated lipid species in the lipid nanoparticle has two or more hydrophobic tails, i.e., two or more terminal hydrocarbon chains that each independently has five or more members, where each of these terminal hydrocarbon chains is not directly bonded to an ester. In some examples, each of the conjugated lipids in the lipid nanoparticle has two or more terminal hydrocarbon chains that each independently has five or more members, and that each is not directly bonded to an ester. The number of such terminal hydrocarbon chains in the conjugated lipid can be, for example, two or more 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. In some examples, at least one of the conjugate lipid species in the lipid nanoparticle includes a carbamate linkage. In some embodiments, each conjugated lipid in the lipid nanoparticle includes a carbamate linkage.

[0069] 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 hydrocarbonchain 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.

[0070] In some embodiments, at least one of the ionizable lipid species in the lipid nanoparticle does not include a disulfide bond. In some embodiments, each ionizable lipid of the lipid nanoparticle lacks a disulfide bond. In some examples, at least one of the ionizable lipid species in the lipid nanoparticle does not include sulfur. In some embodiments, each ionizable lipid of the lipid nanoparticle lacks sulfur.

[0071] 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 5 mol% of the total lipid of the lipid nanoparticle. The molar fraction of the conjugated lipid in the total lipid of the provided lipid nanoparticle can be, for example, between about 0.1 mol% and about 5 mol% of the total lipid of the lipid nanoparticle, e.g., between about 0.1 mol% and about 4 mol%, between about 0.1 mol% and about 3 mol%, between about 0.1 mol% and about 2 mol%, between about 0.1 mol% and about 1 mol%, between about 1 mol% and about 5 mol%, between about 1 mol% and about 4 mol%, between about 1 mol% and about 3 mol%, between about 1 mol% and about 2 mol%, between about 2 mol% and about 5 mol%, between about 2 mol% and about 4 mol%, between about 2 mol% and about 3 mol%, between about 3 mol% and about 5 mol%, between about 3 mol% and about 4 mol%, or between about 4 mol% and about 5 mol%.

[0072] 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 less than about 1 mol% of the total lipid of the lipid nanoparticle. The conjugated lipid can constitute, 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 0.82 mol%, between about 0.1 mol% and about 0.64 mol%, between about 0.1 mol% and about 0.46 mol%, between about 0.1 mol% and about 0.28 mol%, between about 0.28 mol% and about 1 mol%, between about 0.28 mol% and about 0.82 mol%, between about 0.28 mol% and about 0.64 mol%, between about 0.28 mol% and about 0.46 mol%, between about 0.46 mol% and about 1 mol%, between about 0.46 mol% and about 0.82 mol%, between about 0.46 mol% and about 0.64 mol%, between about 0.64 mol% and about 1 mol%, between about 0.64 mol% and about 0.82 mol%, or between about 0.82 mol% and about 1 mol%.

[0073] 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.

[0074] 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.

[0075] 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- NEE), 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.

[0076] 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.

[0077] 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 via a 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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. PCT / US08 / 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.

[0082] In some examples, the provided 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 T cells specific for a protein of interest or a fragment thereof. The conjugated lipid can include or consist of, for example , N - [(methoxypoly (ethylene glycol))carbamoyl] - 1 ,2-dimyristyloxy-propylamine (PEG-c-DMA).

[0083] 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 becomesfusogenic 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.3. Ionizable Lipid

[0084] 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 T cells, e.g., CD8+T cells specific for a protein of interest or a fragment thereof. For example, lipid nanoparticles including ionizable lipids having three or more hydrophobic tail groups are shown herein as eliciting high T cell production. Additionally or alternatively, lipid nanoparticles including ionizable lipids lacking sulfur are shown as enhancing the desired immune response. Also, the amount of the ionizable lipid present in the lipid nanoparticle can 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 T cells, e.g., CD8+T cells specific for a protein of interest or a fragment thereof.

[0085] 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.

[0086] 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 ionizablelipid 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). The number of terminal hydrocarbon chains in the ionizable lipid can be, for example, 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. In some embodiments, at least one of the terminal hydrocarbon chains is alky. In some embodiments, each of the terminal hydrocarbon chains is alkyl. In some embodiments, at least one of the terminal hydrocarbon chains is alkenyl. In some embodiments, each of the terminal hydrocarbon chains is alkenyl.

[0087] In some embodiments, at least one of the ionizable lipid species in the lipid nanoparticle does not include a disulfide bond. In some embodiments, each ionizable lipid of the lipid nanoparticle lacks a disulfide bond. In some examples, at least one of the ionizable lipid species in the lipid nanoparticle does not include sulfur. In some embodiments, each ionizable lipid of the lipid nanoparticle lacks sulfur.

[0088] In some examples, the provided lipid nanoparticles induce strong immune responses when the amount of the ionizable lipid or the pharmaceutically acceptable salt thereof in the lipid nanoparticle is greater than about 1 mol% of the total lipid of the lipid nanoparticle. The molar fraction of the ionizable lipid or the pharmaceutically acceptable salt thereof in the total lipid of the provided lipid nanoparticle can be, for example, between about 1 mol% and about 70 mol% of the total lipid of the lipid nanoparticle, e.g., between about 1 mol% and about 30 mol%, between about 1 mol% and about 13 mol%, between about 1 mol% and about 5.5 mol%, between about 1 mol% and about 2 mol%, between about 2 mol% and about 70 mol%, between about 2 mol% and about 30 mol%, between about 2 mol% and about 13 mol%, between about 2 mol% and about 5.5 mol%, between about 5.5 mol% and about 70 mol%, between about 5.5 mol% and about 30 mol%, between about 5.5 mol% and about 13 mol%, between about 13 mol% and about 70 mol%, between about 13 mol% and about 30 mol%, or between about 30 mol% and about 70 mol%.

[0089] In certain examples, the provided lipid nanoparticles induce strong immune responses when the amount of the ionizable lipid or the pharmaceutically acceptable salt thereof in the lipid nanoparticle is between about 30 mol% and about 70 mol% of the total lipid of the lipid nanoparticle. The molar fraction of the ionizable lipid or the pharmaceutically acceptable salt thereof in the total lipid of the provided lipid nanoparticle can be, for example, between about30 mol% and about 62 mol%, between about 30 mol% and about 54 mol%, between about 30 mol% and about 46 mol%, between about 30 mol% and about 38 mol%, between about 38 mol% and about 70 mol%, between about 38 mol% and about 62 mol%, between about 38 mol% and about 54 mol%, between about 38 mol% and about 46 mol%, between about 46 mol% and about 70 mol%, between about 46 mol% and about 62 mol%, between about 46 mol% and about 54 mol%, between about 54 mol% and about 70 mol%, between about 54 mol% and about 62 mol%, or between about 62 mol% and about 70 mol%.4. Cholesterol

[0090] The lipid nanoparticles disclosed herein can further 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 T cells 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 T cells, e.g., CD8+T cells specific for a protein of interest or a fragment thereof.

[0091] In some examples, the provided lipid nanoparticles and vaccines induce strong immune responses when the amount of the cholesterol or the derivative thereof in the lipid nanoparticle is between about 20 mol% and about 60 mol% of the total lipid of the lipid nanoparticle. The molar fraction of the cholesterol or the derivative thereof in the total lipid of the provided lipid nanoparticle can be, for example, between about 20 mol% and about 52 mol%, between about 20 mol% and about 44 mol%, between about 20 mol% and about 36 mol%, between about 20 mol% and about 28 mol%, between about 28 mol% and about 60 mol%, between about 28 mol% and about 52 mol%, between about 28 mol% and about 44 mol%, between about 28 mol% and about 36 mol%, between about 36 mol% and about 60 mol%, between about 36 mol% and about 52 mol%, between about 36 mol% and about 44 mol%, between about 34 mol% and about 60 mol%, between about 34 mol% and about 52 mol%, or between about 52 mol% and about 60 mol%.

[0092] In some embodiments, the lipid nanoparticle includes one species of sterols or a derivative thereof. 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 ormore, nine or more, or ten or more species of sterols. The lipid nanoparticle can include, for example, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, cholesterol hydroxyethyl ether, cholesterol hydroxybutyl ether, cholesterol hydroxyhexyl ether, cholesterol stearate, cholesterol oleate, 7- betahydroxycholesterol, 7-alphahydroxy cholesterol, 4-betahydroxycholesterol, cholesterol PEG, beta sitosterol, or any combination thereof. In certain examples, the lipid nanoparticle includes cholesterol, but substantially no derivative of cholesterol.5. Nucleic Acid

[0093] 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.

[0094] 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.

[0095] 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 besingle-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).

[0096] 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 related embodiments, oligonucleotides, which can 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.

[0097] 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

[0098] 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. In certain examples, the target cell is a cell of the spleen of a subject.

[0099] In some embodiments, the protein of interest is an antigen. The presence of the antigen in the lipid nanoparticles allows the lipid nanoparticles to induce an immune response in subjects to which the lipid nanoparticles 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 T cells, e.g., CD8+T cells, associated with the antigen encoded by the nucleic acid of the lipid nanoparticles.(1) Antigens Associated with Undesired Cell Proliferation

[0100] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is associated with a disease or disorder characterized by an undesired cell proliferation. For example, the antigen can include or consist of a tumor-specific antigen, a tumor-associated differentiation antigen, a tumor-associated overexpressed antigen, a tumor- associated cancer-testis antigen, a tumor-associated viral-associated antigen, or any variants and / or combination thereof.

[0101] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle includes or consists of a tumor-specific antigen. For example, the antigen can include or consist of at least a fragment of any of B Melanoma Antigen 1 (BAGE-1), Fragile X Mental Retardation 1 Neighbor (FMR1NB), Cyclin Al (CCNA1), G Antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, N- Acetylglucosaminyl-Transferase V (GNT-V), Human Endogenous Retrovirus E (HERV-E), HERV-K-MEL, Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1), Coiled-Coil Domain Containing 110 (KM-HN-1), L Antigen Family Member 1 (LAGE-1), LDL Receptor Related Protein Associated Protein 1 (LRPAP1), Lymphocyte Antigen 6 Family Member K (LYSK), Melanoma Antigen Family Al (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE-CI, MAGE-C2, L Antigen Family Member 2 (LAGE-2), Sarcoma Antigen 1 (SAGE), Sperm Autoantigenic Protein 17 (Spl7), Synovial Sarcoma X Breakpoint 2 (SSX-2), SSX-4, Axonal Glycoprotein 1 (TAG-1), TAG-2, Taxol Resistance Associated Gene 3 (TRAG-3), Tyrosinase-Related Protein 2 (TRP-2), X Antigen Family Member IB (XAGE-lb), or any variants and / or combination thereof.

[0102] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle includes or consists of a tumor-associated differentiation antigen. For example, the antigen can include or consist of at least a fragment of any of Carcinoembryonic Antigen Related Cell Adhesion Molecule (CEA), Premelanosome Protein 17 (PMEL17), Secretoglobin Family 2A Member 2 (SCGB2A2), Melanoma Antigen Recognized By T-Cells 1 (MART-1), Ankyrin Repeat Domain 30A (ANKRD30A), Ocular Albinism 1 (OA1), Prostatic Acid Phosphatase (PAP), Prostate-Specific Antigen (PSA), Ras-Related Protein RAB38, Tyrosinase Related Protein 1 (TRP-1), TRP-2, Tyrosinase (TYR), or any variants and / or combination thereof.

[0103] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle includes or consists of a tumor-associated differentiation antigen. For example, the antigen can include or consist of at least a fragment of any of Perilipin 2 (PLIN2), Aldehyde Dehydrogenase 1 Family Member Al (ALDH1A1), Alpha Fetoprotein (AFP), Apoptosis Regulator BCL-X, WD Repeat Domain 46 (WDR46), Calcitonin Related Polypeptide Alpha (CALCA), CD274, CD45, Cleavage And Polyadenylation Specific Factor 1 (CPSF1), Cyclin DI (CCND1), DickkopfWNT Signaling Pathway Inhibitor 1 (DKK1), Enable Homolog Actin Regulator (ENAH), Epithelial Cell Adhesion Molecule (EpCAM), Ephrin Type-A Receptor 3 (EphA3), Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), Fibroblast Growth Factor 5 (FGF5), Carbonic Anhydrase 9 (CA9), Glypican 3 (GPC3), Hepatic And Glial Cell Adhesion Molecule (HEPACM), Hepsin (HPN), Erb-B2 Receptor Tyrosine Kinase 2 (ErbB2), Heat Shock Protein Family H Member 1 (HSPH1), Indoleamine 2,3-Dioxygenase 1 (IDO1), Glypican 3 (GPC3), Interleukin 13 Receptor Subunit Alpha 2 (IL13RA2), IMP U3 Small Nucleolar Ribonucleoprotein 3 (IMP-3), Carboxylesterase 2 (CES2), Kallikrein Related Peptidase 4 (KLK4), Kinesin Family Member 20A (KIF20A), Lengsin (LGSN), Colony Stimulating Factor 1 (CSF1), Chondroitin Sulfate Proteoglycan 4 (CSPG4), Mouse Double Minute 2 Proto-Oncogene (MDM-2), Melanoma-Overexpressed Antigen (MELOE), Midkine (MDK), Matrix Metallopeptidase 2 (MMP-2), MMP-7, Mucin 1 (MUC1), MUC5AC, Nectin Cell Adhesion Molecule 4 (Nectin-4), Tumor Protein P53, Paired Box 5 (PX5), Zinc Finger Protein 395 (ZNF395), Placenta Enriched 1 (PLAC1), Preferentially Expressed Antigen in Melanoma Nuclear Receptor Transcriptional Regulator (PRAME), Folate Hydrolase 1 (FOLH1), Renal Tumor Antigen 1 (RAGE-1), Regulator Of G Protein Signaling 5 (RGS5), Ras Homolog Family Member C (RHOC), Ring Finger Protein 43 (RNF43), Doublecortin Domain Containing 2 (DCDC2), Secemin 1 (SCRN1), Sex Determining Region Y-Box Transcription Factor 10 (SOX 10), Six-Transmembrane Epithelial Antigen Of Prostate Family Member 1 (STEAP1), Baculoviral IAP Repeat Containing 5 (BIRC5), Telomerase, Telomerase Reverse Transcriptase (TERT), Trophoblast Glycoprotein (TPBG), Vascular Endothelial Growth Factor (VEGF), Wilms Tumor Protein Transcription Factor 1 (WT1), or any variants and / or combination thereof.

[0104] In some embodiments the antigen encoded by the nucleic acid of the provided lipid nanoparticle includes or consists of a subject-specific cancer antigen. The subject-specific cancer antigen can be one identified by analyzing a sample derived from the subject, e.g., a patient suffering from the cancer. For example, the subject-specific cancer antigen can be aprotein or a fragment thereof that is expressed at lower levels in non-cancerous cells or tissues of the subject. In some embodiments, the subject-specific cancer antigen is not detectably expressed in non-cancerous cells or tissues of the subject. Additionally or alternatively, the subject-specific cancer antigen can be a protein or a fragment thereof having a mutated amino acid sequence relative to a corresponding protein or protein fragment of non-cancerous cells or tissues of the subject.(2) Antigens Associated with Infections

[0105] In some examples, the antigen encoded by the nucleic acid of the provided lipid nanoparticle is associated with an infection, e.g., the antigen 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, Clostridium species, Corynebacterium species, Coxiella burnetiid, Ehrlichia species, Eikenella corrodens, Enterobacter species, Enterococcus faecalis, Enterococcus faecium, 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.

[0106] 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, Scedosporiumspecies, Cryptococcus gattii, Lomentospora prolificans, Talaromyces marneffei, Coccidioides species, Pneumocystis jirovecii. Pichia kudriavzeveii (Candida krusei)'. Paracoccidioides species, or a combination thereof.

[0107] 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.

[0108] 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, Fasciola hepatica, Schistosoma mansoni, Toxocara canis, Toxocara cati, Wuchereria bancrofti, 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.

[0109] 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 1, Example2, and Example 3 demonstrate use of provided lipid nanoparticles to encapsulate a nucleic acid, i.e., mRNA, encoding hemagglutinin.

[0110] 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[oni] 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 or consists 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 an immune response, e.g., a CD8+T cell response, against an undesired cell proliferation (e.g., a cancer), an infection, or an autoimmune 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 T cells that target the pathogen.

[0112] 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 ofmRNA 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

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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 .

[0117] 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.

[0118] 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.

[0119] 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

[0120] 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)

[0121] 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

[0122] 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.

[0123] 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 sensenucleotide 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

[0124] 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

[0125] 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.

[0126] 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

[0127] 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).

[0128] 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.

[0129] 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 lipidnanoparticles 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

[0130] 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, e.g., a disease associated with an undesired cell proliferation (e.g., a cancer) or with an infection, or an autoimmune 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 T cells that target the pathogen.

[0131] 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 ofcircRNAs 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.

[0132] 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.

[0133] 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

[0134] 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, e.g., a disease associated with an undesired cell proliferation (e.g., a cancer) or with an infection, or an autoimmune 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 T cells that target the pathogen.

[0135] 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., saRNA having 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.

[0136] 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

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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 oil suspensions, 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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

[0147] 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.

[0148] 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.

[0149] 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

[0150] In other aspects, the disclosure provides several methods for making and / or using the lipid nanoparticles and vaccines described in Section C, the pharmaceutical compositions described 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 T cells, e.g., CD8+T cells specific for a protein of interest or a fragment thereof, in the subject.1. Preparing Lipid Nanoparticles

[0151] 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, themethods 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.

[0152] 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 the aqueous 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.

[0153] 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 phospholipid can be any of those described in Section C.1, the conjugated lipid can be any of those described in Section C.2, the ionizable lipid can be any of those described in Section C.3, and the sterol can be cholesterol or any of its derivatives 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.2. Inducing an Immune Response

[0163] 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 stimulating T cells, e.g., CD8+T cells specific for a protein of interest or a fragment thereof. The provided methods can be used to induce an immune response against, for example, an undesired cell proliferation (e.g., a cancer), an infection, or an autoimmune disease.

[0164] 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 an undesired cell proliferation or pathogenic infection being treated, or to reduce side effects caused by induction of the immune response.

[0165] 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.

[0166] In some embodiments, the immune response is assessed by detecting T cells obtained from the subject, for example using fluorescence-activated cell sorting (FACS). In some embodiments, the immune response includes generation of T cells associated with 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.

[0167] 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.3. Preventing or Treating a Disease or Disorder

[0168] 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.

[0169] In some embodiments, the prevented or treated disease or disorder is an autoimmune disease. Non-limiting examples of autoimmune diseases that can be treated with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions include celiac disease, type 1 diabetes, Graves’ disease, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, acquired aplastic anemia, psoriasis, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, systemic lupus erythematosus, Sjogren’s syndrome, undifferentiated connective tissue disease, Addison disease, dermatomyositis, Hashimoto thyroiditis, myasthenia gravis, ankylosingspondylitis (AS), antiphospholipid syndrome (APS), autoimmune autonomic ganglionopathy (AAG), autoimmune encephalitis, autoimmune gastritis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), autoimmune polyglandular syndromes, autoimmune progesterone dermatitis, Balo disease, Behcet’s disease, bullous pemphigoid, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cold agglutinin disease, CREST syndrome | limited cutaneous systemic sclerosis, dermatitis herpetiformis, discoid lupus, eosinophilic fasciitis, Evans syndrome, glomerulonephritis, Goodpasture’s syndrome, granulomatosis with polyangiitis (GPA), Guillain-Barre syndrome (GBS), Henoch-Schonlein purpura, Hurst’s disease, IgA nephropathy, immune thrombocytopenia (ITP), juvenile idiopathic arthritis, Lambert-Eaton myasthenic syndrome (LEMS), linear IgA disease (LAD), lupus nephritis, lymphocytic hypophystitis, Meniere’s disease, mixed connective tissue disease (MCTD), Devic's disease, ocular cicatricial pemphigoid, palindromic rheumatism, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, paroxysmal nocturnal hemoglobinuria (PNH), Parsonage-Turner syndrome, Pemphigus gestationis, Pemphigus foliaceus, Pemphigus vulgaris, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis (PBC), Palmoplantar Pustulosis, pure red cell aplasia (PRCA), Raynaud’s syndrome, reactive arthritis, relapsing polychondritis, rheumatic fever, sarcoidosis, Schmidt syndrome, scleroderma, small fiber sensory neuropathy, testicular autoimmunity, vitiligo, and combinations thereof.

[0170] In some embodiments, the prevented or treated disease or disorder is one characterized by an undesired cell proliferation. Non-limiting examples of such diseases and disorders that can be treated with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions include cancers, benign prostatic hyperplasia (BPH), psoriasis, endometriosis, polycystic ovary syndrome (PCOS), hypertrophic cardiomyopathy, pulmonary fibrosis, Grave’s disease, fibroadenomas, nodular hyperplasia of the prostate, adenomyosis, uterine fibroids, adenomatous goiter, adenomatous polyps of the colon, non-malignant basal cell carcinoma, cervical dysplasia, cutaneous warts, fibrous dysplasia, gastric hyperplastic polyps, giant cell tumor of bone, glomerulonephritis, hepatic adenoma, hyperplastic polyps of the colon, hyperthyroid goiter, keloids, leiomyomas, mastocytosis, medullary sponge kidney, myeloproliferative disorders, nevi, non-alcoholic fatty liver disease (NAFLD), osteochondromas, osteoid osteoma, osteosclerosis, ovarian cysts, pancreatic pseudocysts,parathyroid hyperplasia, Peutz-Jeghers syndrome, proliferative diabetic retinopathy, and combinations thereof.

[0171] In some embodiments, the prevented or treated disease is a cancer. Non-limiting examples of cancers that can be treated with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions include Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Eymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma,Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast- ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer,Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

[0172] In some embodiments, the prevented or treated disease is a cancerous tumor. The cancerous tumor can be a solid cancerous tumor or a liquid cancerous tumor. The liquid cancerous tumor can be, for example, a lymphoma or a leukemia. A tumor treated with the methods disclosed herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and / or do not metastasize). In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, a tumor is stabilized for at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the tumor is completely eliminated, or reduced below a level of detection. In some embodiments, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

[0173] One skilled in the art will also appreciate that the provided systems and compositions can be co-administered with other therapeutic agents for the treatment of cancer. Suitable anticancer agents for combination therapy include, without limitation, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, interferons, radiopharmaceuticals, peptides with anti-tumor activity such as TNF-a, pharmaceutically acceptable salts thereof; derivatives thereof, prodrugs thereof, and combinations thereof. For example, a pharmaceutical composition comprising the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and / or virus-like particles can be administered to a patient before, during, or after administration of an anti-cancer agent or combination of anti-cancer agents either before, during, or after chemotherapy.

[0174] In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions results in stable disease, partial remission or complete remission in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions that kills or otherwise slows the growth or progression of cancer cells and leads to stable disease or to partial or complete remission of the cancer in the subject). In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions results in a reduction in metastases of the cancer in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions that reduces metastases of the cancer in the subject). In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions results in a reduction in volume, size, or growth of a tumor in the subject (e.g., the methods described herein comprise administering to thesubject a dose of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions that reduces the volume, size, or growth of a tumor in the subject). In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions results in an increased responsiveness of the cancer to a subsequently administered anti -cancer agent (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions that increases responsiveness of the cancer to a subsequently administered anticancer agent).

[0175] In some examples, 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.

[0176] 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.

[0177] 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 species of T cells, e.g., CD8+T cells. 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.

[0178] 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.

[0179] 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.

[0180] 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 obtainedfrom normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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 thepresence 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.

[0185] In some embodiments, the provided method further includes the step of providing to the subject a diagnosis and / or the results of treatment.G. EXEMPLARY EMBODIMENTS

[0186] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0187] Embodiment 1: A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof; a conjugated lipid comprising between 0.1 mol% and 5 mol% of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof, the ionizable lipid comprising three or more terminal hydrocarbon chains that each independently has five or more members; and a nucleic acid encoding a protein of interest or a fragment thereof; wherein the lipid nanoparticle is configured to have a negative zeta potential at physiological pH.

[0188] Embodiment 2: An embodiment of embodiment 1, wherein the phospholipid has a net negative charge at physiological pH.

[0189] Embodiment 3: An embodiment of embodiment 1 or 2, wherein the conjugated lipid comprises between 0.1 mol% and 1 mol% of the total lipid of the lipid nanoparticle.

[0190] Embodiment 4: An embodiment of any one of embodiments 1-3, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the lipid nanoparticle.

[0191] Embodiment 5: An embodiment of any one of embodiments 1-4, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

[0192] Embodiment 6: A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net negative charge at physiological pH; a conjugated lipid comprising less than 1 mol% of the total lipid of the lipidnanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof; and a nucleic acid encoding a protein of interest or a fragment thereof.

[0193] Embodiment 7 : An embodiment of embodiment 6, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the lipid nanoparticle.

[0194] Embodiment 8: An embodiment of embodiment 6 or 7, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

[0195] Embodiment 9: A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net negative charge at physiological pH and comprising at least 8 mol% of the total lipid of the lipid nanoparticle; a conjugated lipid comprising between 0.1 mol % and 5 mol % of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof; and a nucleic acid encoding a protein of interest or a fragment thereof.

[0196] Embodiment 10: An embodiment of embodiment 9, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

[0197] Embodiment 11: A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net negative charge at physiological pH; a conjugated lipid comprising between 0.1 mol% and 5 mol% of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof comprising more than 1 mol% of the total lipid of the lipid nanoparticle, the ionizable lipid not comprising sulfur; and a nucleic acid encoding a protein of interest or a fragment thereof.

[0198] Embodiment 12: An embodiment of any one of embodiments 1-11, wherein the phospholipid comprises a naturally occurring phospholipid.

[0199] Embodiment 13: An embodiment of any one of embodiments 1-12, wherein the phospholipid comprises a phosphatidylserine.

[0200] Embodiment 14: An embodiment of embodiment 13, wherein the phosphatidylserine comprises l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diheptadecanoyl-sn- glycero-3-phospho-L-serine, l,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), l,2-didecanoyl-sn-glycero-3-phospho- L-serine, l,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), l,2-dilauroyl-sn-glycero-3- phospho-L-serine (DLPS), l,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, 1,2- didocosahexaenoyl-sn-glycero-3-phospho-L-serine, l-pentadecanoyl-2-oleoyl-sn-glycero-3- phospho-L-serine, 1 -palmitoyl -2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), l-stearoyl-2- oleoyl-sn-glycero-3-phospho-L-serine (SOPS), 1 -palmitoyl -2 -linoleoyl-sn-glycero-3- phospho-L-serine, l-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, 1 -palmitoyl -2- docosahexaenoyl-sn-glycero-3-phospho-L-serine, l-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, l-stearoyl-2- docosahexaenoyl-sn-glycero-3-phospho-L-serine, or a combination thereof.

[0201] Embodiment 15: An embodiment of any one of embodiments 1-12, wherein the phospholipid comprises a phosphatidylglycerol.

[0202] Embodiment 16: An embodiment of embodiment 15, wherein the phosphatidylglycerol comprises l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG),1.2-diheptadecanoyl-sn-glycero-3-phospho-(l'-rac-glycerol), l,2-didecanoyl-sn-glycero-3- phospho-(l'-rac-glycerol), l,2-dilauroyl-sn-glycero-3-phospho-(l'-rac -glycerol) (DLPG), 1,2- dimyristoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol) (DMPG), 1 ,2-dipentadecanoyl-sn- glycero-3 -phospho-( 1 '-rac-glycerol), 1 ,2-dipalmitoyl-sn-glycero-3 -phospho-( 1' -rac-glycerol) (DPPG), l,2-distearoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DSPG), 1,2-dielaidoyl-sn- glycero-3-phospho-(l'-rac-glycerol), l,2-dilinoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol),1.2-dilinolenoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l,2-diarachidonoyl-sn-glycero-3-[phospho-rac-( 1 -glycerol)], l,2-didocosahexaenoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol) (POPG), 1 -palmitoyl-2- linoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), 1 -palmitoyl -2 -arachidonoyl-sn-glycero-3- phospho-( 1 '-rac-glycerol) (PAPG), 1 -palmitoyl-2-docosahexaenoyl-sn-glycero-3 -phospho-( 1 '- rac-glycerol), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (SOPG), 1-stearoyl- 2 -linoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l-stearoyl-2-arachidonoyl-sn-glycero-3- phospho-( 1 '-rac-glycerol) (S APG), 1 -stearoyl -2 -docosahexaenoyl-sn-glycero-3 -phospho-( 1 '- rac-glycerol), or a combination thereof.

[0203] Embodiment 17: An embodiment of any one of embodiments 1-16, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the lipid nanoparticle.

[0204] Embodiment 18: An embodiment of any one of embodiments 1-17, 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.

[0205] Embodiment 19: An embodiment of any one of embodiments 1-18, wherein the conjugated lipid comprises a carbamate linkage.

[0206] Embodiment 20: An embodiment of any one of embodiments 1-19, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

[0207] Embodiment 21: An embodiment of any one of embodiments 1-20, wherein the conjugated lipid comprises N- [(methoxypoly (ethylene glycol))carbamoyl]-l,2-dimyristyloxy- propylamine (PEG-c-DMA).

[0208] Embodiment 22: An embodiment of any one of embodiments 1-21, wherein the ionizable lipid does not comprise a disulfide bond.

[0209] Embodiment 23: An embodiment of any one of embodiments 1-22, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

[0210] Embodiment 24: An embodiment of any one of embodiments 1-23, 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).

[0211] Embodiment 25: An embodiment of any one of embodiments 1-24, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the lipid nanoparticle.

[0212] Embodiment 26: An embodiment of any one of embodiments 1-25, wherein the lipid nanoparticle further comprises cholesterol or a derivative thereof.

[0213] Embodiment 27 : An embodiment of embodiment 26, wherein the cholesterol or the derivative thereof comprise between 20 mol% and 60 mol% of the total lipid of the lipid nanoparticle.

[0214] Embodiment 28: An embodiment of any one of embodiments 1-27, 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.

[0215] Embodiment 29: An embodiment of any one of embodiments 1-27, wherein the lipid nanoparticle does not comprise a permanently charged cationic lipid.

[0216] Embodiment 30: An embodiment of any one of embodiments 1-29, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 8.5 mol% and 10.5 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprises between 0.25 mol% and 0.9 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 48 mol% and 58 mol% of the total lipid of the lipid nanoparticle; and the lipid nanoparticle further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 35 mol% and 39 mol% of the total lipid of the lipid nanoparticle.

[0217] Embodiment 31: An embodiment of any one of embodiments 1-29, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 9 mol% and 11 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprises between 0.85 mol% and 2.6 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 45 mol% and 55 mol% of the total lipid of the lipid nanoparticle; and the lipid nanoparticle further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 36.5 mol% and 40.5 mol% of the total lipid of the lipid nanoparticle.

[0218] Embodiment 32: An embodiment of embodiment 30 or 31, wherein: the phospholipid comprises DOPG, DOPS. or a combination thereof; the conjugated lipid comprises PEG-c- DMA; and the ionizable lipid comprises 3D-P-DMA.

[0219] Embodiment 33: An embodiment of any one of embodiments 1-32, wherein the nucleic acid comprises RNA.

[0220] Embodiment 34: An embodiment of embodiment 33, wherein the RNA comprises mRNA, saRNA, circRNA or a combination thereof.

[0221] Embodiment 35: An embodiment of any one of embodiments 1-32, wherein the nucleic acid comprises DNA.

[0222] Embodiment 36: An embodiment of any one of embodiments 1-35, wherein the protein of interest comprises an antigen.

[0223] Embodiment 37: An embodiment of embodiment 36, wherein the antigen is associated with a disease characterized by an undesired cell proliferation, with an infection, or with an autoimmune disease.

[0224] Embodiment 38: An embodiment of embodiment 37, wherein the antigen comprises a cancer antigen.

[0225] Embodiment 39: An embodiment of embodiments 38, wherein the cancer antigen comprises a subject-specific cancer antigen.

[0226] Embodiment 40: 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, and a phospholipid or a pharmaceutically acceptable salt thereof; and combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle; wherein the conjugated lipid comprises between 0.1 mol% and 5 mol% of the total lipid of the alcoholic solution, and the composition of the alcoholic solution is configured such that the lipid nanoparticle has a negative zeta potential at physiological pH.

[0227] Embodiment 41 : An embodiment of embodiment 40, wherein the phospholipid has a net negative charge at physiological pH.

[0228] Embodiment 42: An embodiment of embodiment 40 or 41, wherein the conjugated lipid comprises between 0.1 mol% and 1 mol% of the total lipid of the alcoholic solution.

[0229] Embodiment 43: An embodiment of any one of embodiments 40-42, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the alcoholic solution.

[0230] Embodiment 44: An embodiment of any one of embodiments 40-43, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

[0231] Embodiment 45 : 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, and aphospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net negative 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 less than 1 mol% of the total lipid of the alcoholic solution.

[0232] Embodiment 46: An embodiment of embodiment 45, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the lipid nanoparticle.

[0233] Embodiment 47: An embodiment of embodiment 45 or 46, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

[0234] Embodiment 48: 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, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net negative 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 comprising between 0.1 mol% and 5 mol% of the total lipid of the alcoholic solution, and the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the alcoholic solution.

[0235] Embodiment 49: An embodiment of embodiment 48, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

[0236] Embodiment 50: 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, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net negative 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 comprising between 0.1 mol% and 5 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises morethan 1 mol% of the total lipid of the alcoholic solution; and the ionizable lipid does not comprise sulfur.

[0237] Embodiment 51: An embodiment of any one of embodiments 40-50, wherein the method further comprises removing at least a portion of the alcoholic organic solvent from the lipid nanoparticle solution.

[0238] Embodiment 52: An embodiment of embodiment 51, wherein the removing of at least a portion of the alcoholic organic solvent comprises exchanging the alcoholic organic solvent with an aqueous solution.

[0239] Embodiment 53: An embodiment of any one of embodiments 40-52, wherein the method further comprises concentrating the lipid nanoparticle solution.

[0240] Embodiment 54: An embodiment of embodiment 53, wherein the concentrating of the lipid nanoparticle precursor comprises centrifuging the lipid nanoparticle solution.

[0241] Embodiment 55: An embodiment of any one of embodiments 40-54, wherein the method further comprises filtering the lipid nanoparticle solution.

[0242] Embodiment 56: An embodiment of any one of embodiments 40-55, wherein the alcoholic organic solvent comprises ethanol.

[0243] Embodiment 57: An embodiment of any one of embodiments 40-56, 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.

[0244] Embodiment 58: An embodiment of embodiment 57, wherein the nonalcoholic organic solvent comprises tetrahydrofuran.

[0245] Embodiment 59: An embodiment of any one of embodiments 40-58, wherein the phospholipid comprises a naturally occurring phospholipid.

[0246] Embodiment 60: An embodiment of any one of embodiments 40-59, wherein the phospholipid comprises a phosphatidylserine.

[0247] Embodiment 61 : An embodiment of embodiment 60, wherein the phosphatidylserine comprises DOPS, l,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine, DSPS, DMPS, 1,2- didecanoyl-sn-glycero-3-phospho-L-serine, DPPS, DLPS, l,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine, 1 -pentadecanoyl- 2-oleoyl-sn-glycero-3-phospho-L-serine, POPS, SOPS, 1 -palmitoyl -2 -linoleoyl-sn-glycero-3- phospho-L-serine, l-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, 1 -palmitoyl -2- docosahexaenoyl-sn-glycero-3 -phospho-L-serine, l-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, l-stearoyl-2- docosahexaenoyl-sn-glycero-3 -phospho-L-serine, or a combination thereof.

[0248] Embodiment 62 An embodiment of any one of embodiments 40-61, wherein the phospholipid comprises a phosphatidylglycerol.

[0249] Embodiment 63: An embodiment of embodiment 62, wherein the phosphatidylglycerol comprises DOPG, l,2-diheptadecanoyl-sn-glycero-3-phospho-(l'-rac- glycerol), l,2-didecanoyl-sn-glycero-3-phospho-(l'-rac-glycerol), DLPG, DMPG, 1,2- dipentadecanoyl-sn-glycero-3-phospho-(l'-rac -glycerol), DPPG, DSPG, 1,2-dielaidoyl-sn- glycero-3-phospho-(l'-rac-glycerol), l,2-dilinoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l,2-dilinolenoyl-sn-glycero-3-phospho-(l'-rac -glycerol), l,2-diarachidonoyl-sn-glycero-3-[phospho-rac-( 1 -glycerol)], l,2-didocosahexaenoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], POPG, l-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(l'-rac -glycerol), PAPG, 1 -palmitoyl -2- docosahexaenoyl-sn-glycero-3-phospho-(l'-rac-glycerol), SOPG, l-stearoyl-2-linoleoyl-sn- glycero-3-phospho-(l'-rac-glycerol), SAPG, 1 -stearoyl -2 -docosahexaenoyl-sn-glycero-3 - phospho-(l'-rac-glycerol), or a combination thereof.

[0250] Embodiment 64: An embodiment of any one of embodiments 40-63, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the alcoholic solution.

[0251] Embodiment 65: An embodiment of any one of embodiments 40-64, 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.

[0252] Embodiment 66: An embodiment of any one of embodiments 40-65, wherein the conjugated lipid comprises a carbamate linkage.

[0253] Embodiment 67: An embodiment of any one of embodiments 40-66, wherein the conjugated lipid comprises a PEG-lipid conjugate.

[0254] Embodiment 68: An embodiment of any one of embodiments 40-67, wherein the conjugated lipid comprises PEG-c-DMA.

[0255] Embodiment 69: An embodiment of any one of embodiments 40-68, wherein the ionizable lipid does not comprise a disulfide bond.

[0256] Embodiment 70: An embodiment of any one of embodiments 40-69, wherein the ionizable lipid does not comprise sulfur.

[0257] Embodiment 71: An embodiment of any one of embodiments 40-70, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

[0258] Embodiment 72: An embodiment of any one of embodiments 40-71, wherein the ionizable lipid comprises 3D-P-DMA.

[0259] Embodiment 73: An embodiment of any one of embodiments 40-72, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the alcoholic solution.

[0260] Embodiment 74: An embodiment of any one of embodiments 40-73, wherein the alcoholic solution further comprises cholesterol or a derivative thereof.

[0261] Embodiment 75: An embodiment of embodiment 74, wherein the cholesterol or the derivative thereof comprise between 20 mol% and 60 mol% of the total lipid of the alcoholic solution.

[0262] Embodiment 76: An embodiment of any one of embodiments 40-75, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 8.5 mol% and 10.5 mol% of the total lipid of the alcoholic solution; the conjugated lipid comprises between 0.25 mol% and 0.9 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 48 mol% and 58 mol% of the total lipid of the alcoholic solution; and the alcoholic solution further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 35 mol% and 39 mol% of the total lipid of the alcoholic solution.

[0263] Embodiment 77: An embodiment of any one of embodiments 40-75, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 9 mol% and 11 mol% of the total lipid of the alcoholic solution; the conjugated lipid comprises between 0.85 mol% and 2.6 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 45 mol% and 55 mol% of thetotal lipid of the alcoholic solution; and the alcoholic solution further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 36.5 mol% and 40.5 mol% of the total lipid of the alcoholic solution.

[0264] Embodiment 78: An embodiment of embodiment 76 or 77, wherein: the phospholipid comprises DOPG, DOPS. or a combination thereof; the conjugated lipid comprises PEG-c- DMA; and the ionizable lipid comprises 3D-P-DMA.

[0265] Embodiment 79: An embodiment of any one of embodiments 40-78, wherein the nucleic acid comprises RNA.

[0266] Embodiment 80: An embodiment of embodiment 79, wherein the RNA comprises mRNA, saRNA, circRNA or a combination thereof.

[0267] Embodiment 81: An embodiment of any one of embodiments 40-78, wherein the nucleic acid comprises DNA.

[0268] Embodiment 82: An embodiment of any one of embodiments 40-81, wherein the protein of interest comprises an antigen.

[0269] Embodiment 83: An embodiment of embodiment 82, wherein the antigen is associated with a disease characterized by an undesired cell proliferation, with an infection, or with an autoimmune disease.

[0270] Embodiment 84: An embodiment of embodiment 83, wherein the antigen comprises a cancer antigen.

[0271] Embodiment 85: An embodiment of embodiment 84, wherein the cancer antigen comprises a subject-specific cancer antigen.

[0272] Embodiment 86: A lipid nanoparticle obtainable by the method of any one of embodiments 40-85.

[0273] Embodiment 87: A vaccine comprising the lipid nanoparticle of any one of embodiments 1-39 or 86.

[0274] Embodiment 88: An embodiment of embodiment 87, wherein the protein of interest comprises an antigen.

[0275] Embodiment 89: An embodiment of embodiment 88, wherein the antigen comprises a cancer antigen, and wherein the vaccine is a cancer vaccine.

[0276] Embodiment 90: An embodiment of embodiment 89, wherein the cancer antigen comprises a subject-specific cancer antigen, and wherein the cancer vaccine is a personalized cancer vaccine.

[0277] Embodiment 91: An embodiment of any one of embodiments 87-90, wherein the vaccine further comprises a pharmaceutically acceptable excipient.EXAMPLES

[0278] 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.

[0279] Nucleic acid was encapsulated in lipid nanoparticles using a controlled mixing process of the type described in US Patent No. 9,005,654. In this process, an aqueous solution of mRNA in 100 mM acetate buffer at pH 5.0 was combined with an ethanolic solution of lipids. The ethanolic solution included 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 a phospholipid (zwitterionic or negatively charged) at various molar ratios. 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) and filtered 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 Zetasizer Nano-ZS (Malvern Panalytical, Malvern, Worcestershire, United Kingdom). To create empty LNPs, no payload was encapsulated but the LNPs were otherwise prepared using the same process. Characteristics of exemplary and comparative LNPs prepared according to these procedures are outlined in Table 1 (Example 2) and Table 2 (Example 3).Example 2. Measurement of lipid nanoparticle zeta potentials.

[0280] The zeta potentials of lipid nanoparticles described herein were measured at different pH values using the Zetasizer Nano-ZS. Buffers ranging from pH 4.6 to 7.6 were prepared by mixing 0.1 M citric acid and 0.2 M Na2HPC>4 solutions at different ratios. The buffers were diluted 1 / 20 with water for injection (WFI) prior to using for LNP sample preparation. TheZetasizer was set according to the following parameters: Dispersant: water, General options (Model): Smoluchowski, Temperature: 25 °C, Equilibrium time: 120 seconds, Cell type: Disposable Folded Capillary Cell (DTS1070), Measurement: Automatic, Measurement (Minimum Runs): 12, Measurement (Maximum Runs): 12, Measurement (Number of Measurements): 3, Measurement (Delay Between Measurements): 0, Advanced (Measurement setting): Automatic Attenuation selection, Advanced (Voltage): Automatic Voltage selection)

[0281] In the assay procedure, a cell loaded with a standard solution was inserted into the Zetasizer and a measurement was recorded according to the above parameters (Zetapotential transfer standard acceptance range: -40 mV + / - 5.8 mV). The standard was decanted, and the cuvette was flushed with WFI and rinsed twice with the buffer used for measuring LNP Zetapotential. Next, 1 m of LNP sample was prepared by diluting stock LNP to 0.005 mg / mL using the buffer intended for Z-Potential measurement. The Zeta cell was then loaded with 800 pL of the prepared sample. Analysis of results involved plotting Zeta-Potential measurement averages per buffer vs pH values, and / or reporting the average zeta potential at the physiological pH value of 7.4.

[0282] FIG. 1 presents a graph of Zeta-Potential measurements for empty lipid nanoparticles prepared according to the formulations listed in Table 1, where these measurements are plotted over a range of pH values that includes physiological pH 7.4. The data demonstrate that the formulations of LNP C and LNP D each result in a particle exhibiting a negative zeta potential at pH values greater than approximately 6.4.Table 1aZ-average and PDI were measured on a Malvern Zetasizer with dynamic light scattering.bPDI = polydispersity index.cEE = Encapsulation Efficiency.Example 3. CD8+T cell responses in mice treated with lipid nanoparticles.

[0283] Two lipid nanoparticle compositions were prepared following the procedure of Example 1 and using the formulations listed in Table 2 with 3 pg of influenza hemagglutinin (HA) mRNA (SEQ ID NO: 2). The LNP compositions were diluted in phosphate buffered saline (PBS) and injected into the gastrocnemius muscle (25 pL injection volume) of mice witha 3 / 10 mL, 29G x 1 / 2-inch insulin syringe (Covidien #8881600145). Post termination at Day 12 after immunization spleens were mashed in complete Roswell Park Memorial Institute (RPMI) medium 1640 (ATCC) on ice and filtered through a 70 pm cell strainer. Red blood cells (RBCs) were lysed with 2 mL Red Blood Cell Lysing Buffer HYBRI-MAX™ (Sigma R7757) for 5 minutes on ice and the reaction was stopped with ten times the volume of RPMI. Then, 7.5 x 106cells per sample were stimulated for 16 h at 37 °C and 5% CO2, in the presence of HA peptide pool (JPT Peptide Technologies) at 2 pg / mL per peptide. Brefeldin A Solution (Thermofisher 00-4506-52) was added to each sample from one hour after the start of the stimulation. Unstimulated samples for each animal were included. Concanavalin A stimulated samples were included as a positive control.Table 2aZ-average and PDI were measured on a Malvern Zetasizer with dynamic light scattering.bPDI = polydispersity index.cEE = Encapsulation Efficiency.

[0284] After stimulation, cells were washed with PBS and stained for 30 min in the dark at 4 °C with the LIVE / DEAD fixable violet dead cell stain kit (Thermo Fisher Scientific). Samples were incubated in the Fc blocker (Purified Rat Anti -Mouse CD16 / CD32, BD Biosciences) for 10 min in the dark at 4 °C and then surface-stained with the monoclonal antibodies anti-CD3 AF488 (Clone 145-2C11, BD Biosciences), anti-CD4 BUV395 (clone GK1.5, BD Biosciences) and anti-CD8 PE (clone 53-6.7, BD Biosciences) for 30 min at 4 °C. After surface staining, cells were washed with fluorescence-activated cell sorting (FACS) stain buffer, fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (BD Biosciences) and washed again using the permeabilization buffer (Thermo Fisher Scientific). Cells were intracellularly stained with anti-TNF-a allophycocyanin (APC) (clone MP6-XT22, BD Biosciences) anti-IFN-y APC (clone XMG1.2, BD Biosciences), and anti-IL- 2 APC (clone JES6-5H4, BioLegend) monoclonal antibodies for 30 min at 4 °C. Staining of each cytokine was performed in separate wells. Next, the cells were washed once with the permeabilization buffer (Thermo Fisher Scientific) and once with FACS stain buffer. Cells were then resuspended in FACS buffer and analyzed immediately. Splenocytes were analyzed on an LSRFORTESSA™ X-20 Cell Analyzer (BD Biosciences). In all, 150,000 events were collected per specimen. Data were analyzed with the BD FACSDIVA™ program. Data wereexpressed by subtracting the percentages of the unstimulated stained cells from the percentages of the peptide pool-stimulated stained samples.

[0285] Results of this experiment are shown in FIGS. 2-4, which plot the percentages of CD8+IFNg+T cells, CD8+TNF-a+T cells, and CD8+IL-2+T cells, respectively, for mice administered lipid nanoparticles have the LNP A and LNP D formulations listed in Table 2 with 3 pg of HA mRNA. The data demonstrate that lipid nanoparticles with the LNP D formulation stimulated CD8+T cell amounts to a significantly greater extent than did lipid nanoparticles with the LNP A formulation. These findings therefore provide an example of the greater immune response that can be achieved by delivering to a subject a protein of interest that is encapsulated in a lipid nanoparticle that has a negative zeta potential at physiological pH, and that includes a phospholipid or a pharmaceutically acceptable salt thereof where the phospholipid has a net negative charge at physiological pH.

[0286] 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 phospholipid or a pharmaceutically acceptable salt thereof; a conjugated lipid comprising between 0. 1 mol% and 5 mol% of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof, the ionizable lipid comprising three or more terminal hydrocarbon chains that each independently has five or more members; and a nucleic acid encoding a protein of interest or a fragment thereof; wherein the lipid nanoparticle is configured to have a negative zeta potential at physiological pH.

2. The lipid nanoparticle of claim 1, wherein the phospholipid has a net negative charge at physiological pH.

3. The lipid nanoparticle of claim 1 or 2, wherein the conjugated lipid comprises between 0.1 mol% and 1 mol% of the total lipid of the lipid nanoparticle.

4. The lipid nanoparticle of any one of claims 1-3, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the lipid nanoparticle.

5. The lipid nanoparticle of any one of claims 1-4, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

6. A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net negative charge at physiological pH; a conjugated lipid comprising less than 1 mol% of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof; and a nucleic acid encoding a protein of interest or a fragment thereof.

7. The lipid nanoparticle of claim 6. wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the lipid nanoparticle.

8. The lipid nanoparticle of claim 6 or 7, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

9. A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net negative charge at physiological pH and comprising at least 8 mol% of the total lipid of the lipid nanoparticle; a conjugated lipid comprising between 0. 1 mol % and 5 mol % of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof; and a nucleic acid encoding a protein of interest or a fragment thereof.

10. The lipid nanoparticle of claim 9, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

11. A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, the phospholipid having a net negative charge at physiological pH; a conjugated lipid comprising between 0. 1 mol% and 5 mol% of the total lipid of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof comprising more than 1 mol% of the total lipid of the lipid nanoparticle, the ionizable lipid not comprising sulfur; and a nucleic acid encoding a protein of interest or a fragment thereof.

12. The lipid nanoparticle of any one of claims 1-11, wherein the phospholipid comprises a naturally occurring phospholipid.

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

14. The lipid nanoparticle of claim 13, wherein the phosphatidylserine comprises l,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diheptadecanoyl-sn- glycero-3-phospho-L-serine, l,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2- dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), l,2-didecanoyl-sn-glycero-3-phospho- L-serine, l,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), l,2-dilauroyl-sn-glycero-3- phospho-L-serine (DLPS), l,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, 1,2- didocosahexaenoyl-sn-glycero-3-phospho-L-serine, l-pentadecanoyl-2-oleoyl-sn-glycero-3- phospho-L-serine, 1 -palmitoyl -2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), l-stearoyl-2- oleoyl-sn-glycero-3-phospho-L-serine (SOPS), 1 -palmitoyl -2 -linoleoyl-sn-glycero-3- phospho-L-serine, l-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, 1 -palmitoyl -2- docosahexaenoyl-sn-glycero-3-phospho-L-serine, l-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine, l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, l-stearoyl-2- docosahexaenoyl-sn-glycero-3-phospho-L-serine, or a combination thereof.

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

16. The lipid nanoparticle of claim 15, wherein the phosphatidylglycerol comprises l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DOPG), 1,2-diheptadecanoyl- sn-glycero-3-phospho-(l'-rac-glycerol), l,2-didecanoyl-sn-glycero-3-phospho-(l'-rac- glycerol), l,2-dilauroyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DLPG), 1,2-dimyristoyl-sn- glycero-3-phospho-(l'-rac -glycerol) (DMPG), l,2-dipentadecanoyl-sn-glycero-3-phospho-(l'- rac -glycerol), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac -glycerol) (DPPG), 1,2-distearoyl- sn-glycero-3 -phospho-( 1 '-rac -glycerol) (DSPG), 1 ,2-dielaidoyl-sn-glycero-3 -phospho-( 1 '-rac- glycerol), 1 ,2-dilinoleoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol), 1 ,2-dilinolenoyl-sn- glycero-3 -phospho-( 1 '-rac-glycerol), 1 ,2-diarachidonoyl-sn-glycero-3 -[phospho-rac-( 1 - glycerol)] , 1 ,2-didocosahexaenoyl-sn-glycero-3 -[phospho-rac-( 1 -glycerol)] , 1 -palmitoyl -2- oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (POPG), 1 -palmitoyl -2 -linoleoyl-sn-glycero-3- phospho-( 1 '-rac-glycerol), 1 -palmitoyl -2 -arachidonoyl-sn-glycero-3-phospho-( 1 '-rac- glycerol) (PAPG), l-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-(l'-rac-glycerol), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac -glycerol) (SOPG), 1 -stearoyl -2-linoleoyl-sn-glycero-3-phospho-(l'-rac-glycerol), 1 -stearoyl -2 -arachidonoyl-sn-glycero-3-phospho-(l'-rac- glycerol) (SAPG), 1 -stearoyl -2 -docosahexaenoyl-sn-glycero-3-phospho-(l'-rac-glycerol), or a combination thereof.

17. The lipid nanoparticle of any one of claims 1-16, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the lipid nanoparticle.

18. The lipid nanoparticle of any one of claims 1-17, 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.

19. The lipid nanoparticle of any one of claims 1-18, wherein the conj ugated lipid comprises a carbamate linkage.

20. The lipid nanoparticle of any one of claims 1-19, wherein the conjugated lipid comprises a polyethylene glycol (PEG) -lipid conjugate.

21. The lipid nanoparticle of any one of claims 1 -20, wherein the conjugated lipid comprises N- [(methoxypoly (ethylene glycol))carbamoyl]-l,2-dimyristyloxy- propylamine (PEG-c-DMA).

22. The lipid nanoparticle of any one of claims 1-21, wherein the ionizable lipid does not comprise a disulfide bond.

23. The lipid nanoparticle of any one of claims 1-22, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

24. The lipid nanoparticle of any one of claims 1-23, 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).

25. The lipid nanoparticle of any one of claims 1-24, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the lipid nanoparticle.

26. The lipid nanoparticle of any one of claims 1-25, wherein the lipid nanoparticle further comprises cholesterol or a derivative thereof.

27. The lipid nanoparticle of claim 26, wherein the cholesterol or the derivative thereof comprise between 20 mol% and 60 mol% of the total lipid of the lipid nanoparticle.

28. The lipid nanoparticle of any one of claims 1-27, 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.

29. The lipid nanoparticle of any one of claims 1-27, wherein the lipid nanoparticle does not comprise a permanently charged cationic lipid.

30. The lipid nanoparticle of any one of claims 1-29, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 8.5 mol% and 10.5 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprises between 0.25 mol% and 0.9 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 48 mol% and 58 mol% of the total lipid of the lipid nanoparticle; and the lipid nanoparticle further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 35 mol% and 39 mol% of the total lipid of the lipid nanoparticle.

31. The lipid nanoparticle of any one of claims 1-29, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 9 mol% and 11 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprises between 0.85 mol% and 2.6 mol% of the total lipid of the lipid nanoparticle; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 45 mol% and 55 mol% of the total lipid of the lipid nanoparticle; and the lipid nanoparticle further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 36.5 mol% and 40.5 mol% of the total lipid of the lipid nanoparticle.

32. The lipid nanoparticle of claim 30 or 31, wherein: the phospholipid comprises DOPG, DOPS. or a combination thereof; the conjugated lipid comprises PEG-c-DMA; and the ionizable lipid comprises 3D-P-DMA.

33. The lipid nanoparticle of any one of claims 1-32, wherein the nucleic acid comprises RNA.

34. The lipid nanoparticle of claim 33, wherein the RNA comprises mRNA, saRNA, circRNA or a combination thereof.

35. The lipid nanoparticle of any one of claims 1-32, wherein the nucleic acid comprises DNA.

36. The lipid nanoparticle of any one of claims 1 to 35, wherein the protein of interest comprises an antigen.

37. The lipid nanoparticle of claim 36, wherein the antigen is associated with a disease characterized by an undesired cell proliferation, with an infection, or with an autoimmune disease.

38. The lipid nanoparticle of claim 37, wherein the antigen comprises a cancer antigen.

39. The lipid nanoparticle of claim 38, wherein the cancer antigen comprises a subject-specific cancer antigen.

40. 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, and a phospholipid or a pharmaceutically acceptable salt thereof; and combining the aqueous solution and the alcoholic solution to yield a lipid nanoparticle solution comprising the lipid nanoparticle; whereinthe conjugated lipid comprises between 0.1 mol% and 5 mol% of the total lipid of the alcoholic solution, and the composition of the alcoholic solution is configured such that the lipid nanoparticle has a negative zeta potential at physiological pH.

41. The method of claim 40, wherein the phospholipid has a net negative charge at physiological pH.

42. The method of claim 40 or 41, wherein the conjugated lipid comprises between 0.1 mol% and 1 mol% of the total lipid of the alcoholic solution.

43. The method of any one of claims 40-42, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the alcoholic solution.

44. The method of any one of claims 40-43, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

45. 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, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net negative 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 less than 1 mol% of the total lipid of the alcoholic solution.

46. The method of claim 45, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the lipid nanoparticle.

47. The method of claim 45 or 46, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

48. 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, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net negative 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 comprising between 0. 1 mol% and 5 mol% of the total lipid of the alcoholic solution, and the phospholipid or the pharmaceutically acceptable salt thereof comprises at least 8 mol% of the total lipid of the alcoholic solution.

49. The method of claim 48, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the nanoparticle, and the ionizable lipid does not comprise sulfur.

50. 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, and a phospholipid or a pharmaceutically acceptable salt thereof; the phospholipid having a net negative 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 comprising between 0. 1 mol% and 5 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprises more than 1 mol% of the total lipid of the alcoholic solution; and the ionizable lipid does not comprise sulfur.

51. The method of any one of claims 40-50, wherein the method further comprises removing at least a portion of the alcoholic organic solvent from the lipid nanoparticle solution.

52. The method of claim 51, wherein the removing of at least a portion of the alcoholic organic solvent comprises exchanging the alcoholic organic solvent with an aqueous solution.

53. The method of any one of claims 40-52, wherein the method further comprises concentrating the lipid nanoparticle solution.

54. The method of claim 53, wherein the concentrating of the lipid nanoparticle precursor comprises centrifuging the lipid nanoparticle solution.

55. The method of any one of claims 40-54, wherein the method further comprises filtering the lipid nanoparticle solution.

56. The method of any one of claims 40-55, wherein the alcoholic organic solvent comprises ethanol.

57. The method of any one of claims 40-56, 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.

58. The method of claim 57, wherein the nonalcoholic organic solvent comprises tetrahydrofuran.

59. The method of any one of claims 40-58, wherein the phospholipid comprises a naturally occurring phospholipid.

60. The method of any one of claims 40-59, wherein the phospholipid comprises a phosphatidylserine.

61. The method of claim 60, wherein the phosphatidylserine comprises DOPS, l,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine, DSPS, DMPS, 1,2-didecanoyl- sn-glycero-3-phospho-L-serine, DPPS, DLPS, l,2-dilinoleoyl-sn-glycero-3-phospho-L-serine, l,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine, l-pentadecanoyl-2-oleoyl-sn-glycero- 3-phospho-L-serine, POPS, SOPS, l-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine, 1- palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, 1 -palmitoyl -2 -docosahexaenoyl- sn-glycero-3-phospho-L-serine, 1 -stearoyl -2 -linoleoyl-sn-glycero-3-phospho-L-serine, 1- stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine, 1 -stearoyl -2 -docosahexaenoyl-sn- glycero-3-phospho-L-serine, or a combination thereof.

62. The method of any one of claims 40-61, wherein the phospholipid comprises a phosphatidylglycerol.

63. The method of claim 62, wherein the phosphatidylglycerol comprisesDOPG, 1 ,2-diheptadecanoyl-sn-glycero-3 -phospho-( l'-rac -glycerol), 1 ,2-didecanoyl-sn- glycero-3-phospho-(l'-rac-glycerol), DLPG, DMPG, l,2-dipentadecanoyl-sn-glycero-3- phospho-(l'-rac-glycerol), DPPG, DSPG, l,2-dielaidoyl-sn-glycero-3-phospho-(l'-rac- glycerol), 1 ,2-dilinoleoyl-sn-glycero-3 -phospho-( 1 '-rac-glycerol), 1 ,2-dilinolenoyl-sn- glycero-3 -phospho-( 1 '-rac-glycerol), 1 ,2-diarachidonoyl-sn-glycero-3 -[phospho-rac-( 1 - glycerol)], l,2-didocosahexaenoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], POPG, 1- palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(l'-rac-glycerol), PAPG, 1 -palmitoyl -2- docosahexaenoyl-sn-glycero-3-phospho-(l'-rac-glycerol), SOPG, l-stearoyl-2-linoleoyl-sn- glycero-3-phospho-(l'-rac-glycerol), SAPG, 1 -stearoyl -2 -docosahexaenoyl-sn-glycero-3 - phospho-(l'-rac-glycerol), or a combination thereof.

64. The method of any one of claims 40-63, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the alcoholic solution.

65. The method of any one of claims 40-64, 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.

66. The method of any one of claims 40-65, wherein the conjugated lipid comprises a carbamate linkage.

67. The method of any one of claims 40-66, wherein the conjugated lipid comprises a PEG-lipid conjugate.

68. The method of any one of claims 40-67, wherein the conjugated lipid comprises PEG-c-DMA.

69. The method of any one of claims 40-68, wherein the ionizable lipid does not comprise a disulfide bond.

70. The method of any one of claims 40-69, wherein the ionizable lipid does not comprise sulfur.

71. The method of any one of claims 40-70, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains that each have five or more members.

72. The method of any one of claims 40-71, wherein the ionizable lipid comprises 3D-P-DMA.

73. The method of any one of claims 40-72, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the alcoholic solution.

74. The method of any one of claims 40-73, wherein the alcoholic solution further comprises cholesterol or a derivative thereof.

75. The method of claim 74, wherein the cholesterol or the derivative thereof comprise between 20 mol% and 60 mol% of the total lipid of the alcoholic solution.

76. The method of any one of claims 40-75, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 8.5 mol% and 10.5 mol% of the total lipid of the alcoholic solution; the conjugated lipid comprises between 0.25 mol% and 0.9 mol% of the total lipid of the alcoholic solution;the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 48 mol% and 58 mol% of the total lipid of the alcoholic solution; and the alcoholic solution further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 35 mol% and 39 mol% of the total lipid of the alcoholic solution.

77. The method of any one of claims 40-75, wherein: the phospholipid or the pharmaceutically acceptable salt thereof comprise between 9 mol% and 11 mol% of the total lipid of the alcoholic solution; the conjugated lipid comprises between 0.85 mol% and 2.6 mol% of the total lipid of the alcoholic solution; the ionizable lipid or the pharmaceutically acceptable salt thereof comprising between 45 mol% and 55 mol% of the total lipid of the alcoholic solution; and the alcoholic solution further comprises cholesterol or a derivative thereof, the cholesterol or the derivative thereof comprising between 36.5 mol% and 40.5 mol% of the total lipid of the alcoholic solution.

78. The method of claim 76 or 77, wherein: the phospholipid comprises DOPG, DOPS. or a combination thereof; the conjugated lipid comprises PEG-c-DMA; and the ionizable lipid comprises 3D-P-DMA.

79. The method of any one of claims 40-78, wherein the nucleic acid comprises RNA.

80. The method of claim 79, wherein the RNA comprises mRNA, saRNA, circRNA or a combination thereof.

81. The method of any one of claims 40-78, wherein the nucleic acid comprises DNA.

82. The method of any one of claims 40-81, wherein the protein of interest comprises an antigen.

83. The method of claim 82, wherein the antigen is associated with a disease characterized by an undesired cell proliferation, with an infection, or with an autoimmune disease.

84. The method of claim 83, wherein the antigen comprises a cancer antigen.

85. The method of claim 84, wherein the cancer antigen comprises a subjectspecific cancer antigen.

86. A lipid nanoparticle obtainable by the method of any one of claims 40- 85.

87. A vaccine comprising the lipid nanoparticle of any one of claims 1-39 or 86.

88. The vaccine of claim 87, wherein the protein of interest comprises an antigen.

89. The vaccine of claim 88, wherein the antigen comprises a cancer antigen, and wherein the vaccine is a cancer vaccine.

90. The vaccine of claim 89, wherein the cancer antigen comprises a subject-specific cancer antigen, and wherein the cancer vaccine is a personalized cancer vaccine.

91. The vaccine of any one of claims 87-90, wherein the vaccine further comprises a pharmaceutically acceptable excipient.

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