Lipid nanoparticles for stimulating t cells

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 574,479, filed April 4, 2024, the entire disclosure of which is incorporated herein by reference for all purposes. Background Technology

[0003] Lipid nanoparticles (LNPs) have become a key technology in 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 encapsulated drugs. For example, lipid nanoparticles have emerged as a promising technology for vaccine delivery. These particles can, for instance, protect immunogenic nucleic acids (such as mRNA molecules) from degradation, facilitate their intracellular delivery, and promote endosome escape, thus enabling the translation of mRNA into target proteins, such as antigen proteins.

[0004] However, despite their success, there is an urgent need for improved LNPs, including those that can be used to stimulate high CD8 levels in subjects to whom LNPs are administered. + T cell responses are granules. This elevated CD8 content can benefit them. + T-cell stimulation is used for both preventative and curative therapies, including those targeting cancer, infectious diseases, and autoimmune diseases. However, current LNPs may be limited, reducing their efficacy in these therapies. These limitations may include suboptimal biodistribution and / or clearance rates, induction of undesirable immune responses, potential off-target effects, and low expression efficiency of the delivered payload (e.g., mRNA). For example, inflammation-related side effects such as pain, swelling, fever, and drowsiness may be associated with some current RNA-LNP-based vaccines. Additionally, some LNP designs may inadvertently induce high levels of mRNA expression in off-target tissues and organs, thereby reducing preventative and / or therapeutic efficacy and increasing the likelihood of undesirable side effects. Therefore, developing next-generation LNPs with enhanced stability, targeted delivery, and minimized side effects is important for fully realizing the potential of LNPs in nucleic acid delivery for preventative and therapeutic care. This disclosure addresses these needs and provides associated and other advantages. Summary of the Invention

[0005] This summary provides a high-level overview of various aspects of this disclosure and introduces some concepts described and illustrated in this document and the accompanying drawings. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to define the scope of the claimed subject matter. The embodiments covered by this disclosure are defined by the claims rather than by this summary. The subject matter should be understood by referring to appropriate portions of the specification throughout, any or all of the drawings, and each claim. Some exemplary embodiments of this disclosure are described in detail below.

[0006] In one aspect, this disclosure provides a lipid nanoparticle. The lipid nanoparticle comprises phospholipids or pharmaceutically acceptable salts thereof. The lipid nanoparticle further comprises conjugated lipids. The total lipids of the lipid nanoparticle comprise 0.1 mol% to 5 mol% of the conjugated lipids. The lipid nanoparticle further comprises ionizable lipids or pharmaceutically acceptable salts thereof. The ionizable lipid comprises three or more terminal hydrocarbon chains, each of which has five or more members. The lipid nanoparticle further comprises nucleic acids. The nucleic acids encode a protein of interest or a fragment thereof. The lipid nanoparticle is configured to have a negative zeta potential at physiological pH.

[0007] On the other hand, this disclosure provides another type of lipid nanoparticle. The lipid nanoparticle comprises phospholipids or pharmaceutically acceptable salts thereof. The phospholipids have a net negative charge at physiological pH. The lipid nanoparticles further comprise conjugated lipids. The total lipids of the lipid nanoparticles comprise less than 1 mol% of the conjugated lipids. The lipid nanoparticles further comprise ionizable lipids or pharmaceutically acceptable salts thereof. The lipid nanoparticles further comprise nucleic acids encoding a protein of interest or fragments thereof.

[0008] On the other hand, this disclosure provides another type of lipid nanoparticle. The lipid nanoparticle comprises phospholipids or pharmaceutically acceptable salts thereof. The phospholipids have a net negative charge at physiological pH. The total lipids of the lipid nanoparticles comprise at least 8 mol% of the phospholipids or pharmaceutically acceptable salts thereof. The lipid nanoparticles further comprise conjugated lipids. The total lipids of the lipid nanoparticles comprise 0.1 mol% to 5 mol% of the conjugated lipids. The lipid nanoparticles further comprise ionizable lipids or pharmaceutically acceptable salts thereof. The lipid nanoparticles further comprise nucleic acids. The nucleic acids encode a protein of interest or a fragment thereof.

[0009] On the other hand, this disclosure provides another type of lipid nanoparticle. The lipid nanoparticle comprises phospholipids or pharmaceutically acceptable salts thereof. The phospholipids have a net negative charge at physiological pH. The lipid nanoparticle further comprises conjugated lipids. The total lipids of the lipid nanoparticle comprise 0.1 mol% to 5 mol% of the conjugated lipids. The lipid nanoparticle further comprises ionizable lipids or pharmaceutically acceptable salts thereof. The total lipids of the lipid nanoparticle comprise more than 1 mol% of the ionizable lipids or their pharmaceutically acceptable salts. The ionizable lipids do not include sulfur. The lipid nanoparticle further comprises nucleic acids encoding a protein of interest or fragments thereof.

[0010] On the other hand, this disclosure provides a method for generating lipid nanoparticles. The method includes providing an aqueous solution. The aqueous solution includes nucleic acids encoding a protein of interest or a fragment thereof. The method further includes providing an alcohol solution. The alcohol solution includes an alcoholic organic solvent, conjugated lipids, ionizable lipids or pharmaceutically acceptable salts thereof, and phospholipids or pharmaceutically acceptable salts thereof. The total lipids in the alcohol solution include 0.1 mol% to 5 mol% of the conjugated lipids. The method further includes combining the aqueous solution and the alcohol solution to generate a lipid nanoparticle solution containing the lipid nanoparticles. The composition of the alcohol solution is configured such that the lipid nanoparticles have a negative zeta potential at physiological pH.

[0011] On the other hand, this disclosure provides another method for generating lipid nanoparticles. The method includes providing an aqueous solution. The aqueous solution includes nucleic acids encoding a protein of interest or a fragment thereof. The method further includes providing an alcohol solution. The alcohol solution includes an alcoholic organic solvent, conjugated lipids, ionizable lipids or pharmaceutically acceptable salts thereof, and phospholipids or pharmaceutically acceptable salts thereof. The phospholipids have a net negative charge at physiological pH. The total lipids in the alcohol solution include less than 1 mol% of the conjugated lipids. The method further includes combining the aqueous solution and the alcohol solution to generate a lipid nanoparticle solution containing the lipid nanoparticles.

[0012] On the other hand, this disclosure provides another method for generating lipid nanoparticles. The method includes providing an aqueous solution. The aqueous solution includes nucleic acids encoding a protein of interest or a fragment thereof. The method further includes providing an alcohol solution. The alcohol solution includes an alcoholic organic solvent, conjugated lipids, ionizable lipids or pharmaceutically acceptable salts thereof, and phospholipids or pharmaceutically acceptable salts thereof. The phospholipids have a net negative charge at physiological pH. The total lipids of the alcohol solution include 0.1 mol% to 5 mol% of the conjugated lipids. The total lipids of the alcohol solution further include at least 8 mol% of the phospholipids or pharmaceutically acceptable salts thereof. The method further includes combining the aqueous solution and the alcohol solution to generate a lipid nanoparticle solution containing the lipid nanoparticles.

[0013] On the other hand, this disclosure provides another method for generating lipid nanoparticles. The method includes providing an aqueous solution. The aqueous solution includes nucleic acids encoding a protein of interest or a fragment thereof. The method further includes providing an alcohol solution. The alcohol solution includes an alcoholic organic solvent, conjugated lipids, ionizable lipids or pharmaceutically acceptable salts thereof, and phospholipids or pharmaceutically acceptable salts thereof. The ionizable lipids do not include sulfur. The total lipids of the alcohol solution include 0.1 mol% to 5 mol% of the conjugated lipids. The total lipids of the alcohol solution further include more than 1 mol% of the ionizable lipids or their pharmaceutically acceptable salts. The phospholipids have a net negative charge at physiological pH. The method further includes combining the aqueous solution and the alcohol solution to generate a lipid nanoparticle solution containing the lipid nanoparticles.

[0014] On the other hand, this disclosure provides another type of lipid nanoparticle. The lipid nanoparticle can be obtained by any of the methods disclosed herein.

[0015] On the other hand, this disclosure provides a vaccine. The vaccine includes any of the lipid nanoparticles disclosed herein. Attached Figure Description

[0016] Figure 1 A graph showing the variation of zeta potential measurements across the pH range for the four lipid nanoparticle formulations provided in Table 1 is presented.

[0017] Figure 2 The study compared CD8 levels in mice after administration of two different lipid nanoparticle formulations containing HA mRNA. + IFN-γ + A graph showing the relative measured amount of T cells.

[0018] Figure 3The study compared CD8 levels in mice after administration of two different lipid nanoparticle formulations containing HA mRNA. + TNF-α + A graph showing the relative measured amount of T cells.

[0019] Figure 4 The study compared CD8 levels in mice after administration of two different lipid nanoparticle formulations containing HA mRNA. + IL-2 + A graph showing the relative measured amount of T cells. Detailed Implementation

[0020] A. Introduction

[0021] This disclosure generally relates to lipid nanoparticles that, when used in preventative or curative therapies, offer advantageous improvements in inducing immune responses, such as immune responses characterized by enhanced stimulation of specific T cells. For example, given the unique role these cells play in adaptive immunity, antigen-specific CD8... + The initiation of T-cell responses can be a key aspect of targeted immunotherapies. CD8 + T cells, commonly known 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. Following antigen recognition, these CTLs can directly exert cytotoxic effects, leading to the lysis of abnormal cells. Therefore, stimulating antigen-specific CD8+ cells... + T-cell generation therapy can enhance the precision of the immune system in eradicating cells expressing targeted antigens while minimizing collateral damage to healthy cells. This specificity not only enhances therapeutic efficacy but also reduces off-target cytotoxicity, thereby improving the safety profile of the treatment. Therefore, stimulating antigen-specific CD8... + The ability to produce T cells may be key to advancing effective immunotherapy. Furthermore, certain conditions (such as cancer, autoimmune diseases, and some infections) can be treated using T cells involving CD8. + T-cell-stimulated immunotherapy is more effective in preventing or alleviating disease.

[0022] However, it has been shown that lipid nanoparticles are difficult to use as antigen carriers to induce strong CD8 in vaccinated individuals. + T cell response. This difficulty stems from several challenges related to the complex nature of immune activation and the physicochemical properties of LNPs. For example, the necessary CD8 activation... + For a cellular response, the antigen encapsulated by LNPs must be efficiently delivered to the targeted cells or organs, where it can be properly processed and presented to CD8. +T cells. However, many LNPs are preferentially taken up via endocytosis, which often leads to lysosomal degradation of the encapsulated antigen and limits the efficiency of targeted delivery. Furthermore, the charge and lipid composition of LNPs can significantly influence their biodistribution, cellular uptake, and endosome escape, each of which can significantly affect the effectiveness of antigen delivery and presentation.

[0023] The materials and methods disclosed herein overcome these difficulties by using specific lipid nanoparticle compositions that have been demonstrated herein to stimulate CD8 in response to the stimulation of a protein or protein fragment of interest (e.g., an antigen of interest). + Surprisingly effective in T cell generation. These provided lipid nanoparticle formulations comprise selected materials combined with each other in specific relative amounts. Notably, the importance of these formulations, and the resulting configurations and properties of the lipid nanoparticles they produce, was previously not recognized, particularly regarding their beneficial effects on generating enhanced immune responses.

[0024] B. Definition

[0025] Unless otherwise expressly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, any methods or materials similar to or equivalent to those described herein may be used in practice with this disclosure. For the purposes of this disclosure, the following terms are defined.

[0026] The abbreviations used herein have their conventional meanings in the fields of chemistry and biology. The description of the compounds disclosed herein is subject to the principles of chemical bonding known to those skilled in the art. Therefore, where a group can be substituted by one or more of a plurality of substituents, such substitutions are chosen to conform to the principles of chemical bonding and to yield compounds that are not inherently unstable and / or, as known to those skilled in the art, may be unstable under environmental conditions (e.g., aqueous, neutral, or physiological conditions). When a substituent is specified by its conventional chemical formula (written from left to right), it equally encompasses chemically identical substituents produced when the structure is written from right to left; for example, -CH2O- is equivalent to -OCH2-.

[0027] Unless otherwise stated, the compounds disclosed herein may also contain atomic isotopes in non-natural proportions on one or more atoms constituting such compounds. For example, the compounds may use isotopes such as deuterium (… 2 H), tritium ( 3 H), Iodine-125 ( 125 I), carbon-13 ( 13 C) or carbon-14 ( 14C) Marking. All isotopic variations of the compounds disclosed herein, whether radioactive or non-radioactive, are covered within the scope of this disclosure.

[0028] As used herein, when referring to chemical substances, the term "substitution" means replacing a hydrogen atom with a non-hydrogen atom or a group of covalently bonded atoms. The atom or group of atoms that replaces the hydrogen atom is called a "substituent".

[0029] As used herein, when referring to chemical substances, the term "member" refers to a non-hydrogen atom in a covalently bonded group of atoms, such as a compound or its substituents.

[0030] As used herein, unless the context clearly states otherwise, the singular form "a / an" and "described" include plural indicators. Thus, for example, references to "phospholipids" optionally include combinations of two or more phospholipids, etc.

[0031] As used herein, the terms “about” and “approximately” when used to modify a quantity specified as a numerical value or range indicate that the numerical value and a reasonable deviation from a value known to those skilled in the art, such as ±20%, ±10%, or ±5%, are within the intended meaning of the value.

[0032] As used herein, the term “and / or” means and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of such combinations when interpreted in an alternative context (“or”).

[0033] As used herein, the terms “including,” “comprising,” “having,” “containing,” and their variations are inclusive and open-ended, and do not exclude additional, unlisted elements or method steps besides those expressly enumerated. As used herein, the phrase “consisting of” is closed-ended and excludes any element, step, or component not expressly specified. As used herein, the phrase “generally constitutes” limits the scope of the described feature to the specified materials or steps, and those materials or steps that do not materially affect the essential and novel characteristics of the disclosed feature.

[0034] As used herein, the terms “optional” or “optionally” mean that an event, situation, or substituent described below may or may not occur, and the description includes instances where the event or situation occurs and instances where the event or situation does not occur.

[0035] As used herein, when referring to an element or characteristic, the terms “first,” “second,” “third,” etc., are used merely to more clearly distinguish or identify multiple elements or characteristics and are not intended to indicate an order or other sequence or numerical limitation, or to require the presence of each of the multiple elements or characteristics.

[0036] As used herein, the terms "lipid particle," "lipid nanoparticle," and "LNP" refer to particles comprising phospholipids and ionizable lipids. Lipid particles may contain additional lipid components, such as sterols and / or conjugated lipids, and may further contain nucleic acids, wherein the nucleic acids may be encapsulated within the particles. Lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Application Publications Nos. 2004 / 0142025 and 2007 / 0042031 and International Patent Application Publication No. WO2012 / 000104.

[0037] As used herein, the term "phospholipid" refers to a class of lipids having a hydrophilic "headgroup" containing a phosphate ester and a hydrophobic portion. The hydrophobic portion may contain one or more hydrophobic groups, most typically two. The hydrophobic groups are also called hydrophobic "tails" and may be derived from fatty acids and linked by alcohol residues (e.g., glycerol). Exemplary structures of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

[0038] As used herein, the term "ionizable lipid" refers to a type of lipid that carries a net positive charge at a selected pH (e.g., acidic or physiological pH). In some cases, ionizable lipids include ionizable primary, secondary, or tertiary amine head groups (e.g., pH-titrile). In some cases, ionizable lipids promote the encapsulation of negatively charged nucleic acid (e.g., mRNA or siRNA) payloads during particle formation. In some cases, ionizable lipids promote endosome fusion and cytoplasmic release of the payload after cellular uptake of lipid nanoparticles.

[0039] As used herein, the terms “permanently charged lipids,” “permanently charged cationic lipids,” and “permanently charged ionic lipids” generally refer to lipid species that carry a net positive charge regardless of the pH of their biological environment.

[0040] As used herein, the term "conjugated lipid" refers to polymer-conjugated lipids, such as polymer-conjugated lipids that inhibit lipid particle aggregation. 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 with dialkoxypropyl, PEG coupled with diacylglycerol, PEG coupled with cholesterol, PEG coupled with phosphatidylethanolamine, PEG coupled with ceramide (see, for example, U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to lipids or linked to lipids via a linker portion. Any linker portion suitable for coupling polymers to lipids can be used, including, for example, ester-free linker portions and ester-containing linker portions.

[0041] As used herein, the term "salt" refers to the acid salt or base salt of the compounds disclosed herein. A "pharmaceutically acceptable salt" is a salt that is compatible with other components of a formulation containing the compound and is harmless to its recipient (i.e., the subject). Therefore, it should be understood that pharmaceutically acceptable salts do not cause significant adverse toxicological effects on the subject.

[0042] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless specifically defined, the term encompasses nucleic acids containing nucleotide analogs or modified backbone residues or bonds that are synthetic, naturally occurring, or unnatural, and that have binding properties similar to and are metabolized in a similar manner to reference nucleotides. Non-limiting examples of nucleotide analogs are described, for example, in International Patent Application No. WO 2007 / 024708. Examples of nucleic acids including such nucleotide analogs, modified backbone residues, or bonds include, but are not limited to, nucleic acids containing phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses its conserved variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), complementary sequences, and explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine (Batzer et al., (191) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) Journal of Biol. Chem. 260:2605; and Rossolini et al., (1994) Mol. Cell. Probes 8:91).

[0043] Non-limiting examples of polynucleotides or nucleic acids include DNA, RNA, coding or non-coding regions of genes or gene segments, intergenic DNA, loci (locus) as defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), small nucleolar RNA (snoRNA), ribozymes, deoxynucleotides (dNTPs), or dideoxynucleotides (ddNTPs). Polynucleotides may also include complementary DNA (cDNA), which is the DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification. Polynucleotides may 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. Polynucleotides may contain modified nucleotides, such as methylated nucleotides, nucleotide analogs, and / or nucleosides suitable for reducing the immunogenicity of RNA, as described in International Patent Application No. WO 2007 / 024708. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. The nucleotide sequence may be interspersed with non-nucleotide components. Polynucleotides may be further modified post-polymerization, such as by binding to labeled components. Unless otherwise specified, when provided, polynucleotide sequences are listed in a 5' to 3' orientation.

[0044] Nucleic acids or polynucleotides can be double-stranded or triple-stranded nucleic acids, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acids, the nucleic acid strands do not need to be homoelongated; for example, double-stranded nucleic acids do not need to be double-stranded along the entire length of both strands.

[0045] Nucleic acid modification can include the addition of chemical groups that incorporate additional charges, polarizations, hydrogen bonds, electrostatic interactions, and functionalities into individual nucleic acid bases or as a whole nucleic acid. Such modifications include base modifications such as 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications at the amine site outside the cytosine ring, substitution of S-bromouracil, backbone modifications, and uncommon base pairing combinations such as isobasic cytosine and isoguanidine.

[0046] Nucleic acids can be derived from fully chemically synthesized methods, such as solid-phase mediated chemical synthesis, from biological sources, such as by isolation from any species that produces nucleic acids, or from methods involving manipulation of nucleic acids using molecular biological tools, such as DNA replication, PCR amplification, reverse transcription, in vitro transcription, as described, for example, in International Patent Application Publication No. WO 2007 / 024708, or from combinations of these methods.

[0047] As used herein in the context of lipid nanoparticles and nucleic acids, the term "fully encapsulated" means that the nucleic acids in the particles do not significantly degrade upon exposure to serum or upon nuclease assays that would significantly degrade free nucleic acids. In a fully encapsulated system, preferably less than 25% of the particle nucleic acids are degraded in treatments that typically degrade 100% of free nucleic acids, more preferably less than 10%, and most preferably less than 5%. Full encapsulation also indicates that the particles are serum-stable, i.e., they do not rapidly disintegrate into their component parts upon administration in vivo.

[0048] 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, wherein the composition may be used to induce an immune response against the antigen or immunogen in a subject to enhance the subject's immunity to disease and / or infection.

[0049] As used herein, the term "subject" refers to a vertebrate, and preferably a mammal. Mammal subjects to whom the provided compositions are suitable include, but are not limited to, mice, rats, apes, humans, farm animals, sporting animals, and pets. In some embodiments, the subject is a 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 10 years of age or older, for example, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or 80 years of age or older. In some embodiments, the subject is less than 80 years of age, for example, less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0050] As used herein, the terms "pharmaceuticalally acceptable excipient" and "pharmaceuticalally acceptable carrier" refer to substances that facilitate administration of the active agent to a subject and facilitate absorption by the subject, and that such substances may be included in the compositions of this disclosure without causing significant adverse toxicological effects on the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, physiological saline solutions, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, etc. Those skilled in the art will recognize that other pharmaceutically acceptable excipients and carriers are useful in this disclosure.

[0051] As used herein, the term “administration” refers to oral administration to a subject, as a suppository, local contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, subcutaneous administration, intrathecal administration, intraparenchymal administration, subretinal administration, or intravitreal administration, or implantation of a sustained-release device, such as a microosmotic pump.

[0052] As used herein, the term “therapeutic effective amount” means the amount or dose of a compound, composition, or formulation administered to produce a therapeutic effect. The exact amount or dose will depend on the purpose of treatment and will be determined by a person skilled in the art using known techniques (see, for example, 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), edited by Gennaro, Lippincott, Williams & Wilkins).

[0053] As used herein, the term "treatment" refers to any procedure that achieves any sign of success in eliminating or improving an injury, pathology, symptom, or condition (e.g., pain), including any objective or subjective parameters such as the reduction, relief, or decrease of symptoms, or making the symptoms, injury, pathology, or condition more tolerable for the patient; reducing the frequency or duration of symptoms or conditions; or, in some cases, preventing the onset of one or more symptoms. Treatment or improvement of symptoms can be based on any objective or subjective parameters, including, for example, the results of a physical examination or laboratory test.

[0054] C. Lipid nanoparticles

[0055] On the one hand, this disclosure provides various lipid nanoparticles, and vaccines including the provided lipid nanoparticles. Lipid nanoparticles typically comprise conjugated lipids, phospholipids or pharmaceutically acceptable salts thereof, ionizable lipids or pharmaceutically acceptable salts thereof, and nucleic acids encoding a protein of interest or fragments thereof. Specific selection, composition, and amounts (e.g., relative amounts) of these components enable the lipid nanoparticles and vaccines to induce an immune response (e.g., with high levels of T cells (e.g., CD8+)). + A surprising improvement in the ability of lipid nanoparticles to induce a strong immune response characterized by T cell stimulation. For example, lipid nanoparticles have been shown in this paper to induce a strong immune response characterized by T cell stimulation when: (1) the lipid nanoparticles have a negative zeta potential (e.g., a negative zeta potential at physiological pH); (2) the ionizable lipids include a specific configuration of tail groups (e.g., three or more terminal hydrocarbon chains, each having five or more members); (3) the phospholipids have a net negative charge (e.g., a net negative charge at physiological pH); (4) the concentration of conjugated lipids in the lipid nanoparticles is below a certain threshold (e.g., less than 1 mol% of the total lipids in the lipid nanoparticles); (5) the concentration of phospholipids or pharmaceutically acceptable salts thereof is above a certain threshold (e.g., at least 8 mol% of the total lipids in the lipid nanoparticles); and / or (6) the ionizable lipids do not contain sulfur.

[0056] In some cases, and as evidenced by the data provided in Examples 2 and 3, the provided lipid nanoparticles capable of eliciting a strong T-cell response exhibit a negative zeta potential. In some embodiments, the lipid nanoparticles have a negative zeta potential at physiological pH (i.e., at a pH of about 7.4). In preferred embodiments, the lipid nanoparticles have a negative zeta potential at all pH values ​​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. When measured at pH values ​​within this range (e.g., physiological pH), the negative zeta potential of lipid nanoparticles can be, for example, about -1 mV to about -20 mV, such as about -1 mV to about -6 mV, about -1.3 mV to about -8.1 mV, about -1.8 mV to about -11 mV, about -2.5 mV to about -15 mV, or -3.3 mV to about -20 mV. With respect to the lower limit, the negative zeta potential of lipid nanoparticles can be, for example, greater than about -20 mV, such as greater than -15 mV, greater than 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, such as less than about -20 mV, are also contemplated.

[0057] In a preferred embodiment, the zeta potential of the lipid nanoparticles described herein is determined using a measurement of the particle zeta potential at a temperature of about 20°C to about 30°C. In some embodiments, the lipid nanoparticles have a negative zeta potential when measured at physiological pH or within any of the pH ranges listed above, as determined using a measurement performed at a temperature of about 20°C to about 30°C. In a particularly preferred embodiment, the zeta potential of the lipid nanoparticles described herein is determined using a measurement of the zeta potential at a temperature of about 25°C. In a preferred embodiment, the zeta potential of the lipid nanoparticles is determined using the measurement described in Example 2.

[0058] In some examples of the lipid nanoparticles provided, phospholipids or pharmaceutically acceptable salts thereof account for about 8.5 mol% to about 10.5 mol% of the total lipids of the lipid nanoparticles (e.g., about 8.5 mol% to about 10 mol%, about 8.5 mol% to about 9.5 mol%, about 8.5 mol% to about 9 mol%, about 9 mol% to about 10.5 mol%, about 9 mol% to about 10 mol%, about 9 mol% to about 10 mol%, about 9 mol% to about 9.5 mol%, about 9.5 mol% to about 10.5 mol%, or about 10 mol% to about 10.5 mol%), and conjugated lipids account for about 0.25 mol% to 0.9 mol% of the total lipids of the lipid nanoparticles (about 0.25 mol% to about 0.74 mol%, about 0.25 mol% to about 0.58 mol%, about 0.25 mol% to about 0.41 mol%, about 0.41 mol% to about 0.9 mol%). The lipids are approximately 0.41 mol% to about 0.74 mol%, approximately 0.41 mol% to about 0.58 mol%, approximately 0.58 mol% to about 0.9 mol%, approximately 0.58 mol% to about 0.74 mol%, or approximately 0.74 mol% to about 0.9 mol%, and the ionizable lipids or their pharmaceutically acceptable salts comprise approximately 48 mol% to about 58 mol% of the total lipids in the lipid nanoparticles (e.g., approximately 48 mol% to about 55.5 mol%, approximately 48 mol% to about 53 mol%, approximately 48 mol% to about 50.5 mol%, approximately 50.5 mol% to about 58 mol%, approximately 50.5 mol% to about 55.5 mol%, approximately 50.5 mol% to about 53 mol%, approximately 48 mol% to about 50.5 mol%, approximately 50.5 mol% to about 58 mol%, approximately 53 mol% to about 58 mol%, approximately 53 mol% to about 55.5 mol%, or approximately 55.5 mol% to about 58 mol%). In some embodiments, the lipid nanoparticles further comprise cholesterol or a derivative thereof, wherein the cholesterol or the derivative thereof comprises about 35 mol% to 39 mol% of the total lipids in the lipid nanoparticles (e.g., about 35 mol% to about 38 mol%, about 35 mol% to about 37 mol%, about 35 mol% to about 36 mol%, about 36 mol% to about 39 mol%, about 36 mol% to about 38 mol%, about 36 mol% to about 37 mol%, about 37 mol% to about 39 mol%, about 37 mol% to about 38 mol%, or about 38 mol% to about 39 mol%).

[0059] In some examples of the lipid nanoparticles provided, phospholipids or pharmaceutically acceptable salts thereof account for about 9 mol% to about 11 mol% of the total lipids of the lipid nanoparticles (e.g., about 9 mol% to about 10.5 mol%, about 9 mol% to about 10 mol%, about 9 mol% to about 9.5 mol%, about 9.5 mol% to about 11 mol%, about 9.5 mol% to about 10.5 mol%, about 9.5 mol% to about 10 mol%, about 10 mol% to about 11 mol%, about 10 mol% to about 10.5 mol%, or about 10.5 mol% to about 11 mol%), and conjugated lipids account for about 0.85 mol% to 2.6 mol% of the total lipids of the lipid nanoparticles (about 0.85 mol% to about 2.16 mol%, about 0.85 mol% to about 1.73 mol%, about 0.85 mol% to about 1.29 mol%, about 1.29 mol% to about 2.6 mol%). The lipid nanoparticles contain approximately 45 mol% to approximately 55 mol% of total lipids (e.g., approximately 45 mol% to approximately 52.5 mol%, approximately 45 mol% to approximately 50 mol%, approximately 45 mol% to approximately 47.5 mol%, approximately 47.5 mol% to approximately 55 mol%, approximately 47.5 mol% to approximately 52.5 mol%, approximately 47.5 mol% to approximately 50 mol%, approximately 50 mol% to approximately 55 mol%, approximately 50 mol% to approximately 52.5 mol%, or approximately 52.5 mol% to approximately 55 mol%). In some embodiments, the lipid nanoparticles further comprise cholesterol or a derivative thereof, wherein the cholesterol or the derivative thereof comprises about 36.5 mol% to 40.5 mol% of the total lipids in the lipid nanoparticles (e.g., about 36.5 mol% to about 39.5 mol%, about 36.5 mol% to about 38.5 mol%, about 36.5 mol% to about 37.5 mol%, about 37.5 mol% to about 40.5 mol%, about 37.5 mol% to about 39.5 mol%, about 37.5 mol% to about 38.5 mol%, about 38.5 mol% to about 40.5 mol%, about 38.5 mol% to about 39.5 mol%, or about 39.5 mol% to about 40.5 mol%).

[0060] In some instances, the provided lipid nanoparticles further comprise permanently charged cationic lipids. The lipid nanoparticles may include permanently charged cationic lipids comprising, for example, from about 1 mol% to 10 mol% of the total lipids in the lipid nanoparticles, for example, from about 1 mol% to about 5.6 mol%, from about 1 mol% to about 3.2 mol%, from about 1 mol% to about 1.8 mol%, from about 1.8 mol% to about 10 mol%, from about 1.8 mol% to about 5.6 mol%, from about 1.8 mol% to about 3.2 mol%, from about 3.2 mol% to about 10 mol%, or from about 3.2 mol% to 5.6 mol% or from about 5.6 mol% to about 10 mol%. With respect to the upper limit, permanently charged cationic lipids may constitute, for example, no more than about 10 mol% of the total lipids in the lipid nanoparticles, for example, no more than about 6.8 mol%, no more than about 4.6 mol%, no more than about 3.2 mol%, no more than about 2.2 mol%, no more than about 1.5 mol%, or no more than about 1 mol%. With respect to the lower limit, permanently charged cationic lipids may constitute, for example, no less than about 1 mol% of the total lipids in the lipid nanoparticles, for example, 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 instances, the lipid nanoparticles provided do not include permanently charged cationic lipids.

[0061] The properties and relative amounts of lipid nanoparticle components can also be selected to provide lipid nanoparticles whose pKa is favorable for use in stimulating immune responses (e.g., stimulating T cell production). For example, in some embodiments, the pKa of the lipid nanoparticles is about 5.8 to about 6.9, such as about 5.8 to about 6.8, about 5.8 to about 6.7, about 5.8 to about 6.6, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1, about 5.8 to about 6.0, about 5.8 to about 5.9, about 5.9 to about 6.9, about 5.9 to about 6.8, about 5.9 to about 6.7, about 5.9 to about 6.6, about 5.9 to about 6.5, about 5.9 to about 6.4, about 5.9 to about 6.3, about 5.9 to about 6.2, about 5.9 to about 6.1, about 5.9 to about 6.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6. .0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about 6.0 to about 6.1, about 6.1 to about 6.9, about 6.1 to about 6.8, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.1 to about 6.4, about 6.1 to about 6.3, about 6.1 to about 6.2, about 6.2 to about 6.9, about 6.2 to about 6.8, about 6.2 to about 6.7, about 6.2 to about 6.6, about 6.2 to about 6.5, about 6.2 to about 6.4, about 6.2 to about 6.3, about 6.3 to about 6.9, about 6.3 to about 6.8, about 6.3 to about 6.7, about 6.3 to about 6.6, about 6.3 to about 6.5, or about 6.3 to about 6.4.

[0062] The properties and relative amounts of the lipid nanoparticle components can also be selected to provide lipid nanoparticles whose size is beneficial to stimulating an immune response characterized by enhanced T cell production. The average diameter of the lipid nanoparticles can be, for example, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm.

[0063] The provided lipid nanoparticles can be further configured to be substantially non-toxic, for example, by selecting their components. When the lipid nanoparticles are used in a vaccine, the vaccine may additionally include pharmaceutically acceptable excipients.

[0064] 1. Phospholipids

[0065] The lipid nanoparticles disclosed herein typically comprise at least one phospholipid or a pharmaceutically acceptable salt thereof. Phospholipids may be selected or designed to provide the lipid nanoparticles with improved properties in inducing desired immune responses, such as increased T-cell stimulation. In some embodiments, the lipid nanoparticles comprise one type of phospholipid. In other embodiments, the lipid nanoparticles comprise two or more types of phospholipids, 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 types of phospholipids.

[0066] In some instances, the provided lipid nanoparticles have exhibited the ability to elicit strong T-cell responses, partly because the phospholipids in the lipid nanoparticles have a net negative charge at physiological pH. Lipid nanoparticles comprising such negatively charged phospholipids have been shown in this paper to advantageously elicit high levels of CD8 specific for the protein or fragment thereof of interest, for example. + T cells. Phospholipids may include, for example, phosphatidylserine, phosphatidylglycerol, or combinations thereof, or consist of thereof. In some instances, at least one phospholipid species (e.g., a negatively charged phospholipid species) in the lipid nanoparticles is a naturally occurring phospholipid. In some embodiments, each phospholipid in the lipid nanoparticles is a naturally occurring phospholipid.

[0067] Phosphatidylserine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-L-serine), 1,2-distearate-sn-glycerol-3-phosphate-L-serine (DSPS), and 1,2-dimyristoyl-sn-glycerol-3-phosphate-L-serine (DM). PS), 1,2-dicepanoyl-sn-glycerol-3-phosphate-L-serine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (DPPS), 1,2-dilauroyl-sn-glycerol-3-phosphate-L-serine (DLPS), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-L-serine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate-L-serine) 1-Pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (SOPS), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-arachidonicoyl-sn -Glyceryl-3-phosphate-L-serine, 1-palmitoyl-2-docosahexaenoyl-sn-glyceryl-3-phosphate-L-serine, 1-stearoyl-2-linoleoyl-sn-glyceryl-3-phosphate-L-serine, 1-stearoyl-2-arachidonicyl-sn-glyceryl-3-phosphate-L-serine, 1-stearoyl-2-docosahexaenoyl-sn-glyceryl-3-phosphate-L-serine and combinations thereof.

[0068] Phosphatidylglycerol lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-decanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilauroyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DLPG), and 1,2-dimyristoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (D... MPG), 1,2-bispentadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DPPG), 1,2-distearatel-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DSPG), 1,2-ditransoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinolenoyl-sn-glycerol-3-phosphate-(1'- 1,2-Diarachidonic-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1,2-bis(docosahexaenoic-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-palmitoyl-2-arachidonic-sn-glycerol-3-phosphate-(1'-rac-glycerol) (PAPG), 1- Palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (SOPG), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (SAPG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) and combinations thereof.

[0069] In addition to the type of phospholipid, the amount of phospholipid included in the provided lipid nanoparticles has also been shown to provide the particles with their advantageous properties. In some instances, when the amount of phospholipids or their pharmaceutically acceptable salts in the lipid nanoparticles is about 1 mol% to about 20 mol% of the total lipids of the lipid nanoparticles, the provided lipid nanoparticles and vaccines induce a strong immune response. The molar fraction of phospholipids or their pharmaceutically acceptable salts in the total lipids of the provided lipid nanoparticles may be, for example, about 1 mol% to about 16.2 mol%, about 1 mol% to about 12.4 mol%, about 1 mol% to about 8.6 mol%, about 1 mol% to about 4.8 mol%, about 4.8 mol% to about 20 mol%, about 4.8 mol% to about 16.2 mol%, about 4.8 mol% to about 12.4 mol%, about 4.8 mol% to about 8.6 mol%, about 8.6 mol% to about 20 mol%, about 8.6 mol% to about 16.2 mol%, or about 8.6 mol% to about 12.4 mol%.

[0070] In some instances, when the amount of phospholipids or their pharmaceutically acceptable salts in the lipid nanoparticles is additionally or alternatively at least about 8 mol% of the total lipids of the lipid nanoparticles, the provided lipid nanoparticles and vaccines induce a strong immune response. The molar fraction of phospholipids or their pharmaceutically acceptable salts in the total lipids of the provided lipid nanoparticles can be, for example, from about 8 mol% to about 20 mol% of the total lipids of the lipid nanoparticles, such as from about 8 mol% to about 17.6 mol%, from about 8 mol% to about 15.2 mol%, from about 8 mol% to about 12.8 mol%, from about 8 mol% to about 10.4 mol%, from about 10.4 mol% to about 20 mol%, from about 10.4 mol% to about 17.6 mol%, from about 10.4 mol% to about 15.2 mol%, from about 10.4 mol% to about 12.8 mol%, from about 12.8 mol% to about 20 mol%, from about 12.8 mol% to about 17.6 mol%, from about 12.8 mol% to about 15.2 mol%, from about 15.2 mol% to about 20 mol%, from about 15.2 mol% to about 17.6 mol%, or from about 17.6 mol% to about 20 mol%.

[0071] 2. Conjugated lipids

[0072] The lipid nanoparticles disclosed herein typically include at least one conjugated lipid. The conjugated lipids can be selected or engineered to provide improved performance in inducing desired immune responses in lipid nanoparticles or vaccines comprising lipid nanoparticles, such as increasing T cells (e.g., CD8+ cells specific to the protein or fragment thereof of interest). + The generation of T cells. For example, conjugated lipids having two or more hydrophobic tail groups that are not directly bonded to esters are shown herein as inducing high T cell generation. Furthermore, some lipid nanoparticles that advantageously provide a high immune response include conjugated lipids comprising urethane bonds. Moreover, the amount of conjugated lipids present in lipid nanoparticles has been shown in this disclosure to influence the ability of lipid nanoparticles to induce a strong immune response. For example, conjugated lipid masses below certain thresholds and optionally also above certain other thresholds have been shown to beneficially generate T cell-stimulating (e.g., CD8+ specific for the protein or fragment thereof) cells. + Lipid nanoparticles for T cells.

[0073] In some embodiments, the lipid nanoparticles comprise one type of conjugated lipid. In other embodiments, the lipid nanoparticles comprise two or more types of conjugated lipids, 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 types of conjugated lipids. One or more conjugated lipids may be independently selected or configured to control particle size during formation and / or to prevent particle aggregation by spatially stabilizing the lipid nanoparticles. The conjugated lipids in the lipid nanoparticles may be located at the surface of the particles, wherein the hydrophilic polymer of the conjugated lipids is outwardly oriented and binds to an aqueous environment, and the lipid components of the conjugated lipids are embedded in the particles to anchor them in place.

[0074] In some instances, at least one conjugated lipid species in the lipid nanoparticles has two or more hydrophobic tails, i.e., two or more terminal hydrocarbon chains, each independently having five or more members, wherein each of these terminal hydrocarbon chains is not directly bonded to an ester. In some instances, each conjugated lipid in the lipid nanoparticles has two or more terminal hydrocarbon chains, each independently having five or more members, and each terminal hydrocarbon chain is not directly bonded to an ester. The number of such terminal hydrocarbon chains in the conjugated lipids 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 instances, at least one conjugated lipid species in the lipid nanoparticles includes a urethane bond. In some embodiments, each conjugated lipid in the lipid nanoparticles includes a urethane bond.

[0075] The terminal hydrocarbon chains, such as alkyl chains, of the conjugated lipids in lipid nanoparticles are not particularly limited in length. Each terminal hydrocarbon chain of the conjugated lipid can independently have a length of, for example, C12, C14, C16, C18, C20, C22, or C24. In some instances, the conjugated lipids comprise one or two terminal hydrocarbon chains, each independently being C12, C14, C16, or C18.

[0076] In some embodiments, at least one ionizable lipid species in the lipid nanoparticles does not include disulfide bonds. In some embodiments, each ionizable lipid in the lipid nanoparticles lacks disulfide bonds. In some instances, at least one ionizable lipid species in the lipid nanoparticles does not include sulfur. In some embodiments, each ionizable lipid in the lipid nanoparticles lacks sulfur.

[0077] In some instances, when the amount of conjugated lipids in the lipid nanoparticles is less than about 5 mol% of the total lipids in the lipid nanoparticles, the provided lipid nanoparticles and vaccines induce a strong immune response. The molar fraction of conjugated lipids in the total lipids of the provided lipid nanoparticles can be, for example, about 0.1 mol% to about 5 mol% of the total lipids in the lipid nanoparticles, such as about 0.1 mol% to about 4 mol%, about 0.1 mol% to about 3 mol%, about 0.1 mol% to about 2 mol%, about 0.1 mol% to about 1 mol%, about 1 mol% to about 5 mol%, about 1 mol% to about 4 mol%, about 1 mol% to about 3 mol%, about 1 mol% to about 2 mol%, about 2 mol% to about 5 mol%, about 2 mol% to about 4 mol%, about 2 mol% to about 3 mol%, about 3 mol% to about 5 mol%, about 3 mol% to about 4 mol%, or about 4 mol% to about 5 mol%.

[0078] In some instances, when the amount of conjugated lipids in the lipid nanoparticles is less than about 1 mol% of the total lipids in the lipid nanoparticles, the lipid nanoparticles and vaccines provided induce a strong immune response. The conjugated lipids can constitute, for example, about 0.1 mol% to about 1 mol% of the total lipids in lipid nanoparticles, for example, about 0.1 mol% to about 0.82 mol%, about 0.1 mol% to about 0.64 mol%, about 0.1 mol% to about 0.46 mol%, about 0.1 mol% to about 0.28 mol%, about 0.28 mol% to about 1 mol%, about 0.28 mol% to about 0.82 mol%, about 0.28 mol% to about 0.64 mol%, about 0.28 mol% to about 0.46 mol%, about 0.46 mol% to about 1 mol%, about 0.46 mol% to about 0.82 mol%, about 0.46 mol% to about 0.64 mol%, about 0.64 mol% to about 1 mol%, or about 0.64 mol% to about 0.82 mol%.

[0079] Suitable conjugated lipids for use with the provided lipid nanoparticles include, but are not limited to, polyethylene glycol (PEG)-lipid conjugations, ATTA-lipid conjugations, cationic polymer-lipid conjugations (CPL), polyoxazoline (POZ)-lipids, polysarcosine (pSAR)-lipids, and mixtures thereof. In some embodiments, the lipid nanoparticles comprise PEG-lipid conjugations or ATTA-lipid conjugations, optionally together with CPL.

[0080] In a preferred embodiment, the conjugated lipid comprises or is composed of PEG-lipids. Examples of PEG-lipids include, but are not limited to, PEG coupled with a dialkoxypropyl group (PEG-DAA) (as described, for example, in International Patent Application Publication No. WO 05 / 026372), PEG coupled with a diacylglycerol (PEG-DAG) (as described, for example, in U.S. Patent Application Publications Nos. 2003 / 0077829 and 2005 / 0008689), PEG coupled with a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG coupled with a ceramide (as described, for example, in U.S. Patent No. 5,885,613), PEG coupled with cholesterol or a derivative thereof, mPEG(2000)-N,N-bistetradecylacetamide (ALC-0159), mPEG(2000)-stearate, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG). The PEG-C-DOMG, R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine, and mixtures thereof are all of the PEG lipids described in International Patent Application Publication No. WO 2022 / 133344. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. The lipid nanoparticles provided may comprise one or more methyl-terminated PEG-lipids, one or more unterminated PEG-lipids, or combinations thereof.

[0081] PEG is a linear, water-soluble polymer of repeating ethylene glycol units. PEG is classified according to its molecular weight; 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. Due to its polydispersity, these molecular weights are average molecular weights. PEG is commercially available from Sigma Chemical Co. and other companies, and includes, for example, the following: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol succinimide succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trifluoroethyl sulfonate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs, such as those described in U.S. Patents 6,774,180 and 7,053,150 (e.g., mPEG (20 kDa)amine), can also be used to prepare the PEG-lipid conjugates disclosed herein. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. Additionally, monomethoxy polyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0082] The PEG moiety of the PEG-lipid conjugate described herein may contain an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In some cases, the average molecular weight of the PEG moiety is about 750 Daltons to about 5,000 Daltons (e.g., about 1,000 Daltons to about 5,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons to about 3,000 Daltons, about 750 Daltons to about 2,000 Daltons, etc.). In preferred embodiments, the average molecular weight of the PEG moiety is about 2,000 Daltons or about 750 Daltons.

[0083] In some cases, PEG may optionally be substituted with alkyl, alkoxy, acyl, or aryl groups. PEG may be directly conjugated to lipids or linked to lipids via a linker motif. Any linker motif suitable for coupling PEG to lipids may be used, including, for example, ester-free linker motifs and ester-containing linker motifs. In a preferred embodiment, the linker motif is an ester-free linker motif. As used herein, the term "ester-free linker motif" refers to a linker motif that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable ester-free linker motifs include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), urethane (NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl ((O)CCH2CH2C(O)-), succinimide (-NHC(O)CH2CH2C(O)NH-), and combinations thereof (such as linkers containing both urethane and amide linker motifs). In a preferred embodiment, a urethane linker is used to couple PEG to lipids.

[0084] In other embodiments, the ester-containing linker portion is used to couple PEG to the lipid. Suitable ester-containing linker portions include, for example, carbonates (-OC(O)O-), succinyl groups, phosphate esters (-O-(O)POH-O-), sulfonates, and combinations thereof.

[0085] In addition to the foregoing, it will be apparent to those skilled in the art that other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used to replace PEG include, but are not limited to, ATTA, cationic polymers, polyoxazoline (POZ), polysarcosine, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, poly(ethyl ethylene phosphate) (PEEP), and derivatized cellulose (such as hydroxymethyl cellulose or hydroxyethyl cellulose).

[0086] The conjugated lipids of the provided lipid nanoparticles may include, for example, one or more of, or consist of, the following: polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, or mixtures thereof. In a preferred embodiment, the nucleic acid-lipid particles comprise PEG-lipid conjugates or ATTA-lipid conjugates. The conjugated lipids may comprise PEG-lipids, including, for example, PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dispalmityloxypropyl (C16), PEG-distearateloxypropyl (C18), or mixtures thereof.

[0087] Other PEG-lipid conjugates suitable for use in the provided lipid nanoparticles include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol ester (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Other PEG-lipid conjugates suitable for use in this disclosure include, but are not limited to, 1-[8'-(1,2-dimyristoyl-3-propoxy)-formamido-3',6'-dioxaoctyl]carbamoyl-w-methylpoly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0088] In some instances, the provided lipid nanoparticles have exhibited the ability to induce strong immune responses, partly because the conjugated lipids in the lipid nanoparticles are lipids having two or more terminal hydrocarbon chains (e.g., terminal alkyl chains or terminal alkenyl chains), each terminal hydrocarbon chain having five or more members, and each terminal hydrocarbon chain not being directly bonded to an ester. Lipid nanoparticles including conjugated lipids with such hydrophobic tails have been shown herein to advantageously stimulate high yields of T cells specific to the protein of interest or fragments thereof. Conjugated lipids may include, for example, N-[(methoxypoly(ethylene glycol))carbamoyl]-1,2-dimyristyloxypropylamine (PEG-c-DMA) or be composed thereof.

[0089] By controlling the composition and concentration of the conjugated lipids in the provided lipid nanoparticles, the rate of exchange of the conjugated lipids from the lipid nanoparticles can be controlled, and consequently, the rate at which the lipid nanoparticles become fusion-promoting. For example, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as the conjugated lipid, the rate at which the lipid nanoparticles become fusion-promoting can be altered, for example, by changing the concentration of the lipid conjugate, by changing the molecular weight of the PEG, or by changing the chain length and saturation of the acyl chain groups on the phosphatidylethanolamine or ceramide. Additionally, other variables, including, for example, pH, temperature, ionic strength, etc., can be used to alter and / or control the rate at which the lipid nanoparticles become fusion-promoting. Other methods that can be used to control the rate at which lipid nanoparticles become fusion-promoting will become apparent to those skilled in the art upon reading this disclosure.

[0090] 3. Ionizable lipids

[0091] The lipid nanoparticles disclosed herein typically comprise at least one ionizable lipid or a pharmaceutically acceptable salt thereof. The ionizable lipid may be selected or engineered to provide improved performance in inducing desired immune responses in lipid nanoparticles or vaccines comprising lipid nanoparticles, such as increasing T cells (e.g., CD8+ cells specific to the protein of interest or fragments thereof). + The generation of T cells. For example, lipid nanoparticles comprising ionizable lipids having three or more hydrophobic tail groups are shown herein to induce high T cell generation. Alternatively or concurrently, lipid nanoparticles comprising sulfur-deficient ionizable lipids have shown enhanced desired immune responses. Furthermore, the amount of ionizable lipids present in the lipid nanoparticles can affect the ability of the lipid nanoparticles to induce strong immune responses. For example, ionizable lipid masses greater than certain thresholds have been shown to beneficially generate stimulating T cells (e.g., CD8+ specific for proteins or fragments thereof). + Lipid nanoparticles for T cells.

[0092] In some embodiments, the lipid nanoparticles comprise one type of ionizable lipid. In other embodiments, the lipid nanoparticles comprise two or more types of ionizable lipids, such as 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 types of ionizable lipids.

[0093] In some instances, the provided lipid nanoparticles and vaccines comprising lipid nanoparticles have exhibited the ability to induce strong immune responses, in part because the ionizable lipids in the lipid nanoparticles are lipids having three or more terminal hydrocarbon chains (e.g., terminal alkyl chains or terminal alkenyl chains), each terminal hydrocarbon chain having five or more members. For example, the lipid nanoparticles may comprise trialkyl cationic lipids as described in International Patent Application Publication WO 2013 / 126803, ionizable lipids as described in International Patent Application Publication WO 2017 / 049245, or ionizable lipids as described in International Patent Application Publication WO2017 / 075531. Ionizable lipids may comprise, for example, 5-(dimethylamino)valerate (6Z,16Z)-12-((Z)-dec-4-en-1-yl) dodecano-6,16-dien-11-yl ester (3D-P-DMA) or compositions thereof. The number of terminal hydrocarbon chains in an 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 terminal hydrocarbon chain is an alkyl group. In some embodiments, each terminal hydrocarbon chain is an alkyl group. In some embodiments, at least one terminal hydrocarbon chain is an alkenyl group. In some embodiments, each terminal hydrocarbon chain is an alkenyl group.

[0094] In some embodiments, at least one ionizable lipid species in the lipid nanoparticles does not include disulfide bonds. In some embodiments, each ionizable lipid in the lipid nanoparticles lacks disulfide bonds. In some instances, at least one ionizable lipid species in the lipid nanoparticles does not include sulfur. In some embodiments, each ionizable lipid in the lipid nanoparticles lacks sulfur.

[0095] In some instances, when the amount of ionizable lipids or their pharmaceutically acceptable salts in the lipid nanoparticles is greater than about 1 mol% of the total lipids in the lipid nanoparticles, the lipid nanoparticles provide a strong immune response. The molar fraction of ionizable lipids or their pharmaceutically acceptable salts in the total lipids of the provided lipid nanoparticles can be, for example, from about 1 mol% to about 70 mol% of the total lipids of the lipid nanoparticles, such as from about 1 mol% to about 30 mol%, from about 1 mol% to about 13 mol%, from about 1 mol% to about 5.5 mol%, from about 1 mol% to about 2 mol%, from about 2 mol% to about 70 mol%, from about 2 mol% to about 30 mol%, from about 2 mol% to about 13 mol%, from about 2 mol% to about 5.5 mol%, from about 5.5 mol% to about 70 mol%, from about 5.5 mol% to about 30 mol%, from about 5.5 mol% to about 13 mol%, from about 13 mol% to about 70 mol%, from about 13 mol% to about 30 mol%, or from about 30 mol% to about 70 mol%.

[0096] In some instances, when the amount of ionizable lipids or their pharmaceutically acceptable salts in the lipid nanoparticles is about 30 mol% to about 70 mol% of the total lipids in the lipid nanoparticles, the provided lipid nanoparticles induce a strong immune response. The molar fraction of ionizable lipids or their pharmaceutically acceptable salts in the total lipids of the provided lipid nanoparticles can be, for example, about 30 mol% to about 62 mol%, about 30 mol% to about 54 mol%, about 30 mol% to about 46 mol%, about 30 mol% to about 38 mol%, about 38 mol% to about 70 mol%, about 38 mol% to about 62 mol%, about 38 mol% to about 54 mol%, about 38 mol% to about 46 mol%, about 46 mol% to about 70 mol%, about 46 mol% to about 62 mol%, about 46 mol% to about 54 mol%, about 54 mol% to about 70 mol%, about 54 mol% to about 62 mol%, or about 62 mol% to about 70 mol%.

[0097] 4. Cholesterol

[0098] The lipid nanoparticles disclosed herein may further include sterols, such as cholesterol, or one or more derivatives thereof. Sterols can be selected or engineered to provide lipid nanoparticles or vaccines comprising lipid nanoparticles, exhibiting improved performance in inducing desired immune responses, such as increasing the generation of T cells associated with the target antigen. Furthermore, the amount of sterols present in the lipid nanoparticles has been shown in this disclosure to influence the ability of the lipid nanoparticles to induce a strong immune response. For example, amounts of sterols below certain thresholds have been shown to beneficially generate stimulating T cells (e.g., CD8+ specific for the protein of interest or fragments thereof). + Lipid nanoparticles for T cells.

[0099] In some instances, when the amount of cholesterol or its derivatives in the lipid nanoparticles is about 20 mol% to about 60 mol% of the total lipids in the lipid nanoparticles, the provided lipid nanoparticles and vaccines induce a strong immune response. The molar fraction of cholesterol or its derivatives in the total lipids of the provided lipid nanoparticles can be, for example, about 20 mol% to about 52 mol%, about 20 mol% to about 44 mol%, about 20 mol% to about 36 mol%, about 20 mol% to about 28 mol%, about 28 mol% to about 60 mol%, about 28 mol% to about 52 mol%, about 28 mol% to about 44 mol%, about 28 mol% to about 36 mol%, about 36 mol% to about 60 mol%, about 36 mol% to about 52 mol%, about 36 mol% to about 44 mol%, about 34 mol% to about 60 mol%, about 34 mol% to about 52 mol%, or about 52 mol% to about 60 mol%.

[0100] In some embodiments, the lipid nanoparticles comprise one class of sterols or derivatives thereof. In other embodiments, the lipid nanoparticles comprise two or more classes of sterols, 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 classes of sterols. The lipid nanoparticles may include, for example, cholesterol, cholesterol ketones, cholesterolenones, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, cholesterol hydroxyethyl ether, cholesterol hydroxybutyl ether, cholesterol hydroxyhexyl ether, cholesterol stearate, cholesterol oleate, 7-β-hydroxycholesterol, 7-α-hydroxycholesterol, 4-β-hydroxycholesterol, cholesterol PEG, β-sitosterol, or any combination thereof. In some instances, the lipid nanoparticles comprise cholesterol, but substantially do not include cholesterol derivatives.

[0101] 5. Nucleic acid

[0102] The lipid nanoparticles disclosed herein typically include nucleic acids. The composition of the lipid nanoparticles can be selected or configured such that the nucleic acids present in the lipid nanoparticles are resistant to degradation by nucleases in aqueous solutions. In some embodiments, at least 50% of the nucleic acid is encapsulated within the lipid nanoparticles; in one embodiment, at least 75% of the nucleic acid is encapsulated within the lipid nanoparticles; in one embodiment, at least 90% of the nucleic acid is encapsulated within the lipid nanoparticles; and in one embodiment, the nucleic acid is completely encapsulated within the lipid nanoparticles.

[0103] In some embodiments, this disclosure provides lipid nanoparticle formulations comprising a plurality of lipid nanoparticles or groups of lipid nanoparticles. In some embodiments, nucleic acids are completely encapsulated within the lipid portions of the lipid nanoparticles, such that the plurality of lipid nanoparticles or groups of lipid nanoparticles comprises approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, approximately 90% to approximately 100%, approximately 30% to approximately 95%, approximately 40% to approximately 95%, approximately 50% to approximately 95%, approximately 60% to approximately 95%, approximately 70% to approximately 95%, approximately 80% to approximately 95%, and approximately 85% to approximately 100%. Nucleic acid is encapsulated in lipid nanoparticles of 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, 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 part thereof or range thereof).

[0104] Nucleic acids present in the lipid nanoparticles described herein may include or be composed of any known form of nucleic acid. Nucleic acids as used herein may be single-stranded DNA or RNA (e.g., ssDNA or ssRNA), or double-stranded DNA or RNA (e.g., dsDNA or dsRNA), or DNA-RNA hybrids. Single-stranded nucleic acids include, for example, mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA).

[0105] Nucleic acids can have various lengths, typically depending on their specific form. For example, in certain embodiments, the length of mRNA, plasmid, or gene can be from about 500 to about 100,000 nucleotides. In certain embodiments, the length of oligonucleotides can range from about 10 to about 100 nucleotides. In various related embodiments, oligonucleotides, which can be single-stranded, double-stranded, or triple-stranded, can 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.

[0106] In some embodiments, the nucleic acids in the provided lipid nanoparticles comprise or consist of modified or substituted polynucleotides or oligonucleotides. In some cases, modified or substituted polynucleotides and oligonucleotides may be preferred over natural forms due to their properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0107] a) Proteins of interest

[0108] The nucleic acids in the provided lipid nanoparticles typically 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., the cell or organism to which the lipid nanoparticles are applied. 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 some instances, the target cell is a cell of the spleen of a subject.

[0109] In some embodiments, the protein of interest is an antigen. The presence of an antigen in the lipid nanoparticles allows the lipid nanoparticles to induce an immune response in the subject to which the lipid nanoparticles are administered. For example, after administration of the provided lipid nanoparticles or vaccine to a subject, and as a result of this administration, the subject may generate T cells (e.g., CD8+) associated with an antigen encoded by a nucleic acid in the lipid nanoparticles. + T cells).

[0110] (1) Antigens associated with unwanted cell proliferation

[0111] In some instances, antigens encoded by nucleic acids in the provided lipid nanoparticles are associated with diseases or conditions characterized by undesirable cell proliferation. For example, antigens may include or consist of tumor-specific antigens, tumor-associated differentiation antigens, tumor-associated overexpression antigens, tumor-associated cancer-testis antigens, tumor-associated virus-associated antigens, or any variants and / or combinations thereof.

[0112] In some instances, the antigens encoded by the nucleic acids in the provided lipid nanoparticles include or constitute tumor-specific antigens. For example, the antigens may include or constitute at least one fragment of any of the following: B melanoma antigen 1 (BAGE-1), Fragile X Intellectual Disability 1 Neighbor (FMR1NB), Cyclin A1 (CCNA1), G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, N-acetylglucosamine transferase V (GNT-V), human endogenous retrovirus E (HERV-E), HER... V-K-MEL, Kitakyushu lung cancer antigen 1 (KK-LC-1), coiled-coil domain-containing protein 110 (KM-HN-1), L antigen family member 1 (LAGE-1), LDL receptor-associated protein-associated protein 1 (LRPAP1), lymphocyte antigen 6 family member K (LYSK), melanoma antigen family A1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE-C1, MAGE-C2, L antigen family member 2 (LAGE-2), sarcoma antigen 1 (SAGE), sperm autoantigen protein 17 (Sp17), synovial sarcoma X breakpoint 2 (SSX-2), SSX-4, axon glycoprotein 1 (TAG-1), TAG-2, paclitaxel resistance-associated gene 3 (TRAG-3), tyrosinase-associated protein 2 (TRP-2), X antigen family member 1B (XAGE-1b), or any variant and / or combination thereof.

[0113] In some instances, the antigens encoded by the nucleic acids in the provided lipid nanoparticles include or constitute tumor-associated differentiation antigens. For example, the antigen may include or constitute at least one fragment of any of the following: carcinoembryonic antigen-associated cell adhesion molecule (CEA), promelanosome protein 17 (PMEL17), secretory globin family 2A member 2 (SCGB2A2), T-cell recognized melanoma antigen 1 (MART-1), ankyrin repeat domain 30A (ANKRD30A), ocular albinism 1 (OA1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), Ras-associated protein RAB38, tyrosinase-associated protein 1 (TRP-1), TRP-2, tyrosinase (TYR), or any variants and / or combinations thereof.

[0114] In some instances, the antigens encoded by nucleic acids in the provided lipid nanoparticles include or constitute tumor-associated differentiation antigens. For example, the antigen may include or constitute at least one fragment of any of the following: perilipin 2 (PLIN2), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), alpha-fetoprotein (AFP), apoptosis regulator BCL-X, WD repeat domain 46 (WDR46), calcitonin-associated polypeptide α (CALCA), CD274, CD45, cleavage and polyadenylation specific factor 1 (CPSF1), cyclin D1 (CCND1), Dickkopf... Inhibitors of the WNT signaling pathway 1 (DKK1), Enable homolog actin regulatory factor (ENAH), epithelial cell adhesion molecule (EpCAM), hepatocyte ligand A receptor 3 (EphA3), Zeste enhancer 2 multicomb inhibitory complex 2 subunit (EZH2), fibroblast growth factor 5 (FGF5), carbonic anhydrase 9 (CA9), phosphatidylinositol polysaccharide 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), phosphatidylinositol glycan 3 (GPC3), interleukin-13 receptor subunit α2 (IL13RA2), IMP U3 small nucleolar ribonucleoprotein 3 (IMP-3), carboxylesterase 2 (CES2), kallikrein-associated peptidase 4 (KLK4), kinin family member 20A (KIF20A), lensin (LGSN), colony-stimulating factor 1 (CSF1), chondroitin sulfate proteoglycan 4 (CSPG4), mouse two-microsome 2 proto-oncogene (MDM-2), melanoma overexpression antigen (MELOE), metaphase factor (MDK), matrix metallopeptidase 2 (MMP-2), MMP-7, mucin 1 (MUC1), MUC5AC, The following proteins are included: cell adhesion molecule 4 (adhesion protein-4), tumor protein P53, pairing box 5 (PX5), zinc finger protein 395 (ZNF395), placental enrichment 1 (PLAC1), melanoma nuclear receptor transcription factor preferential expression antigen (PRAME), folate hydrolase 1 (FOLH1), renal tumor antigen 1 (RAGE-1), G protein signaling regulator 5 (RGS5), Ras homolog family member C (RHOC), ring finger protein 43 (RNF43), 2 containing bicortin domain (DCDC2), secernin 1 (SCRN1), sex-determining region Y box transcription factor 10 (SOX10), six-transmembrane prostate epithelial antigen family member 1 (STEAP1), 5 containing baculovirus IAP repeat sequence (BIRC5), telomerase, telomerase reverse transcriptase (TERT), trophoblast glycoprotein (TPBG), vascular endothelial growth factor (VEGF), nephroblastoma protein transcription factor 1 (WT1), or any variants and / or combinations thereof. ,

[0115] In some embodiments, the antigen encoded by the nucleic acids in the provided lipid nanoparticles includes or is composed of a subject-specific cancer antigen. The subject-specific cancer antigen may be an antigen identified by analyzing a sample derived from a subject (e.g., a patient with cancer). For example, the subject-specific cancer antigen may be a protein or fragment thereof expressed at low levels in the subject's non-cancerous cells or tissues. In some embodiments, the subject-specific cancer antigen is undetectably expressed in the subject's non-cancerous cells or tissues. Additionally or alternatively, the subject-specific cancer antigen may be a protein or fragment thereof having a mutated amino acid sequence relative to the corresponding protein or protein fragment in the subject's non-cancerous cells or tissues.

[0116] (2) Infection-associated antigens

[0117] In some instances, the antigens encoded by nucleic acids in the provided lipid nanoparticles are infection-related; for example, the antigens are derived from pathogens. Pathogens could be bacterial pathogens. For example, pathogens can be *Achromobacter xylosoxidans*, *Acinetobacter baumannii*, *Actinomyces* such as *Actinomyces israelii*, *Aeromonas*, *Bacillus*, *Bacteroides fragilis*, *Bacteroides melaninogenicus*, *Bartonella*, *Bordetella pertussis*, *Borrelia*, *Brucella*, *Burkholderia*, *Campylobacter*, *Capnocytophaga*, *Chlamydia pneumoniae*, and *Chlamydia psittaci*. psittaci, Citrobacter, Clostridium, Corynebacterium, Coxiella burnetii, Ehrlichia, Eikenella corrodens, Enterobacter, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Fusobacterium necrophorum, Gardnerella vaginalis, Haemophilus, Helicobacter pylori pylori, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria monocytogenes, Moraxella*Catarrhalis*, *Morganella*, *Mycoplasma pneumoniae*, *Neisseria*, *Nocardia*, *Pasteurella multocida*, *Peptostreptococcus*, *Porphyromonas gingivalis*, *Propionibacterium acnes*, *Proteus*, *Providencia*, *Pseudomonas aeruginosa*, *Salmonella*, *Serratia marcescens*, *Shigella*, *Staphylococcus epidermidis*, *Staphylococcus hominis*, *Staphylococcus hemolyticus*. Staphylococcus 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, or combinations thereof.

[0118] In some instances, the antigens encoded by the nucleic acids in the provided lipid nanoparticles are derived from fungal pathogens. For example, pathogens can include *Cryptococcus neoformans*, *Candida auris*, *Aspergillus fumigatus*, *Candida albicans*, *Nakaseomyces glabrata* (Candida glabrata), *Histoplasma*, fungal mycomas, *Mucorales*, *Fusarium*, *Candida tropicalis*, *Candida parapsilosis*, *Scedosporium*, *Cryptococcus gattii*, *Lomentosporaprolificans*, *Talaromyces marneffei*, *Coccidioides*, and *Pneumocystis*. jirovecii), Pichiakudriavzeveii (Candida krusei), Paracoccidioides, or combinations thereof.

[0119] In some instances, the antigens encoded by the nucleic acids in the provided lipid nanoparticles are derived from protozoan pathogens. For example, the pathogens could be *Cryptosporidium*; *Toxoplasma gondii*; *Plasmodium* species such as *Plasmodium malariae*, *Plasmodium falciparum*, *Plasmodium vivax*, and *Plasmodium ovale*; *Trypanosoma cruzi* and *Trypanosoma brucei*; *Leishmania*; *Giardia duodenalis*; *Entamoeba histolytica*; *Naegleria fowleri*; *Babesia microti*; and *Balamuthia*. The genera *Mandrillaris*, *Blastocystishominis*, *Trichomonas vaginalis*, *Schistosoma*, or combinations thereof.

[0120] In some instances, the antigens encoded by the nucleic acids in the provided lipid nanoparticles are derived from worm pathogens. For example, pathogens can include Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Strongyloides stercoralis, Ancylostoma duodenale, Hymenolepis nana, Taeniasaginata, Enterobius, Fasciola hepatica, Schistosoma mansoni, Toxocara canis, Toxocara cati, Wuchereria bancrofti, Brugia malayi, Onchocercavolvulus, Trichostrongylus, and Dracunculus. The following are listed: *Medinensis*, *Baylisascaris*, *Echinococcus*, *Taenia multiceps*, *Taenia serialis*, *Taenia glomerata*, *Taenia brauna*, *Clonorchis sinensis*, *Fasciolopsis buski*, *Opisthorchis*, *Paragonimus*, *Schistosoma*, *Moniliformis*, or combinations thereof.

[0121] In some instances, the antigens encoded by the nucleic acids in the provided lipid nanoparticles are derived from viral pathogens. For example, the antigens may be derived from coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), influenza viruses (e.g., influenza A, B, and C viruses), filoviruses (e.g., Ebola virus, Marburg virus), arenaviruses (e.g., Lassa virus, Junin virus, Machupo virus, Guanarito virus, Sabiavirus), Zika virus, rabies virus, rhinovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), hepatitis viruses (e.g., hepatitis C virus), herpes simplex virus, human papillomavirus (HPV), or Epstein-Barr virus. In a specific embodiment, the infectious disease antigen is a SARS-CoV-2 protein selected from the group consisting of: S (spike) protein, E (enveloped) protein, M (membrane) protein, N (nucleocapsid) protein, and antigenic fragments thereof. In other embodiments, the infectious disease antigen is a hemagglutinin (HA) protein from a Paramyxoviridae or Orthomyxoviridae virus (e.g., influenza virus). Examples 1, 2, and 3 demonstrate the use of the provided lipid nanoparticles to encapsulate the nucleic acid encoding hemagglutinin, i.e., mRNA.

[0122] In some instances, the antigen encoded by the nucleic acid in the provided lipid nanoparticles is derived from a pathogenic toxin. The antigen may be associated with any non-enzymatic product of any pathogen (e.g., any of the bacterial, fungal, protozoan, worm, or viral pathogens described above), wherein the pathogen product is harmful to the subject infected with the pathogen. In some instances, the antigen is derived from a pathogenic adhesion element, i.e., any product of any pathogen (e.g., any of the bacterial, fungal, protozoan, worm, or viral pathogens described above), wherein the pathogen product participates in the adhesion of the pathogen to the cells of the subject infected with the pathogen.

[0123] b) mRNA

[0124] The nucleic acid encoding the protein of interest in the provided lipid nanoparticles may include or be composed of an RNA molecule encoding or a fragment thereof. In some instances, the nucleic acid in the provided lipid nanoparticles includes or is composed of one or more messenger RNA (mRNA) molecules (e.g., a cocktail of mRNA molecules). The mRNA molecule may be selected or engineered for transfer to cells in a subject (e.g., a mammal, such as a human) to which the provided lipid nanoparticles or vaccine are administered. The mRNA molecule may encode an immune response expressed intracellularly to elicit an immune response against undesired cell proliferation (e.g., cancer), infection, or autoimmune disease (e.g., CD8). + One or more antigens (T cell responses). Therefore, the compositions and methods described herein can be used, for example, to prevent infectious diseases caused by pathogens such as viruses (e.g., coronaviruses such as SARS-CoV-2) by expressing antigenic peptides (e.g., mRNA molecules encoding viral proteins such as S (spike), E (enveloping), M (membrane), or N (nucleocapsid) proteins or antigenic fragments thereof) to stimulate the adaptive immune system to generate pathogen-targeting T cells in an organism (e.g., a mammal, such as a human).

[0125] In some embodiments, the mRNA molecules are completely encapsulated within lipid particles. In some instances, the RNA in the provided lipid nanoparticles or the provided population of nanoparticles comprises a mixture of one or more types of mRNA with one or more other types of RNA (e.g., gRNA, siRNA, miRNA, etc.). Regarding formulations containing mRNA mixtures, different types of mRNA species present in the mixture (e.g., mRNAs with different sequences) may be co-encapsulated in the same particle, or each type of mRNA present in the mixture may be encapsulated in a separate particle. The mRNA mixture may be formulated in the particles described herein using a mixture of two or more separate mRNAs (each having a unique sequence) at the same, similar, or different concentrations or molar ratios. In one embodiment, a mixture of mRNAs (corresponding to multiple mRNAs with different sequences) is formulated using each type of mRNA at the same, similar, or different concentrations or molar ratios, and different types of mRNAs are co-encapsulated in the same particle. In another embodiment, each type of mRNA present in the mixture is encapsulated in different particles at the same, similar, or different mRNA concentrations or molar ratios, and the resulting particles (each containing a different mRNA payload) are administered individually (e.g., at different times depending on a prophylactic or therapeutic regimen) or in combination as a single unit dose (e.g., with a pharmaceutically acceptable carrier). In a particular embodiment, the lipid particles are serum stable, resistant to nuclease degradation, and substantially nontoxic to mammals (such as humans).

[0126] (1) Modification of mRNA

[0127] The mRNA molecules present in the provided lipid nanoparticles may include one, two, or more nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in the cells into which it is introduced, relative to the corresponding unmodified mRNA.

[0128] In some embodiments, the modified nucleoside comprises or is composed of the following: pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurylmethyluridine, 1-taurylmethyl-pseudouridine, 5-taurylmethyl-2-thio-uridine, 1-tauryl... Sulfonylmethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-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.

[0129] In some embodiments, the modified nucleosides include or consist of the following: 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolocytidine, pyrrolopseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4- Thio-1-methyl-1-denitro-pseudo-isocytidine, 1-methyl-1-denitro-pseudo-isocytidine, 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-pseudo-isocytidine, 4-methoxy-1-methyl-pseudo-isocytidine, or any combination thereof.

[0130] In other embodiments, the modified nucleosides include or consist of the following: 2-aminopurine, 2,6-diaminopurine, 7-deazo-adenine, 7-deazo-8-aza-adenine, 7-deazo-2-aminopurine, 7-deazo-8-aza-2-aminopurine, 7-deazo-2,6-diaminopurine, 7-deazo-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine. Glycosides, N6-isopentenyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyl adenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine.

[0131] In some embodiments, the modified nucleosides comprise or consist of the following: 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseuuridine, or combinations thereof. An α-thiosubstituted phosphate moiety is provided to impart stability to the RNA polymer via a non-natural thiophosphate backbone bond. The thiophosphate RNA polymer exhibits increased nuclease resistance and thus a longer half-life in the cellular environment. The thiophosphate-linked nucleic acids are also expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0132] In some embodiments, for example, if precise timing of protein production is required, it is necessary to degrade the modified nucleic acid introduced into the cell within the cell. Therefore, this disclosure provides modified nucleic acids containing degradation domains that can be directed within the cell.

[0133] In other embodiments, the modified nucleosides include or consist of the following: inosine, 1-methyl-inosine, wyoside, wyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-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 combinations thereof.

[0134] (2) Optional mRNA components

[0135] In another embodiment, the mRNA molecule 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 sites (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 translatable regions. mRNA molecules containing Kozak sequences are also provided. Additionally, mRNA molecules containing one or more intronic nucleotide sequences that can be excised from the mRNA sequence are provided herein.

[0136] (a) Untranslated Region (UTR)

[0137] The untranslated region (UTR) of a gene is transcribed but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. Increasing evidence suggests that the UTR plays a regulatory role in the stability and translation of mRNA molecules. Regulatory features of the UTR can be incorporated into the mRNA used in the lipid particles described in this paper to increase molecular stability. Specific features can also be incorporated to ensure controlled downregulation of the transcript, preventing it from being misdirected to unwanted tissue or organ sites.

[0138] (b) 5' with cap

[0139] The 5' cap structure of mRNA participates in nuclear export, thereby increasing mRNA stability. It also binds to mRNA cap-binding protein (CBP), which, through association with poly(A)-binding proteins, forms mature circular mRNA species responsible for mRNA stability and translation capacity within the cell. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

[0140] mRNA molecules can be 5'-capped, thereby creating a 5'-ppp-5'-triphosphate bond between the terminal guanylic acid cap residue and the 5'-terminal sense nucleotide. This 5'-guanylic acid cap can then be methylated to produce an N7-methyl-guanylic acid residue. The ribose of the terminal and / or anti-terminal transcribed nucleotide of the 5' end of the mRNA can also optionally be 2'-O-methylated. 5'-decapping via hydrolysis and cleavage of the guanylic acid cap structure can target the mRNA molecule for degradation.

[0141] (c) IRES sequence

[0142] mRNAs containing an internal ribosome entry site (IRES) can also be used in the lipid nanoparticles described herein. The IRES can act as a single ribosome binding site or as one of multiple ribosome binding sites for the mRNA. mRNAs containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by the ribosome (“polycistronic mRNA”). When the mRNA has an IRES, a second translatable region is optionally provided further. Examples of IRES sequences include, but are not limited to, those from picornaviruses (e.g., FMDV), pest viruses (e.g., CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (S1V), and cricket paralysis virus (CrPV).

[0143] (d) Poly-A tail

[0144] During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to polynucleotide molecules such as mRNA to increase stability. Post-transcriptional, the 3' end of the transcript can be immediately cleaved to release the 3' hydroxyl group. Then, poly-A polymerase adds the adenine nucleotide chain to the RNA. This process, known as polyadenylation, adds a poly-A tail of between 100 and 250 residues in length.

[0145] Typically, the poly-A tail is longer than 30 nucleotides. In some embodiments, the poly-A tail is longer than 35 nucleotides (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 length of the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the mRNA. In other embodiments, the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the total length of the mRNA.

[0146] (3) mRNA molecule production

[0147] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, *Gene*, 25:263-269 (1983); Sambrook et al., *Molecular Cloning, A Laboratory Manual* (2nd edition, 1989)). PCR methods are also well known in the art (see, for example, U.S. Patents 4,683,195 and 4,683,202; *PCR Protocols: A Guide to Methods and Applications* (edited by Innis et al., 1990)). Expression libraries are also well known to those skilled in the art. Further foundational texts disclosing the general methods of use in this 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 produced by in vitro transcription and then purified. Standard techniques for purifying mRNA (e.g., mRNA produced by in vitro transcription) are well known in the art and include, for example, oligomeric (dT) affinity purification, cellulose-based purification, and other chromatographic methods. Examples of available mRNA purification techniques are provided by Zhang, J. et al., *Pharmaceutics*, 15:2182 (2023).

[0148] In some embodiments, the provided lipid nanoparticles contain mRNA synthesized from a DNA template. Methods for synthesizing mRNA from a DNA template are well known and generally involve the use of an enzyme (e.g., T7 RNA polymerase and / or its derivatives, such as optimized alternative enzymes), nucleotides (optionally including modified nucleotides as described herein), and optional mRNA components (e.g., a 5' cap). In some embodiments, the lipid nanoparticles contain mRNA generated by in vitro transcription using a commercially available kit. Commercially available kits suitable for generating mRNA contained in the provided lipid nanoparticles include, for example, the MEGASCRIPT™ T7 Transcription Kit available from Thermo Fisher Scientific. Alternatively or additionally, the mRNA used with the provided lipid nanoparticles can be generated using chemical synthesis methods, such as those described by Abe et al., ACS Chem. Biol. 2022, 17, 6, 1308-1314. In another embodiment, the lipid nanoparticles comprise mRNA produced using an mRNA printer (e.g., any of those mRNA printers reviewed in Sheridan, Nature Biotechnol, 2022, 40, 1160-1162).

[0149] In some embodiments, the mRNA generated by any one or more of the methods described herein is purified prior to its use in forming one or more of the provided lipid nanoparticles. Suitable mRNA purification techniques for use with the mRNA in the provided lipid nanoparticles include, but are not limited to, HPLC purification, cellulose purification, tangential flow filtration, bead-based purification (e.g., purification with Dynabead), silica-based purification, precipitation, and combinations thereof. Non-limiting examples of mRNA purification are described, for example, in International Patent Application Publication No. WO 2018 / 006052 A1.

[0150] c) circRNA

[0151] In some instances, the nucleic acids in the provided lipid nanoparticles comprise one or more circular RNA (circRNA) molecules, such as mixtures of circRNA molecules, or are composed of them. circRNA molecules can be selected or engineered for transfer to cells within the body of a subject (e.g., a mammal, such as a human) to which the provided lipid nanoparticles or vaccine are administered. circRNA molecules may encode one or more antigens expressed intracellularly to elicit immunity against a disease (e.g., a disease associated with unwanted cell proliferation (e.g., cancer) or infection, or an autoimmune disease). Therefore, the compositions and methods described herein can be used, for example, to prevent infectious diseases caused by pathogens such as viruses (e.g., coronaviruses, such as SARS-CoV-2) by expressing antigenic peptides (e.g., circRNA molecules encoding viral proteins such as S (spike), E (enveloping), M (membrane), or N (nucleocapsid) proteins or antigenic fragments thereof) to stimulate the adaptive immune system to generate pathogen-targeting T cells in an organism (e.g., a mammal, such as a human).

[0152] In some embodiments, the circRNA molecules are completely encapsulated within lipid particles. In some instances, the RNA in the provided lipid nanoparticles or the provided population of nanoparticles comprises a mixture of one or more types of circRNAs with one or more other types of RNA (e.g., gRNA, siRNA, miRNA, etc.). Regarding formulations containing circRNA mixtures, different types of circRNAs present in the mixture (e.g., circRNAs with different sequences) may be co-encapsulated in the same particle, or each type of circRNA present in the mixture may be encapsulated in a separate particle. CircRNA mixtures can be formulated in particles described herein using mixtures of two or more separate circRNAs (each having a unique sequence) at the same, similar, or different concentrations or molar ratios. In one embodiment, a mixture of circRNAs (corresponding to multiple circRNAs with different sequences) is formulated using each type of circRNA at the same, similar, or different concentrations or molar ratios, and different types of circRNAs are co-encapsulated in the same particle. In another embodiment, each type of circRNA present in the mixture is encapsulated in different particles at the same, similar, or different circRNA concentrations or molar ratios, and the resulting particles (each containing a different circRNA payload) are administered individually (e.g., at different times depending on a prophylactic or therapeutic regimen) or in combination as a single unit dose (e.g., with a pharmaceutically acceptable carrier). In a particular embodiment, the lipid particles are serum stable, resistant to nuclease degradation, and substantially nontoxic to mammals (e.g., humans).

[0153] In another embodiment, the circRNA molecule 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 sites (IRES), in-frame 2A peptide coding sequences, and m6A modified 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, such as any of the nucleoside modifications described in sections C.5.b and C.5.b(1). In such embodiments, nucleoside modifications may also be present in the translatable region. CircRNA molecules containing Kozak sequences are also provided. Additionally, circRNA molecules containing one or more intronic nucleotide sequences that can be excised from the circRNA sequence are provided herein.

[0154] Various methods available in the art can be used to generate circRNA. For example, U.S. Patent No. 6,210,931 teaches a method for synthesizing circRNA by inserting a DNA fragment into a plasmid containing a sequence capable of spontaneous cutting and self-circularization. U.S. Patent No. 5,773,244 teaches the generation of circRNA by preparing a DNA construct encoding an RNA cyclase ribozyme, expressing the DNA construct as RNA, and then subjecting the RNA to self-splicing, which produces intron-free circRNA in vitro. International Patent Application Publication WO 1992 / 001813 teaches a method for preparing single-stranded circular nucleic acids by synthesizing a linear polynucleotide, combining the linear polynucleotide with a complementary linking oligonucleotide under hybridization conditions, and linking the linear polynucleotide. Other methods for generating circRNA are provided, for example, in International Patent Application Publications WO 2015 / 034925 and WO 2016 / 011222.

[0155] d) saRNA

[0156] In some instances, the nucleic acids in the provided lipid nanoparticles comprise one or more self-amplifying RNA (saRNA) molecules, such as mixtures of saRNA molecules, or are composed of them. The saRNA molecules can be selected or engineered for transfer to cells within the body of a subject (e.g., a mammal, such as a human) to which the provided lipid nanoparticles or vaccine are administered. The saRNA molecules may encode one or more antigens expressed intracellularly to elicit immunity against a disease (e.g., a disease associated with unwanted cell proliferation (e.g., cancer) or infection, or an autoimmune disease). Therefore, the compositions and methods described herein can be used, for example, to prevent infectious diseases caused by pathogens such as viruses (e.g., coronaviruses, such as SARS-CoV-2) by expressing antigenic peptides (e.g., saRNA molecules encoding viral proteins such as S (spike), E (enveloping), M (membrane), or N (nucleocapsid) proteins or antigenic fragments thereof) to stimulate the adaptive immune system to generate pathogen-targeting T cells in an organism (e.g., a mammal, such as a human).

[0157] In some embodiments, the saRNA molecules are completely encapsulated within lipid particles. In some instances, the RNA in the provided lipid nanoparticles or the provided population of nanoparticles comprises a mixture of one or more types of saRNA with one or more other types of RNA (e.g., gRNA, siRNA, miRNA, etc.). Regarding formulations containing a mixture of saRNAs, different types of saRNAs present in the mixture (e.g., saRNAs with different sequences) may be co-encapsulated in the same particle, or each type of saRNA present in the mixture may be encapsulated in a separate particle. The saRNA mixture may be formulated in the particles described herein using a mixture of two or more separate saRNAs (each having a unique sequence) at the same, similar, or different concentrations or molar ratios. In one embodiment, a mixture of saRNAs (corresponding to multiple saRNAs with different sequences) is formulated using each saRNA type at the same, similar, or different concentrations or molar ratios, and different types of saRNAs are co-encapsulated in the same particle. In another embodiment, each type of saRNA present in the mixture is encapsulated in different particles at the same, similar, or different saRNA concentrations or molar ratios, and the resulting particles (each containing a different saRNA payload) are administered individually (e.g., at different times depending on a prophylactic or therapeutic regimen) or in combination as a single unit dose (e.g., with a pharmaceutically acceptable carrier). In a particular embodiment, the lipid particles are serum stable, resistant to nuclease degradation, and substantially nontoxic to mammals (e.g., humans).

[0158] In another embodiment, the saRNA molecule 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 sites (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, such as any of the nucleoside modifications described in sections C.5.b and C.5.b(1). In such embodiments, nucleoside modifications may also be present in translatable regions. saRNA molecules containing Kozak sequences are also provided. Additionally, saRNA molecules containing one or more intronic nucleotide sequences that can be excised from the saRNA sequence are provided herein.

[0159] D. Pharmaceutical compositions and formulations

[0160] On the other hand, this disclosure provides a pharmaceutical composition comprising one or more of the lipid nanoparticles or vaccines described in Section C herein. In some embodiments, the pharmaceutical composition further comprises a therapeutically effective amount of a pharmaceutically acceptable excipient. Additionally or alternatively, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0161] Pharmaceutically acceptable carriers (e.g., physiological saline or phosphate-buffered saline) can be selected based on the route of administration and standard pharmaceutical practice. Physiologically buffered saline (e.g., 135–150 mM NaCl) is typically used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and glycoproteins such as albumin, lipoproteins, and globulins, which enhance stability. Further suitable carriers are described, for example, in *Remington: The Science and Practice of Pharmacy*, 23rd edition (2020). Pharmaceutically acceptable carriers are typically added after particle formation. Therefore, after particle formation, the particles can be diluted with a pharmaceutically acceptable carrier (such as physiologically buffered saline).

[0162] Examples of suitable excipients for use with the provided pharmaceutical composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, and polyacrylic acid (such as carbopol, e.g., carbopol 941, carbopol 980, carbopol 981, etc.).

[0163] The provided pharmaceutical compositions may additionally include: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives such as methylparaben, ethylparaben, and propylparaben (i.e., para-hydroxybenzoate); pH adjusters such as inorganic and organic acids and bases; sweeteners; colorants; and / or flavoring agents. The compositions may also contain biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0164] Pharmaceutical compositions that are liquid compositions, whether or not they are solutions, suspensions, or other similar forms, may include one or more of the following: sterile diluents, such as water for injection, saline solutions (preferably physiological saline), Ringer's solution, or isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides of glycerol, polyethylene glycol, glycerol, cyclodextrin, propylene glycol, or other solvents that can act as solvents or suspension media; antimicrobial agents, such as benzyl alcohol or methylparaben; 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 or polyols; and agents for adjusting the tension, such as sodium chloride or dextrose. Liquid compositions may include aqueous or oily suspensions or emulsions containing sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions and similar pharmaceutical mediators. Aqueous solutions in saline are also commonly used for injection. Ethanol, glycerol, propylene glycol, and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. The desired particle size can be maintained in the case of a dispersion system by using coatings such as lecithin, and appropriate flowability can be maintained by using surfactants.

[0165] The provided pharmaceutical composition for administration is preferably sterile. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The composition for administration can be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials.

[0166] For oral administration, the provided pharmaceutical compositions may 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-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0167] For topical application, the provided pharmaceutical composition may be in the form of emulsion, lotion, gel, cream, jelly, solution, suspension, ointment, and / or transdermal patch. For inhalation delivery, the composition may be delivered as a dry powder or in liquid form via a nebulizer. For parenteral application, the composition may be in the form of a sterile injectable solution and / or sterile packaged powder. Preferably, the injectable solution is prepared at a pH of about 4.5 to about 7.5.

[0168] The concentration of lipid nanoparticles in the provided drug formulation can vary widely, for example, from less than about 0.05% by weight, typically or at least about 2% to about 5%, up to about 10% to about 90%, and can be selected primarily by fluid volume, viscosity, etc., depending on the specific administration modality chosen. For example, the concentration can be increased to reduce the treatment-related fluid load. This may be particularly desirable in patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, lipid nanoparticles composed of irritating lipids can be diluted to low concentrations to reduce inflammation at the application site.

[0169] The provided pharmaceutical compositions can be sterilized using conventional, well-known sterilization techniques. Aqueous solutions can be packaged for use or filtered and lyophilized under aseptic conditions; the lyophilized formulation is combined with the sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable excipients close to those required for physiological conditions, such as pH adjusters and buffers, tonicants, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and / or calcium chloride. Additionally, lipid nanoparticle suspensions may include lipid protectants that protect lipids from free radical and lipid peroxidation damage during storage. Lipophilic free radical quenchers, such as α-tocopherol, and water-soluble iron-specific chelating agents (such as ferrioxamine) are suitable.

[0170] E. Reagent kit

[0171] On the other hand, this disclosure provides kits to facilitate and / or standardize the use of the materials described herein (i.e., lipid nanoparticles and vaccines described in Section C and / or pharmaceutical compositions described in Section D), and to facilitate the methods described herein (i.e., the methods described in Section F). Materials and reagents for carrying out these various methods may be provided in the kits to facilitate the execution of the methods.

[0172] The kits provided may contain chemical reagents and other components. Additionally, kits containing the provided lipid nanoparticles, ionizable lipids, and / or pharmaceutical compositions may include, but are not limited to, instructions for use by the kit user. The kits may also be packaged for easy storage and safe transport, for example, as ampoules or other vials packaged in a capped box.

[0173] The provided kit may comprise one or more containers, each of which is separated to contain various elements of the lipid nanoparticles (e.g., nucleic acids and individual lipid components within the nanoparticles). In some embodiments, the kit may further comprise an endosome membrane destabilizer (e.g., calcium ions). The kit typically contains the lipid nanoparticle composition of this disclosure, preferably in a dehydrated form, along with instructions on its rehydration and application.

[0174] F. Method

[0175] In other respects, this disclosure provides several methods for preparing 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 the materials provided, such as properties that facilitate the induction of an immune response in a subject. These improved properties include, for example, stimulation of T cells in the subject, such as CD8 cells specific to the protein of interest or fragments thereof. + T cells.

[0176] 1. Preparation of lipid nanoparticles

[0177] Some methods provided in this disclosure can be used to prepare one or more lipid nanoparticles, wherein the lipid nanoparticles include those described in Section C. Lipid nanoparticles can be prepared by, for example, sequential mixing methods and / or direct dilution methods. In some embodiments, the methods include selecting, designing, and / or preparing one or more components of the prepared lipid nanoparticles. For example, the methods may include selecting, designing, and / or preparing phospholipids, ionizable lipids, cholesterol, or derivatives thereof, conjugated lipids, and / or nucleic acids.

[0178] In some embodiments, the lipid nanoparticles described herein are produced by a continuous mixing method, such as a method comprising: providing an aqueous solution containing 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 is mixed with the aqueous solution, thereby substantially instantaneously producing lipid particles encapsulating the nucleic acid. Examples of this method and apparatus for carrying out this method are described in U.S. Patent Application Publication No. 2004 / 0142025.

[0179] In some instances, the organic lipid solution is an alcoholic organic solvent comprising an alcoholic solution of lipids that will form lipid nanoparticles. These lipids may 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 may be any of the phospholipids described in Section C.1, the conjugated lipid may be any of the conjugated lipids described in Section C.2, the ionizable lipid may be any of the ionizable lipids described in Section C.3, and the sterol may be any of the cholesterol or derivatives thereof described in Section C.4. In some embodiments, the alcoholic organic solvent of the alcoholic solution comprises or is composed of ethanol. In some instances, the alcoholic solution is a product of combining an alcoholic organic solvent and a phospholipid solution, wherein the phospholipid solution comprises a non-alcoholic organic solvent and one or more phospholipids or pharmaceutically acceptable salts thereof. In some embodiments, the non-alcoholic organic solvent comprises or is composed of tetrahydrofuran.

[0180] The act of continuously introducing lipids and a buffer solution into a mixing environment (e.g., in a mixing chamber) causes the lipid solution to be continuously diluted with the buffer solution, thereby generating lipid particles essentially immediately upon mixing. As used herein, the phrase “continuously diluting a lipid solution with a buffer solution” (and its variations) generally refers to a lipid solution being diluted sufficiently rapidly and forcefully during hydration to achieve vesicle generation. By mixing an aqueous solution containing nucleic acids with an organic lipid solution, the organic lipid solution undergoes continuous stepwise dilution in the presence of a buffer solution (i.e., an aqueous solution) to generate lipid particles.

[0181] The lipid nanoparticles formed using a continuous mixing method are typically about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. The resulting particles do not aggregate, and the size is optionally set to achieve a uniform particle size.

[0182] In some embodiments, the lipid nanoparticles described herein are produced by a direct dilution method comprising forming a lipid particle solution and immediately and directly introducing the lipid particle solution into a collection container containing a controlled amount of dilution buffer. In some embodiments, the collection container includes one or more elements configured to agitate the contents of the collection container to facilitate dilution. In one embodiment, the amount of dilution buffer present in the collection container is substantially equal to the volume of the lipid particle solution introduced therein. As a non-limiting example, introducing a lipid particle solution containing 45% ethanol into a collection container containing an equal volume of dilution buffer will advantageously produce smaller particles.

[0183] In some embodiments, the lipid nanoparticles described herein are produced via a direct dilution method, wherein a third reservoir containing a dilution buffer is fluidly coupled to a second mixing region. In this embodiment, the lipid particle solution formed in the first mixing region is immediately and directly mixed with the dilution buffer in the second mixing region. In some embodiments, the second mixing region includes a T-connector arranged such that the lipid particle solution and the dilution buffer flow meet as opposing 180° flows; however, connectors providing a shallower angle, for example, about 27° to about 180°, may be used. A pump mechanism delivers a controlled flow of buffer to the second mixing region. In one embodiment, the flow rate of the dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of the lipid particle solution introduced therein from the first mixing region. This embodiment advantageously allows for greater control over the flow of the dilution buffer mixed with the lipid particle solution in the second mixing region, and therefore also allows for greater control over the concentration of the lipid particle solution in the buffer throughout the second mixing process. This control over the dilution buffer flow rate advantageously allows for the formation of smaller particle sizes at reduced concentrations. Examples of these methods and apparatuses for implementing these direct dilution methods are described in U.S. Patent No. 9,005,654.

[0184] The lipid particles formed using the direct dilution method are typically about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. The resulting particles do not aggregate, and the size is optionally set to achieve a uniform particle size.

[0185] If desired, the lipid particles described herein can be sizing using any of the methods available to those skilled in the art. Sizing can be performed to achieve a desired particle size range and a relatively narrow particle size distribution. Several techniques can be used to sizing the particle size to the desired size, for example, as described in Lam et al. 2023, *Advanced Materials*, 35:26, 2211420. One sizing method for liposomes and equally applicable to the particles of this invention is described in U.S. Patent No. 4,737,323. Sonicating the particle suspension by bath or probe sonication produces a progressive size reduction to a particle size of less than about 50 nm. Homogenization is another method that relies on shear energy to break larger particles into smaller ones. In a typical homogenization procedure, the particles are recycled through a standard emulsion homogenizer until a selected particle size is observed, typically between about 60 nm and about 80 nm. In both methods, the particle size distribution can be monitored using conventional laser beam particle size discrimination or QELS.

[0186] Extruding particles through a microporous polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle size to a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired particle size distribution is achieved. Particles can be extruded through membranes with progressively smaller pores to achieve a gradual reduction in size.

[0187] In some embodiments, the nucleic acid to lipid ratio (mass / mass ratio) in the formed lipid particles is in the range of about 0.01 to about 0.2, about 0.02 to about 0.1, about 0.03 to about 0.1, or about 0.01 to about 0.08. The ratio of starting materials may also fall within this range. In other embodiments, the lipid particle formulation uses a nucleic acid to lipid mass ratio of about 400 µg nucleic acid / 10 mg total lipids or about 0.01 to about 0.08 (e.g., about 0.04, which corresponds to 1.25 mg total lipids / 50 µg nucleic acid). In some embodiments, the nucleic acid:lipid mass ratio of the particles is about 0.08.

[0188] In other embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio) in the formed lipid particles ranges from about 1 (1:1) to about 100 (100:1), about 5 (5:1) to about 100 (100:1), about 1 (1:1) to about 50 (50:1), about 2 (2:1) to about 50 (50:1), about 3 (3:1) to about 50 (50:1), about 4 (4:1) to about 50 (50:1), about 5 (5:1) to about 50 (50:1), about 1 (1:1) to about 25 (25:1), about 2 (2:1) to about 25 (25:1). 1) Approximately 3 (3:1) to approximately 25 (25:1), approximately 4 (4:1) to approximately 25 (25:1), approximately 5 (5:1) to approximately 25 (25:1), approximately 5 (5:1) to approximately 20 (20:1), approximately 5 (5:1) to approximately 15 (15:1), approximately 5 (5:1) to approximately 10 (10:1), approximately 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 starting materials may also fall within this range.

[0189] 2. Inducing an immune response

[0190] Some methods provided by this disclosure can be used to induce an immune response in a subject. These methods generally involve administering to a subject one or more lipid nanoparticles, one or more vaccines, or one or more pharmaceutical compositions, 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, inducing an immune response includes stimulating T cells, such as CD8 cells specific for a protein or fragment thereof. + T cells. The provided methods can be used to induce immune responses against, for example, unwanted cell proliferation (e.g., cancer), infection, or autoimmune diseases.

[0191] The disclosed lipid nanoparticles, vaccines, and pharmaceutical compositions can be administered as a single dose or as multiple doses using the provided methods, for example, by administering two doses at intervals 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 dosing regimens can be determined by a practicing physician. In some embodiments, additional compounds or drugs may be co-administered to the subject. Such compounds or drugs may be co-administered to, for example, alleviate signs or symptoms of disease caused by undesirable cell proliferation or the treated pathogenic infection, or reduce side effects caused by inducing an immune response.

[0192] The provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions can be administered via any of the generally accepted modes of administration. Thus, administration can be, for example, intramuscular, intradermal, intravenous, topical, subcutaneous, percutaneous, transdermal, oral, intra-articular, parenteral, intra-arterial, intracardiac, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, or by inhalation. In a preferred embodiment, the lipid nanoparticles, vaccines, and / or pharmaceutical compositions are administered intramuscularly or intradermally. In a particularly preferred embodiment, the lipid nanoparticles, vaccines, and / or pharmaceutical compositions are administered intramuscularly.

[0193] 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 generating antigen-associated T cells, wherein the antigen or a fragment thereof is encoded by a nucleic acid in a lipid nanoparticle or vaccine. The antigen can be any of the antigens described herein, such as any of the antigens described in section C.5.a.

[0194] In some embodiments, the immune response induced by the provided method is greater than the immune response induced by a corresponding composition that does indeed include lipid nanoparticles having the formulations described in Section C.

[0195] 3. To prevent or treat diseases or symptoms

[0196] Some methods provided by this disclosure can be used to prevent or treat a disease or condition in a subject. These methods generally involve administering to the subject one or more lipid nanoparticles, one or more vaccines, or one or more pharmaceutical compositions, 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, treating a subject's disease includes reducing or eliminating one or more signs or symptoms of the disease.

[0197] In some embodiments, the disease or condition being prevented or treated 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, Sjögren's syndrome, undifferentiated connective tissue disease, Addison's disease, dermatomyositis, and Hashimoto's thyroiditis. Thyroiditis, myasthenia gravis, ankylosing spondylitis (AS), antiphospholipid syndrome (APS), autoimmune autonomic ganglion disease (AAG), autoimmune encephalitis, autoimmune gastritis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), autoimmune polyglandular syndrome, autoimmune progesterone dermatitis, Balo disease, Behçet's disease, bullous pemphigoid, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cold agglutinin disease, CREST syndrome, localized systemic sclerosis of the cutaneous region, herpetic dermatitis, discoid lupus, eosinophilic fasciitis, Evans syndrome Glomerulonephritis, Goodpasture's syndrome, granulomatous disease with polyangiitis (GPA), Guillain-Barré syndrome (GBS), Henoch-Schönlein purpura, Hurst's disease, IgA nephropathy, immune thrombocytopenic purpura (ITP), juvenile idiopathic arthritis, Lambert-Eaton myasthenic syndrome,LEMS, linear IgA disease (LAD), lupus nephritis, lymphocytic hypophysitis, Ménière's disease, mixed connective tissue disease (MCTD), Devic's disease, ocular cicatricial pemphigoid, relapsing rheumatic disease, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, paroxysmal nocturnal hemoglobinuria (PNH), Parsonage-Turner syndrome, gestational pemphigus, pemphigus foliaceus, pemphigus vulgaris, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis polymyositis, primary biliary cirrhosis (PBC), palmoplantar pustulosis, pure red cell aplasia (PRCA), Raynaud's syndrome, reactive arthritis, relapsing polychondritis, rheumatic fever, sarcoidosis, Schmidt syndrome Syndrome), scleroderma, small fiber sensory neuropathy, testicular autoimmune disease, vitiligo, and combinations thereof.

[0198] In some embodiments, the disease or condition being prevented or treated is a disease or condition characterized by undesirable cell proliferation. Non-limiting examples of such diseases and conditions that can be treated with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions include cancer, benign prostatic hyperplasia (BPH), psoriasis, endometriosis, polycystic ovary syndrome (PCOS), hypertrophic cardiomyopathy, pulmonary fibrosis, Graves' disease, fibroadenoma, prostatic nodular hyperplasia, adenomyosis, uterine fibroids, adenomatous goiter, colonic adenomatous polyps, non-malignant basal cell carcinoma, and cervical dysplasia. Skin warts, fibrous dysplasia, gastric hyperplastic polyps, giant cell tumor of bone, glomerulonephritis, hepatic adenoma, colonic hyperplastic polyps, hyperthyroid goiter, keloids, leiomyomas, mastocytosis, medullary sponge kidney, myeloproliferative disorders, nevi, non-alcoholic fatty liver disease (NAFLD), osteochondroma, osteoid osteoma, osteosclerosis, ovarian cysts, pancreatic pseudocysts, parathyroid hyperplasia, Peutz-Jeghers syndrome, proliferative diabetic retinopathy, and combinations thereof.

[0199] In some embodiments, the disease being prevented or treated is cancer. Non-limiting examples of cancers that can be treated with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions include acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentigines-like melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, mature acute myeloid leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, amelomas, adenocarcinoma, adenoid cystic carcinoma, adenoma, and adenoma-like odontogenesis. Sexually transmitted tumors, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancers, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdomyosarcoma, basal cell carcinoma, basoid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma. Duct carcinoma, biliary tract cancer, bladder cancer, germ cell tumor, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt's lymphoma, cancer of unknown primary location, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinosarcoma, Castleman's disease, embryonal tumor of the central nervous system, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct cancer, chondroma, chondrosarcoma, chordoma, choroidal carcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease. Diseases, including dermatofibrosarcoma protuberans, dermoid cysts, desmoplastic small round cell tumors, diffuse large B-cell lymphomas, dysembryonic neuroepithelial tumors, embryonal carcinomas, endodermal sinus tumors, endometrial cancer, endometrial uterine cancer, endometrioid tumors, enteropathy-associated T-cell lymphomas, ependymoblastomas, ependymomas, epithelioid sarcomas, erythroleukemia, esophageal cancer, olfactory neuroblastomas, Ewing family tumors, Ewing family sarcomas, Ewing's sarcoma, extracranial germ cell tumors, gonadal germ cell tumors, extrahepatic bile duct carcinomas, and extramammary Paget's disease.Diseases, fallopian tube cancer, parasitic twin, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglion cell glioma, ganglion neuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, embryonic tissue tumor, gestational choriocarcinoma, gestational trophoblastoma, giant cell tumor of bone, glioblastoma multiforme, glioma, cerebral glioma, glomus tumor, glucagonoma, gonadal blastoma, ovarian granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer. Cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer-ovarian cancer syndrome, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, renal cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer. Cancer), malignant lentigines, melanoma, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibroushistiocytoma, bone malignant fibrous histiocytoma, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdomyosarcoma, malignant salamander tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma.Mesothelioma, mesothelioma, metastatic squamous neck carcinoma with occult primary lesion, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, myxoid tumor, multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disorders, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorders, myxoma, nasal cavity carcinoma, nasopharyngeal carcinoma, nasopharyngeal carcinoma, vegetation, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma. Lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular tumors, oligodendroastrocytoma, oligodendroglioma, large eosinophilia, optic nerve sheath meningioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, low-grade malignant potential ovarian tumors, Paget's disease of the breast, superior sulcus tumor of the lung, pancreatic cancer. Cancer), papillary thyroid carcinoma, papilloma, paraganglioma, sinus carcinoma, parathyroid carcinoma, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineal blastoma, pituitary cell tumor, pituitary adenoma, pituitary adenoma, plasma cell vegetation, pleural pulmonary blastoma, polyembryoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal carcinoma, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, rhabdomyosarcoma, Richter's transformationTransformation), sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, Schwannomatosis, sebaceous gland carcinoma, secondary growths, 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 intestinal cancer, soft tissue sarcoma, somatostatinoma, sootypic warts, spinal cord tumors, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial diffuse melanoma, supratentorial primitive neuroectodermal tumor. Tumors, surface epithelial-stromal tumors, synovial sarcomas, T-cell acute lymphoblastic leukemia, T-cell large granular lymphoblastic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphoblastic leukemia, teratomas, advanced lymphomas, testicular cancer, theca cell tumors, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, urogenital vegetations, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

[0200] In some embodiments, the disease being prevented or treated is a cancerous tumor. The cancerous tumor can be a solid tumor or a liquid-filled tumor. A liquid-filled tumor can be, for example, lymphoma or leukemia. Tumors treated with the methods disclosed herein can achieve stable tumor growth (e.g., the size of one or more tumors increases by no more than 1%, 5%, 10%, 15%, or 20%, and / or there is no metastasis). In some embodiments, the tumor remains stable for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more. In some embodiments, the tumor remains stable for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. In some embodiments, the tumor remains stable for at least about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more. In some embodiments, the size of the 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 the detectable level. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or more after treatment. In some embodiments, the subject remains tumor-free for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more after treatment. In some embodiments, the subject remains tumor-free for at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more after treatment.

[0201] Those skilled in the art will also understand that the provided systems and compositions can be co-administered with other therapeutic agents used to treat cancer. Suitable anticancer agents for combination therapy include, but are not limited to, cytotoxins and pharmaceuticals such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, interferon, radiopharmaceuticals, peptides with antitumor activity (such as TNF-α), their pharmaceutically acceptable salts, their derivatives, their prodrugs, and combinations thereof. For example, pharmaceutical compositions comprising the provided retroviral vector system, retroviral packaging cells, retroviruses, and / or virus-like particles can be administered to patients before, during, or after chemotherapy, or before, during, or after the administration of one or more anticancer agents in combination.

[0202] In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions achieves disease stabilization, partial remission, or complete remission in a subject (e.g., the methods described herein comprise administering to a subject a dose of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions that kills cancer cells or otherwise slows the growth or progression of cancer cells and causes disease stabilization or partial or complete remission of cancer in the subject). In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions achieves a reduction in cancer metastasis in a subject (e.g., the methods described herein comprise administering to a subject a dose of the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions that reduces cancer metastasis in the subject). In some embodiments, treatment with the provided lipid nanoparticles, vaccines, and / or pharmaceutical compositions achieves a reduction in the volume, size, or growth of a tumor in a subject (e.g., the methods described herein comprise administering to a subject 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, vaccine and / or pharmaceutical composition achieves an increased responsiveness of cancer to a subsequently administered anticancer agent (e.g., the methods described herein involve administering to a subject a dose of the provided lipid nanoparticles, vaccine and / or pharmaceutical composition that increases the responsiveness of cancer to a subsequently administered anticancer agent).

[0203] In some instances, the methods provided can be used to prevent or treat infections or infectious diseases caused by pathogens such as viruses, bacteria, fungi, parasites, or any other infectious agents. Non-limiting examples of infectious diseases for which the provided methods may be used for treatment include acquired immunodeficiency syndrome (AIDS / HIV) or HIV-related conditions, Alpers syndrome, anthrax, bovine spongiform encephalopathy (mad cow disease), varicella, 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), kuru, leprosy, Lyme disease, malaria, and hemorrhagic fever (e.g., Rift Valley fever). Fever, Crimean-Congo hemorrhagic fever, Lassa fever, Marburg virus disease and Ebola hemorrhagic fever, measles, meningitis (viral and bacterial), mononucleosis, hospital-acquired infections, otitis media, pelvic inflammatory disease (PID), plague, pneumonia, poliomyelitis, prions, rabies, rheumatic fever, roseola, Ross River virus infection, rubella, salmonellosis, pyogenic arthritis, sexually transmitted diseases (STDs), herpes zoster, smallpox, streptococcal pharyngitis, tetanus, toxic shock syndrome, toxoplasmosis, trachoma, tuberculosis, tularemia, typhoid fever, grain fever, pertussis and yellow fever. The methods provided can be used for the prevention or treatment of infections caused by any of the pathogens described in section C.5.a.

[0204] In some embodiments, the provided method further includes obtaining a test sample from a subject. The test sample may 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. As a non-limiting example, determining the presence or level of a biomarker may be used to determine a response to treatment or to select an appropriate composition for the prevention or treatment of a disease.

[0205] In instances where the lipid nanoparticles, vaccines, or pharmaceutical compositions provided therein comprise nucleic acids encoding a protein of interest or a fragment thereof, the measured biomarker may be an indicator of the expression level of a protein or fragment in a target cell or target organism (e.g., the cell or organism to which the lipid nanoparticles, vaccine, or pharmaceutical composition is administered). For example, the biomarker may be the protein or fragment itself, such that the measured level of the protein or fragment indicates its expression level. When the protein of interest is an antigen, the measured biomarker may include or be composed of one or more components of the immune system of the target cell or target organism. For example, the biomarker may include one or more types of T cells (e.g., CD8+). + (T cells) or are composed of them. In these ways, the determination of biomarker levels in a test sample can provide information related to the efficiency and / or effectiveness of applying lipid nanoparticles in the prevention or treatment of diseases or conditions.

[0206] In some embodiments, the provided method further includes comparing determined levels of one or more biomarkers in the obtained test sample with levels of one or more biomarkers in a reference sample. The reference sample may be obtained, for example, from the subject, wherein the reference sample is obtained prior to obtaining the test sample, e.g., prior to administering a therapeutically effective amount of the provided material to the subject. In this way, the reference sample can provide information about baseline levels of biomarkers in the pre-treatment sample, and the test sample can provide information about post-treatment levels of biomarkers.

[0207] Alternatively, reference samples can be obtained, for example, from different subjects, such as subjects who were not treated according to the provided method. In this way, reference samples can provide information about baseline levels of the biomarker in the absence of treatment, and test samples can provide information about the levels of the biomarker in the presence of treatment. Reference samples can also be obtained, for example, from a group of subjects, such as subjects who were not treated according to the provided method. In this way, reference samples can provide population-average information about baseline levels of the biomarker in the absence of treatment, and test samples can provide information about the levels of the biomarker in the presence of treatment.

[0208] Reference samples may also be obtained from individuals or groups of individuals after treatment has been administered according to the provided method, and may serve as, for example, a positive control sample. In some embodiments, reference samples are obtained from normal tissue. In some embodiments, reference samples are obtained from abnormal tissue.

[0209] An increase or decrease in a biomarker relative to a normal control or reference sample can indicate the presence of a disease or a response to treatment. In some embodiments, an increase in the level of a biomarker in a test sample is determined when the biomarker level is at least 1.1 times higher than a control, for example, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times higher than a control, and thus the presence of a disease (e.g., an infectious disease or cancer), an increased risk of disease, or a response to treatment is determined. In other embodiments, a reduction in the level of a biomarker in the test sample is determined when the biomarker level is at least 1.1 times lower than that of a control, for example, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times lower, and thus the presence of disease, an increased risk of disease, or a response to treatment is determined.

[0210] Biomarker levels can be detected using any method known in the art, including the use of antibodies specific to the biomarker. Exemplary methods include, but are not limited to, polymerase chain reaction (PCR), Western blotting, dot blotting, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays (e.g., using Luminex or fluorescent microbeads). In some cases, nucleic acid sequencing is employed.

[0211] In some embodiments, the presence of a reduced or increased level of one or more biomarkers is indicated by a detectable signal (e.g., blot, fluorescence, chemiluminescence, color, or radioactivity) in an immunoassay or PCR reaction (e.g., quantitative PCR). This detectable signal can be compared to a signal or threshold from a reference sample.

[0212] In some embodiments, the results of biomarker level determination are recorded in a tangible medium. For example, the results of diagnostic assays, such as the presence or absence of one or more biomarkers or observations of a decrease or increase in their presence, the presence of an increased risk or disease (e.g., an infectious disease or cancer), or a diagnosis of whether a subject responds to treatment, may be recorded on, for example, paper or electronic media, such as audio tapes, computer disks, CD-ROMs, or flash drives.

[0213] In some embodiments, the provided method further includes the step of providing the subject with diagnostic and / or treatment results.

[0214] G. Exemplary Embodiment

[0215] The following embodiments are envisioned. All combinations of features and embodiments are envisioned.

[0216] Example 1: A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof; a conjugated lipid comprising 0.1 mol% to 5 mol% of the total lipids of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof comprising three or more terminal hydrocarbon chains, each of the three or more terminal hydrocarbon chains independently having 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.

[0217] Example 2: According to the example described in Example 1, wherein the phospholipid has a net negative charge at physiological pH.

[0218] Example 3: According to the example described in Example 1 or 2, the conjugated lipid accounts for 0.1 mol% to 1 mol% of the total lipids in the lipid nanoparticles.

[0219] Example 4: According to any one of Examples 1 to 3, wherein the phospholipid or its pharmaceutically acceptable salt accounts for at least 8 mol of the total lipids of the lipid nanoparticles.

[0220] Example 5: According to any one of Examples 1 to 4, wherein the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids of the nanoparticles, and the ionizable lipid does not contain sulfur.

[0221] Example 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 lipids 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.

[0222] Example 7: According to the example described in Example 6, wherein the phospholipid or its pharmaceutically acceptable salt accounts for at least 8 mol of the total lipids of the lipid nanoparticles.

[0223] Example 8: According to the example described in Example 6 or 7, wherein: the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids of the nanoparticles, and the ionizable lipid does not contain sulfur.

[0224] Example 9: A lipid nanoparticle comprising: phospholipids or pharmaceutically acceptable salts thereof, the phospholipids having a net negative charge at physiological pH and comprising at least 8 mol% of the total lipids of the lipid nanoparticles; conjugated lipids comprising 0.1 mol% to 5 mol% of the total lipids of the lipid nanoparticles; ionizable lipids or pharmaceutically acceptable salts thereof; and nucleic acids encoding a protein of interest or a fragment thereof.

[0225] Example 10: According to the example described in Example 9, wherein: the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids of the nanoparticles, and the ionizable lipid does not contain sulfur.

[0226] Example 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 0.1 mol% to 5 mol% of the total lipids of the lipid nanoparticle; an ionizable lipid or a pharmaceutically acceptable salt thereof comprising more than 1 mol% of the total lipids of the lipid nanoparticle, the ionizable lipid being sulfur-free; and a nucleic acid encoding a protein of interest or a fragment thereof.

[0227] Example 12: An example according to any one of Examples 1 to 11, wherein the phospholipid comprises naturally occurring phospholipids.

[0228] Example 13: An example according to any one of Examples 1 to 12, wherein the phospholipid comprises phosphatidylserine.

[0229] Example 14: According to the embodiment described in Example 13, wherein the phosphatidylserine comprises 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-L-serine), 1,2-distearateyl-sn-glycerol-3-phosphate-L-serine (DSPS), and 1,2-dimyristoyl-sn-glycerol-3-phosphate-L-serine (…). DMPS), 1,2-dicepanoyl-sn-glycerol-3-phosphate-L-serine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (DPPS), 1,2-dilauroyl-sn-glycerol-3-phosphate-L-serine (DLPS), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-L-serine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate-L-serine) Acids, 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (SOPS), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-arachidonicoyl-s n-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-L-serine, or combinations thereof.

[0230] Example 15: An example according to any one of Examples 1 to 12, wherein the phospholipid comprises phosphatidylglycerol.

[0231] Example 16: According to the embodiment described in Example 15, wherein the phosphatidylglycerol comprises 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-decanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilauroyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DLPG), 1,2-dimyristoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DO ... DMPG), 1,2-bispentadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DPPG), 1,2-distearyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DSPG), 1,2-ditransoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinolenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) 1,2-Diarachidonic-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1,2-bis(docohexanoyl-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-palmitoyl-2-arachidonic-sn-glycerol-3-phosphate-(1'-rac-glycerol) (PAPG), 1 - Palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (SOPG), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (SAPG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) or combinations thereof.

[0232] Example 17: According to any one of Examples 1 to 16, wherein the phospholipid or its pharmaceutically acceptable salt accounts for 1 mol% to 20 mol% of the total lipids of the lipid nanoparticles.

[0233] Example 18: According to any one of Examples 1 to 17, the conjugated lipid comprises two or more terminal hydrocarbon chains, each of which has five or more members and is not directly bonded to an ester.

[0234] Example 19: An example according to any one of Examples 1 to 18, wherein the conjugated lipid comprises a carbamate bond.

[0235] Example 20: According to any one of Examples 1 to 19, wherein the conjugated lipid comprises a polyethylene glycol (PEG) lipid conjugate.

[0236] Example 21: According to any one of Examples 1 to 20, the conjugated lipid comprises N-[(methoxypoly(ethylene glycol))carbamoyl]-1,2-dimyristyloxypropylamine (PEG-c-DMA).

[0237] Example 22: An example according to any one of Examples 1 to 21, wherein the ionizable lipid does not contain disulfide bonds.

[0238] Example 23: An embodiment according to any one of Examples 1 to 22, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains, each of the three or more terminal hydrocarbon chains having five or more members.

[0239] Example 24: According to any one of Examples 1 to 23, the ionizable lipid comprises 5-(dimethylamino)valerate (6Z,16Z)-12-((Z)-dec-4-en-1-yl) dodec-6,16-dien-11-yl ester (3D-P-DMA).

[0240] Example 25: According to any one of Examples 1 to 24, wherein the ionizable lipid or its pharmaceutically acceptable salt accounts for 30 mol% to 70 mol% of the total lipids in the lipid nanoparticles.

[0241] Example 26: According to any one of Examples 1 to 25, wherein the lipid nanoparticles further comprise cholesterol or a derivative thereof.

[0242] Example 27: According to the example described in Example 26, wherein the cholesterol or its derivatives account for 20 mol% to 60 mol% of the total lipids in the lipid nanoparticles.

[0243] Example 28: According to any one of Examples 1 to 27, wherein the lipid nanoparticles further comprise permanently charged cationic lipids, wherein the permanently charged cationic lipids account for no more than 10 mol of the total lipids of the lipid nanoparticles.

[0244] Example 29: An example according to any one of Examples 1 to 27, wherein the lipid nanoparticles do not contain permanently charged cationic lipids.

[0245] Example 30: An embodiment according to any one of Examples 1 to 29, wherein: the phospholipid or its pharmaceutically acceptable salt accounts for 8.5 mol% to 10.5 mol% of the total lipids of the lipid nanoparticles; the conjugated lipid accounts for 0.25 mol% to 0.9 mol% of the total lipids of the lipid nanoparticles; the ionizable lipid or its pharmaceutically acceptable salt accounts for 48 mol% to 58 mol% of the total lipids of the lipid nanoparticles; and the lipid nanoparticles further comprise cholesterol or its derivatives, the cholesterol or its derivatives accounting for 35 mol% to 39 mol% of the total lipids of the lipid nanoparticles.

[0246] Example 31: An embodiment according to any one of Examples 1 to 29, wherein: the phospholipid or its pharmaceutically acceptable salt accounts for 9 mol% to 11 mol% of the total lipids of the lipid nanoparticles; the conjugated lipid accounts for 0.85 mol% to 2.6 mol% of the total lipids of the lipid nanoparticles; the ionizable lipid or its pharmaceutically acceptable salt accounts for 45 mol% to 55 mol% of the total lipids of the lipid nanoparticles; and the lipid nanoparticles further comprise cholesterol or its derivatives, the cholesterol or its derivatives accounting for 36.5 mol% to 40.5 mol% of the total lipids of the lipid nanoparticles.

[0247] Example 32: According to the example of Example 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.

[0248] Example 33: An example according to any one of Examples 1 to 32, wherein the nucleic acid comprises RNA.

[0249] Example 34: According to the embodiment described in Example 33, the RNA comprises mRNA, saRNA, circRNA, or a combination thereof.

[0250] Example 35: An example according to any one of Examples 1 to 32, wherein the nucleic acid comprises DNA.

[0251] Example 36: An example according to any one of Examples 1 to 35, wherein the protein of interest comprises an antigen.

[0252] Example 37: According to the example described in Example 36, wherein the antigen is associated with a disease, infection, or autoimmune disease characterized by undesirable cell proliferation.

[0253] Example 38: According to the example described in Example 37, the antigen comprises a cancer antigen.

[0254] Example 39: According to the example described in Example 38, wherein the cancer antigen comprises a subject-specific cancer antigen.

[0255] Example 40: A method for generating lipid nanoparticles, the method comprising: providing an aqueous solution containing a nucleic acid encoding a protein of interest or a fragment thereof; providing an alcohol solution containing an alcohol 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 alcohol solution to generate a lipid nanoparticle solution containing the lipid nanoparticles; wherein the conjugated lipid comprises 0.1 mol% to 5 mol% of the total lipids in the alcohol solution; and the composition of the alcohol solution is configured such that the lipid nanoparticles have a negative zeta potential at physiological pH.

[0256] Example 41: According to the example described in Example 40, wherein the phospholipid has a net negative charge at physiological pH.

[0257] Example 42: According to the example of Example 40 or 41, wherein the conjugated lipid accounts for 0.1 mol% to 1 mol% of the total lipids in the alcohol solution.

[0258] Example 43: An example according to any one of Examples 40 to 42, wherein the phospholipid or its pharmaceutically acceptable salt accounts for at least 8 mol of the total lipids in the alcohol solution.

[0259] Example 44: An embodiment according to any one of Examples 40 to 43, wherein the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids of the nanoparticles, and the ionizable lipid does not contain sulfur.

[0260] Example 45: A method for producing lipid nanoparticles, the method comprising: providing an aqueous solution containing a nucleic acid encoding a protein of interest or a fragment thereof; providing an alcohol solution containing 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; wherein the phospholipid has a net negative charge at physiological pH; and combining the aqueous solution and the alcohol solution to produce a lipid nanoparticle solution containing the lipid nanoparticles; wherein the conjugated lipid comprises less than 1 mol of the total lipids in the alcohol solution.

[0261] Example 46: According to the example described in Example 45, wherein the phospholipid or its pharmaceutically acceptable salt accounts for at least 8 mol of the total lipids of the lipid nanoparticles.

[0262] Example 47: An example according to Example 45 or 46, wherein: the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids of the nanoparticles, and the ionizable lipid does not contain sulfur.

[0263] Example 48: A method for producing lipid nanoparticles, the method comprising: providing an aqueous solution containing a nucleic acid encoding a protein of interest or a fragment thereof; providing an alcohol solution containing 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 alcohol solution to produce a lipid nanoparticle solution containing the lipid nanoparticles, wherein: the conjugated lipid accounts for 0.1 mol% to 5 mol% of the total lipids in the alcohol solution; and the phospholipid or its pharmaceutically acceptable salt accounts for at least 8 mol% of the total lipids in the alcohol solution.

[0264] Example 49: According to the example described in Example 48, wherein: the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids of the nanoparticles, and the ionizable lipid does not contain sulfur.

[0265] Example 50: A method for generating lipid nanoparticles, the method comprising: providing an aqueous solution containing a nucleic acid encoding a protein of interest or a fragment thereof; providing an alcohol solution containing an alcohol 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 alcohol solution to generate a lipid nanoparticle solution containing the lipid nanoparticles, wherein: the conjugated lipid accounts for 0.1 mol% to 5 mol% of the total lipids in the alcohol solution; the ionizable lipid or its pharmaceutically acceptable salt accounts for more than 1 mol% of the total lipids in the alcohol solution; and the ionizable lipid does not contain sulfur.

[0266] Example 51: An embodiment according to any one of Examples 40 to 50, wherein the method further comprises removing at least a portion of the alcohol organic solvent from the lipid nanoparticle solution.

[0267] Example 52: According to the embodiment described in Example 51, wherein removing at least a portion of the alcohol organic solvent comprises exchanging the alcohol organic solvent with an aqueous solution.

[0268] Example 53: An example according to any one of Examples 40 to 52, wherein the method further comprises concentrating the lipid nanoparticle solution.

[0269] Example 54: According to the example described in Example 53, the concentration of the lipid nanoparticle precursor includes centrifuging the lipid nanoparticle solution.

[0270] Example 55: An example according to any one of Examples 40 to 54, wherein the method further comprises filtering the lipid nanoparticle solution.

[0271] Example 56: An example according to any one of Examples 40 to 55, wherein the alcohol organic solvent comprises ethanol.

[0272] Example 57: According to any one of Examples 40 to 56, the provision of the alcohol solution comprises: forming a phospholipid solution comprising a non-alcoholic organic solvent and the phospholipid or a pharmaceutically acceptable salt thereof; and combining the phospholipid solution and the alcoholic organic solvent.

[0273] Example 58: According to the example described in Example 57, the non-alcoholic organic solvent comprises tetrahydrofuran.

[0274] Example 59: An example according to any one of Examples 40 to 58, wherein the phospholipid comprises naturally occurring phospholipids.

[0275] Example 60: An example according to any one of Examples 40 to 59, wherein the phospholipid comprises phosphatidylserine.

[0276] Example 61: According to the embodiment described in Example 60, wherein the phosphatidylserine comprises DOPS, 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-L-serine), DSPS, DMPS, 1,2-decanoyl-sn-glycerol-3-phosphate-L-serine, DPPS, DLPS, 1,2-dilinoleyl-sn-glycerol-3-phosphate-L-serine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate-L-serine), 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, POPS, S OPS, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-L-serine, or combinations thereof.

[0277] Example 62: An example according to any one of Examples 40 to 61, wherein the phospholipid comprises phosphatidylglycerol.

[0278] Example 63: According to the embodiment described in Example 62, wherein the phosphatidylglycerol comprises DOPG, 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-decanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), DLPG, DMPG, 1,2-bis(pentadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), DPPG, DSPG, 1,2-ditransoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), and 1,2-dilinolenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol). 1,2-Diarachidonico-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1,2-bis(docosahexaenooyl-sn-glycerol-3-[phosphate-rac-(1-glycerol)], POPG, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), PAPG, 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), SOPG, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), SAPG, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), or combinations thereof.

[0279] Example 64: An example according to any one of Examples 40 to 63, wherein the phospholipid or its pharmaceutically acceptable salt accounts for 1 mol% to 20 mol% of the total lipids in the alcohol solution.

[0280] Example 65: An embodiment according to any one of Examples 40 to 64, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains, each of the two or more terminal hydrocarbon chains having five or more members and each not being directly bonded to an ester.

[0281] Example 66: An example according to any one of Examples 40 to 65, wherein the conjugated lipid comprises a carbamate bond.

[0282] Example 67: An example according to any one of Examples 40 to 66, wherein the conjugated lipid comprises a PEG-lipid conjugate.

[0283] Example 68: An example according to any one of Examples 40 to 67, wherein the conjugated lipid comprises PEG-c-DMA.

[0284] Example 69: An example according to any one of Examples 40 to 68, wherein the ionizable lipid does not contain disulfide bonds.

[0285] Example 70: An example according to any one of Examples 40 to 69, wherein the ionizable lipid does not contain sulfur.

[0286] Example 71: An embodiment according to any one of Examples 40 to 70, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains, each of the three or more terminal hydrocarbon chains having five or more members.

[0287] Example 72: An embodiment according to any one of Examples 40 to 71, wherein the ionizable lipid comprises 3D-P-DMA.

[0288] Example 73: An example according to any one of Examples 40 to 72, wherein the ionizable lipid or its pharmaceutically acceptable salt accounts for 30 mol% to 70 mol% of the total lipids in the alcohol solution.

[0289] Example 74: An example according to any one of Examples 40 to 73, wherein the alcohol solution further comprises cholesterol or a derivative thereof.

[0290] Example 75: According to the example described in Example 74, wherein the cholesterol or its derivatives account for 20 mol% to 60 mol% of the total lipids in the alcohol solution.

[0291] Example 76: An example according to any one of Examples 40 to 75, wherein: the phospholipid or its pharmaceutically acceptable salt accounts for 8.5 mol% to 10.5 mol% of the total lipids in the alcoholic solution; the conjugated lipid accounts for 0.25 mol% to 0.9 mol% of the total lipids in the alcoholic solution; the ionizable lipid or its pharmaceutically acceptable salt accounts for 48 mol% to 58 mol% of the total lipids in the alcoholic solution; and the alcoholic solution further comprises cholesterol or its derivatives, the cholesterol or its derivatives accounting for 35 mol% to 39 mol% of the total lipids in the alcoholic solution.

[0292] Example 77: An example according to any one of Examples 40 to 75, wherein: the phospholipid or its pharmaceutically acceptable salt accounts for 9 mol% to 11 mol% of the total lipids in the alcoholic solution; the conjugated lipid accounts for 0.85 mol% to 2.6 mol% of the total lipids in the alcoholic solution; the ionizable lipid or its pharmaceutically acceptable salt accounts for 45 mol% to 55 mol% of the total lipids in the alcoholic solution; and the alcoholic solution further comprises cholesterol or its derivatives, the cholesterol or its derivatives accounting for 36.5 mol% to 40.5 mol% of the total lipids in the alcoholic solution.

[0293] Example 78: According to the example of Example 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.

[0294] Example 79: An example according to any one of Examples 40 to 78, wherein the nucleic acid comprises RNA.

[0295] Example 80: According to the embodiment described in Example 79, the RNA comprises mRNA, saRNA, circRNA, or a combination thereof.

[0296] Example 81: An example according to any one of Examples 40 to 78, wherein the nucleic acid comprises DNA.

[0297] Example 82: An example according to any one of Examples 40 to 81, wherein the protein of interest comprises an antigen.

[0298] Example 83: According to the example described in Example 82, wherein the antigen is associated with a disease, infection, or autoimmune disease characterized by undesirable cell proliferation.

[0299] Example 84: According to the embodiment described in Example 83, wherein the antigen comprises a cancer antigen.

[0300] Example 85: According to the example described in Example 84, the cancer antigen comprises a subject-specific cancer antigen.

[0301] Example 86: A lipid nanoparticle that can be obtained by the method according to any one of Examples 40 to 85.

[0302] Example 87: A vaccine comprising lipid nanoparticles according to any one of Examples 1 to 39 or 86.

[0303] Example 88: According to the example described in Example 87, wherein the protein of interest comprises an antigen.

[0304] Example 89: According to the embodiment described in Example 88, wherein the antigen comprises a cancer antigen, and wherein the vaccine is a cancer vaccine.

[0305] Example 90: According to the embodiment described in Example 89, wherein the cancer antigen comprises a subject-specific cancer antigen, and wherein the cancer vaccine is a personalized cancer vaccine.

[0306] Example 91: An example according to any one of Examples 87 to 90, wherein the vaccine further comprises a pharmaceutically acceptable excipient.

[0307] Example

[0308] This disclosure will be described in more detail through specific examples. The following examples are for illustrative purposes only and are not intended to limit this disclosure in any way.

[0309] Example 1. Preparation of lipid nanoparticles.

[0310] Nucleic acids were encapsulated in lipid nanoparticles using a controlled mixing method of the type described in U.S. Patent No. 9,005,654. In this method, an aqueous solution of mRNA at pH 5.0 in 100 mM acetate buffer was combined with an ethanol solution of lipids. The ethanol solution comprised PEG-conjugated lipids, ionizable lipids (3D-P-DMA: 5-(dimethylamino)valerate (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl ester; WO 2013 / 126803), cholesterol, and phospholipids (zwitterionic or negatively charged) in varying molar ratios. The ethanol was then removed, and the external buffer was replaced with Tris / NaCl buffer via dialysis. After dialysis, the formulation was concentrated using a VivaSpin concentrator unit (MWCO 100,000) and filtered through a 0.2 µm pore sterile filter. Subsequently, aliquots were frozen at -80°C in Tris-sucrose buffer at pH 8.0. Nucleic acid concentrations were determined using the RiboGreen assay. Particle size and polydispersity were determined using a Zetasizer Nano-ZS (Malvern Panalytical, Malvern, Worcestershire, United Kingdom). To generate empty LNPs, no encapsulation payload was used, but LNPs were prepared using the same method. The characteristics of exemplary and comparative LNPs prepared according to these procedures are summarized in Table 1 (Example 2) and Table 2 (Example 3).

[0311] Example 2. Measurement of zeta potential of lipid nanoparticles.

[0312] The zeta potential of the lipid nanoparticles described herein was measured at different pH values ​​using a 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 Na₂HPO₄ solutions at varying ratios. The buffers were diluted 1 / 20 with water for injection (WFI) before use in LNP sample preparation. The Zetasizer was set according to the following parameters: Dispersant: Water; General Options (Model): Smoluchowski model; Temperature: 25 °C; Equilibration time: 120 s; Cell type: Disposable folded capillary cell (DTS1070); Measurement: Automatic; Minimum runs: 12; Maximum runs: 12; Number of measurements: 3; Delay between measurements: 0; Advanced (Measurement Settings): Automatic attenuation selection; Advanced (Voltage): Automatic voltage selection.

[0313] In the assay procedure, a sample cell containing the standard solution was inserted into the Zetasizer, and the measurement results were recorded according to the parameters described above (acceptable range for zeta potential transfer standard: -40 mV + / - 5.8 mV). The standard was decanted, and the cuvette was rinsed with WFI and twice with buffer for measuring the zeta potential of LNPs. Next, a 1 mL LNP sample was prepared by diluting the stock LNP to 0.005 mg / mL using buffer designed for zeta potential measurement. Then, 800 µL of the prepared sample was loaded into the zeta sample cell. Results analysis involved plotting the mean zeta potential measurement for each buffer relative to pH, and / or reporting the mean zeta potential at physiological pH 7.4.

[0314] Figure 1 A graph showing the zeta potential measurements of empty lipid nanoparticles prepared according to the formulations listed in Table 1 is presented, with these measurements plotted over a pH range including physiological pH 7.4. The data confirm that formulations of LNP C and LNP D each produce particles exhibiting a negative zeta potential at pH values ​​greater than approximately 6.4.

[0315] Table 1

[0316]

[0317] Example 3. CD8 in mice treated with lipid nanoparticles + T cell response.

[0318] Two lipid nanoparticle compositions were prepared following the procedure of Example 1 using the formulations listed in Table 2 and 3 μg 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 of mice (25 µL injection volume) using a 3 / 10 mL, 29 G × 1 / 2 inch insulin syringe (Covidien #8881600145). After sacrifice on day 12 post-immunization, spleens were homogenized on ice in complete Roswell Park Memorial Institute (RPMI) 1640 (ATCC) medium and filtered through a 70 µm cell filter. Red blood cells (RBCs) were lysed on ice for 5 min with 2 mL of erythrocyte lysis buffer HYBRI-MAX™ (Sigma R7757) and the reaction was terminated with ten times the volume of RPMI. Then, at 37°C and 5% CO2, in the presence of a HA peptide library (JPT Peptide Technologies) at 2 µg / mL for each peptide, 7.5 × 10⁻⁶ samples were subjected to treatment. 6 Cells were stimulated for 16 hours. Starting one hour after stimulation, Brefeldin A Solution (Thermofisher 00-4506-52) was added to each sample. Unstimulated samples from each animal were included. Samples stimulated with concanavalin A were included as positive controls.

[0319] Table 2

[0320]

[0321] Following stimulation, cells were washed with PBS and stained in the dark at 4°C for 30 minutes using a LIVE / DEAD fixable violet dead cell staining kit (Thermo Fisher Scientific). Samples were incubated in Fc blocking agent (purified rat anti-mouse CD16 / CD32, BD Biosciences) at 4°C for 10 minutes in the dark, and then surface-stained for 30 minutes at 4°C with monoclonal antibodies against 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). After surface staining, cells were washed with fluorescence-activated cell sorting (FACS) staining buffer, fixed and permeabilized using an intracellular fixation and permeabilization buffer kit (BD Biosciences), and washed again with permeabilization buffer (Thermo Fisher Scientific). Cells were stained intracellularly for 30 min at 4°C with monoclonal antibodies against anti-TNF-α allophycocyanin (APC) (clone MP6-XT22, BD Biosciences), anti-IFN-γ APC (clone XMG1.2, BD Biosciences), and anti-IL-2 APC (clone JES6-5H4, BioLegend). Staining for each cytokine was performed in separate wells. Cells were then washed once with permeation buffer (Thermo Fisher Scientific) and once with FACS staining buffer. Cells were then resuspended in FACS buffer and analyzed immediately. Spleen cells were analyzed on an LSRFORTESSA™ X-20 cell analyzer (BD Biosciences). A total of 150,000 events were collected per sample. Data were analyzed using the BD FACSDIVA™ program. Data are expressed as the percentage of unstimulated stained cells subtracted from the percentage of stained samples stimulated by the peptide library.

[0322] The results of this experiment are shown in Figure 2-4 In the figure, the CD8+ of mice administered lipid nanoparticles with LNP A and LNP D formulations listed in Table 2 and 3 µg of HA mRNA were plotted. + IFNg + T cells, CD8 + TNF-α + T cells and CD8 + IL-2 + The percentage of T cells. Data confirm that lipid nanoparticles with LNP D modulators stimulate CD8 compared to lipid nanoparticles with LNP A modulators. +The extent of T cell quantity was significantly greater. Therefore, these findings provide an example of achieving a greater immune response by delivering to subjects proteins of interest encapsulated in lipid nanoparticles having a negative zeta potential at physiological pH and comprising phospholipids or pharmaceutically acceptable salts thereof, wherein the phospholipids have a net negative charge at physiological pH.

[0323] Although the foregoing disclosure has been described in detail by way of illustration and example for purposes of clarity, those skilled in the art will understand that certain changes and modifications within the spirit and scope of this disclosure may be practiced, for example, within the scope of the appended claims. It should also be understood that various aspects of this disclosure, as well as portions of the various described embodiments and features, may be combined or interchanged in whole or in part. In the foregoing description of the various embodiments, those embodiments referenced to another embodiment may be suitably combined with other embodiments, as will be understood by those skilled in the art. Furthermore, those skilled in the art will understand that the above description is merely illustrative and not intended to limit the scope of this disclosure. Additionally, each reference provided herein is incorporated herein by reference in its entirety for all purposes, to the same extent that each reference is individually incorporated by reference.

[0324] sequence list

[0325]

[0326]

[0327]

Claims

1. A lipid nanoparticle comprising: Phospholipids or their pharmaceutically acceptable salts; Conjugated lipids, wherein the conjugated lipids comprise 0.1 mol% to 5 mol% of the total lipids in the lipid nanoparticles; An ionizable lipid or a pharmaceutically acceptable salt thereof, said ionizable lipid comprising three or more terminal hydrocarbon chains, each of said three or more terminal hydrocarbon chains having five or more members independently; as well as Nucleic acid, which encodes a protein or a fragment thereof of interest; The lipid nanoparticles are configured to have a negative zeta potential at physiological pH.

2. The lipid nanoparticles according to claim 1, wherein the phospholipids have a net negative charge at physiological pH.

3. The lipid nanoparticles according to claim 1 or 2, wherein the conjugated lipids account for 0.1 mol% to 1 mol% of the total lipids of the lipid nanoparticles.

4. The lipid nanoparticles according to any one of claims 1 to 3, wherein the phospholipid or its pharmaceutically acceptable salt constitutes at least 8 mol of the total lipids of the lipid nanoparticles.

5. The lipid nanoparticles according to any one of claims 1 to 4, wherein: The ionizable lipids or their pharmaceutically acceptable salts comprise more than 1 mol% of the total lipids in the nanoparticles; and The ionizable lipids do not contain sulfur.

6. A lipid nanoparticle comprising: Phospholipids or pharmaceutically acceptable salts thereof, wherein the phospholipids have a net negative charge at physiological pH; Conjugated lipids, wherein the conjugated lipids constitute less than 1 mol% of the total lipids in the lipid nanoparticles. Ionizable lipids or their pharmaceutically acceptable salts; as well as Nucleic acid, which encodes a protein or fragment thereof of interest.

7. The lipid nanoparticles of claim 6, wherein the phospholipid or its pharmaceutically acceptable salt constitutes at least 8 mol of the total lipids of the lipid nanoparticles.

8. The lipid nanoparticles according to claim 6 or 7, wherein: The ionizable lipid or its pharmaceutically acceptable salt constitutes more than 1 mol% of the total lipids in the nanoparticles; and The ionizable lipids do not contain sulfur.

9. A lipid nanoparticle comprising: Phospholipids or pharmaceutically acceptable salts thereof, wherein the phospholipids have a net negative charge at physiological pH and constitute at least 8 mol% of the total lipids in the lipid nanoparticles. Conjugated lipids, wherein the conjugated lipids comprise 0.1 mol% to 5 mol% of the total lipids in the lipid nanoparticles; Ionizable lipids or their pharmaceutically acceptable salts; as well as Nucleic acid, which encodes a protein or fragment thereof of interest.

10. The lipid nanoparticles according to claim 9, wherein: The ionizable lipids or their pharmaceutically acceptable salts comprise more than 1 mol% of the total lipids in the nanoparticles; and The ionizable lipids do not contain sulfur.

11. A lipid nanoparticle comprising: Phospholipids or pharmaceutically acceptable salts thereof, wherein the phospholipids have a net negative charge at physiological pH; Conjugated lipids, wherein the conjugated lipids comprise 0.1 mol% to 5 mol% of the total lipids in the lipid nanoparticles; An ionizable lipid or a pharmaceutically acceptable salt thereof, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof constitutes more than 1 mol% of the total lipids in the lipid nanoparticles, and the ionizable lipid does not contain sulfur; and Nucleic acid, which encodes a protein or fragment thereof of interest.

12. The lipid nanoparticles according to any one of claims 1 to 11, wherein the phospholipids comprise naturally occurring phospholipids.

13. The lipid nanoparticles according to any one of claims 1 to 12, wherein the phospholipid comprises phosphatidylserine.

14. The lipid nanoparticles of claim 13, wherein the phosphatidylserine comprises 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-L-serine), 1,2-distearateyl-sn-glycerol-3-phosphate-L-serine (DSPS), or 1,2-dimyristoyl-sn-glycerol-3-phosphate-L-serine. (DMPS), 1,2-dicepanoyl-sn-glycerol-3-phosphate-L-serine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine (DPPS), 1,2-dilauroyl-sn-glycerol-3-phosphate-L-serine (DLPS), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-L-serine, 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphate-L-serine) Amino acids, 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (SOPS), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-arachidonicoyl-s n-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-L-serine, or combinations thereof.

15. The lipid nanoparticles according to any one of claims 1 to 12, wherein the phospholipid comprises phosphatidylglycerol.

16. The lipid nanoparticles of claim 15, wherein the phosphatidylglycerol comprises 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-decanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilauroyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DLPG), or 1,2-dimyristoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DMPG), 1,2-bispentadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DPPG), 1,2-distearatel-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DSPG), 1,2-ditransoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinolenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) 1,2-Diarachidonic-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1,2-bis(docohexanoyl-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-palmitoyl-2-arachidonic-sn-glycerol-3-phosphate-(1'-rac-glycerol) (PAPG), 1 - Palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (SOPG), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (SAPG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) or combinations thereof.

17. The lipid nanoparticles according to any one of claims 1 to 16, wherein the phospholipid or its pharmaceutically acceptable salt accounts for 1 mol% to 20 mol% of the total lipids of the lipid nanoparticles.

18. The lipid nanoparticles according to any one of claims 1 to 17, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains, each of the two or more terminal hydrocarbon chains having five or more members and each not being directly bonded to an ester.

19. The lipid nanoparticles according to any one of claims 1 to 18, wherein the conjugated lipid comprises a carbamate bond.

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

21. The lipid nanoparticles according to any one of claims 1 to 20, wherein the conjugated lipid comprises N-[(methoxypoly(ethylene glycol))carbamoyl]-1,2-dimyristyloxypropylamine (PEG-c-DMA).

22. The lipid nanoparticles according to any one of claims 1 to 21, wherein the ionizable lipid does not contain disulfide bonds.

23. The lipid nanoparticles according to any one of claims 1 to 22, wherein the ionizable lipid comprises three or more terminal hydrocarbon chains, each of the three or more terminal hydrocarbon chains having five or more members.

24. The lipid nanoparticles according to any one of claims 1 to 23, wherein the ionizable lipid comprises 5-(dimethylamino)valerate (6Z,16Z)-12-((Z)-dec-4-en-1-yl) dodecano-6,16-dien-11-yl ester (3D-P-DMA).

25. The lipid nanoparticles according to any one of claims 1 to 24, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof accounts for 30 mol% to 70 mol% of the total lipids of the lipid nanoparticles.

26. The lipid nanoparticles according to any one of claims 1 to 25, wherein the lipid nanoparticles further comprise cholesterol or a derivative thereof.

27. The lipid nanoparticles of claim 26, wherein the cholesterol or its derivatives comprise 20 mol% to 60 mol% of the total lipids in the lipid nanoparticles.

28. The lipid nanoparticles according to any one of claims 1 to 27, wherein the lipid nanoparticles further comprise permanently charged cationic lipids, the permanently charged cationic lipids comprising no more than 10 mol of the total lipids of the lipid nanoparticles.

29. The lipid nanoparticles according to any one of claims 1 to 27, wherein the lipid nanoparticles do not contain permanently charged cationic lipids.

30. The lipid nanoparticles according to any one of claims 1 to 29, wherein: The phospholipid or its pharmaceutically acceptable salt constitutes 8.5 mol% to 10.5 mol% of the total lipids in the lipid nanoparticles. The conjugated lipids comprise 0.25 mol% to 0.9 mol% of the total lipids in the lipid nanoparticles. The ionizable lipid or its pharmaceutically acceptable salt constitutes 48 mol% to 58 mol% of the total lipids in the lipid nanoparticles; and The lipid nanoparticles further comprise cholesterol or its derivatives, wherein the cholesterol or its derivatives comprise 35 mol% to 39 mol% of the total lipids in the lipid nanoparticles.

31. The lipid nanoparticles according to any one of claims 1 to 29, wherein: The phospholipid or its pharmaceutically acceptable salt constitutes 9 mol% to 11 mol% of the total lipids in the lipid nanoparticles. The conjugated lipids comprise 0.85 mol% to 2.6 mol% of the total lipids in the lipid nanoparticles. The ionizable lipid or its pharmaceutically acceptable salt constitutes 45 mol% to 55 mol% of the total lipids in the lipid nanoparticles. Furthermore, the lipid nanoparticles further comprise cholesterol or its derivatives, wherein the cholesterol or its derivatives comprise 36.5 mol% to 40.5 mol% of the total lipids in the lipid nanoparticles.

32. The lipid nanoparticles according to claim 30 or 31, wherein: The phospholipids include DOPG, DOPS, or a combination thereof; The conjugated lipid comprises PEG-c-DMA; and The ionizable lipid contains 3D-P-DMA.

33. The lipid nanoparticles according to any one of claims 1 to 32, wherein the nucleic acid comprises RNA.

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

35. The lipid nanoparticles according to any one of claims 1 to 32, wherein the nucleic acid comprises DNA.

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

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

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

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

40. A method for producing lipid nanoparticles, the method comprising: Provide an aqueous solution containing nucleic acids, said nucleic acids encoding the protein of interest or a fragment thereof; An alcohol solution is provided, the alcohol solution comprising an alcohol organic solvent, a conjugated lipid, an ionizable lipid or a pharmaceutically acceptable salt thereof, and a phospholipid or a pharmaceutically acceptable salt thereof; as well as The aqueous solution and the alcohol solution are combined to produce a lipid nanoparticle solution containing the lipid nanoparticles; in The conjugated lipids comprise 0.1 mol% to 5 mol% of the total lipids in the alcohol solution; and The composition of the alcohol solution is configured such that the lipid nanoparticles have 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 according to claim 40 or 41, wherein the conjugated lipid accounts for 0.1 mol% to 1 mol% of the total lipids in the alcohol solution.

43. The method according to any one of claims 40 to 42, wherein the phospholipid or its pharmaceutically acceptable salt constitutes at least 8 mol of the total lipids in the alcoholic solution.

44. The method according to any one of claims 40 to 43, wherein: The ionizable lipids or their pharmaceutically acceptable salts comprise more than 1 mol% of the total lipids in the nanoparticles; and The ionizable lipids do not contain sulfur.

45. A method for producing lipid nanoparticles, the method comprising: Provide an aqueous solution containing nucleic acids, said nucleic acids encoding the protein of interest or a fragment thereof; Provides an alcohol 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; wherein the phospholipid has a net negative charge at physiological pH; and The aqueous solution and the alcoholic solution are combined to produce a lipid nanoparticle solution containing the lipid nanoparticles; wherein The conjugated lipids account for less than 1 mol of the total lipids in the alcohol solution.

46. ​​The method of claim 45, wherein the phospholipid or its pharmaceutically acceptable salt constitutes at least 8 mol of the total lipids of the lipid nanoparticles.

47. The method according to claim 45 or 46, wherein: The ionizable lipids or their pharmaceutically acceptable salts comprise more than 1 mol% of the total lipids in the nanoparticles; and The ionizable lipids do not contain sulfur.

48. A method for producing lipid nanoparticles, the method comprising: Provide an aqueous solution containing nucleic acids, said nucleic acids encoding the protein of interest or a fragment thereof; Provides an alcohol 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; wherein the phospholipid has a net negative charge at physiological pH; and The aqueous solution and the alcoholic solution are combined to produce a lipid nanoparticle solution containing the lipid nanoparticles, wherein: The conjugated lipids comprise 0.1 mol% to 5 mol% of the total lipids in the alcohol solution; and The phospholipid or its pharmaceutically acceptable salt constitutes at least 8 mol of the total lipids in the alcoholic solution.

49. The method according to claim 48, wherein: The ionizable lipids or their pharmaceutically acceptable salts comprise more than 1 mol% of the total lipids in the nanoparticles; and The ionizable lipids do not contain sulfur.

50. A method for producing lipid nanoparticles, the method comprising: Provide an aqueous solution containing nucleic acids, said nucleic acids encoding the protein of interest or a fragment thereof; Provides an alcohol 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; wherein the phospholipid has a net negative charge at physiological pH; and The aqueous solution and the alcoholic solution are combined to produce a lipid nanoparticle solution containing the lipid nanoparticles, wherein: The conjugated lipids comprise 0.1 mol% to 5 mol% of the total lipids in the alcohol solution. The ionizable lipid or its pharmaceutically acceptable salt constitutes more than 1 mol% of the total lipids in the alcoholic solution; and The ionizable lipids do not contain sulfur.

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

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

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

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

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

56. The method according to any one of claims 40 to 55, wherein the alcohol organic solvent comprises ethanol.

57. The method according to any one of claims 40 to 56, wherein the provision of the alcohol solution comprises: To form a phospholipid solution comprising a non-alcoholic organic solvent and the phospholipid or a pharmaceutically acceptable salt thereof; and The phospholipid solution and the alcohol organic solvent are combined.

58. The method of claim 57, wherein the non-alcoholic organic solvent comprises tetrahydrofuran.

59. The method according to any one of claims 40 to 58, wherein the phospholipid comprises naturally occurring phospholipids.

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

61. The method of claim 60, wherein the phosphatidylserine comprises DOPS, 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-L-serine), DSPS, DMPS, 1,2-decanoyl-sn-glycerol-3-phosphate-L-serine, DPPS, DLPS, 1,2-dilinoleyl-sn-glycerol-3-phosphate-L-serine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate-L-serine), 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, POPS, SO PS, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-L-serine, 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-L-serine, or combinations thereof.

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

63. The method of claim 62, wherein the phosphatidylglycerol comprises DOPG, 1,2-bis(heptadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-decanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), DLPG, DMPG, 1,2-bis(pentadecanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), DPPG, DSPG, 1,2-ditransoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1 2-Diarachidonico-sn-glycerol-3-[phosphate-rac-(1-glycerol)], 1,2-bis(docosahexaenooyl-sn-glycerol-3-[phosphate-rac-(1-glycerol)], POPG, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), PAPG, 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), SOPG, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), SAPG, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), or combinations thereof.

64. The method according to any one of claims 40 to 63, wherein the phospholipid or its pharmaceutically acceptable salt accounts for 1 mol% to 20 mol% of the total lipids in the alcoholic solution.

65. The method according to any one of claims 40 to 64, wherein the conjugated lipid comprises two or more terminal hydrocarbon chains, each of the two or more terminal hydrocarbon chains having five or more members and each not being directly bonded to an ester.

66. The method according to any one of claims 40 to 65, wherein the conjugated lipid comprises a carbamate bond.

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

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

69. The method according to any one of claims 40 to 68, wherein the ionizable lipid does not contain disulfide bonds.

70. The method according to any one of claims 40 to 69, wherein the ionizable lipid does not contain sulfur.

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

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

73. The method according to any one of claims 40 to 72, wherein the ionizable lipid or its pharmaceutically acceptable salt accounts for 30 mol% to 70 mol% of the total lipids in the alcoholic solution.

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

75. The method of claim 74, wherein the cholesterol or its derivatives comprise 20 mol% to 60 mol% of the total lipids in the alcohol solution.

76. The method according to any one of claims 40 to 75, wherein: The phospholipid or its pharmaceutically acceptable salt constitutes 8.5 mol% to 10.5 mol% of the total lipids in the alcoholic solution. The conjugated lipids comprise 0.25 mol% to 0.9 mol% of the total lipids in the alcohol solution. The ionizable lipid or its pharmaceutically acceptable salt constitutes 48 mol% to 58 mol% of the total lipids in the alcoholic solution; and The alcohol solution further comprises cholesterol or its derivatives, the cholesterol or its derivatives comprising 35 mol% to 39 mol% of the total lipids in the alcohol solution.

77. The method according to any one of claims 40 to 75, wherein: The phospholipid or its pharmaceutically acceptable salt constitutes 9 mol% to 11 mol% of the total lipids in the alcoholic solution. The conjugated lipids comprise 0.85 mol% to 2.6 mol% of the total lipids in the alcohol solution. The ionizable lipid or its pharmaceutically acceptable salt constitutes 45 mol% to 55 mol% of the total lipids in the alcoholic solution; and The alcohol solution further comprises cholesterol or its derivatives, the cholesterol or its derivatives comprising 36.5 mol% to 40.5 mol% of the total lipids in the alcohol solution.

78. The method according to claim 76 or 77, wherein: The phospholipids include DOPG, DOPS, or a combination thereof; The conjugated lipid comprises PEG-c-DMA; and The ionizable lipid contains 3D-P-DMA.

79. The method according to any one of claims 40 to 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 according to any one of claims 40 to 78, wherein the nucleic acid comprises DNA.

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

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

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 subject-specific cancer antigen.

86. A lipid nanoparticle that can be obtained by the method according to any one of claims 40 to 85.

87. A vaccine comprising lipid nanoparticles according to any one of claims 1 to 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 according to any one of claims 87 to 90, wherein the vaccine further comprises a pharmaceutically acceptable excipient.

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