Compositions and methods for the prevention and / or treatment of covid-19

Abstract

The present disclosure relates to compositions and methods for the preparation, manufacture and therapeutic use of nucleic acid vaccines comprising polynucleotide sequences encoding one or more structural proteins of SARS-CoV-2 and variants for the treatment, mitigation, amelioration and / or prevention of COVID-19.

Description

[Technical Field]

[0001] This disclosure generally relates to nucleic acid vaccines, specifically to the composition, formulation methods, and / or uses of nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, peptides, fragments, or variants of SARS-CoV-2, for the prevention, mitigation, and / or treatment and / or prevention of COVID-19, including physiological effects of reducing infection and / or symptoms. Cross-reference to related applications.

[0002] This application claims priority to Canadian Application No. 3,132,191, filed September 28, 2021, entitled "Composition and Methods for the Prevention and / or Treatment of COVID-19," and Canadian Application No. 3,146,392, filed January 21, 2022, also entitled "Composition and Methods for the Prevention and / or Treatment of COVID-19," the contents of which are incorporated herein by reference in their entirety. Sequence List

[0003] This application is filed together with an electronic sequence list. The sequence list file, titled 20921007PCTSEQLST.xml, was created on September 8, 2022, and has a size of 130,296 bytes. Information from the electronic format of the sequence list is incorporated herein by reference in its entirety. [Previous Technology]

[0004] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel coronavirus strain that began infecting mammals in China in 2019 and has spread to a pandemic. SARS-CoV-2 infection causes coronavirus disease 2019 (known as "COVID-19"), which affects mammals in various ways, ranging from asymptomatic individuals to those with a wide range of symptoms from mild to severe illness or death.

[0005] Vaccines are an effective way to provide preventative protection against infectious diseases. Currently, there are limited vaccines available for the prevention, mitigation, and / or treatment of COVID-19. Treatment for COVID-19 is limited to managing the symptoms and / or side effects of the disease. Therefore, there remains a strong need for COVID-19 vaccines, including formulations that deliver vaccines to a range of different target T cells. [Summary of the Invention]

[0006] This disclosure provides nucleic acid vaccines, components and formulations comprising nucleic acid vaccines, and methods of using them to prevent, mitigate, and treat COVID-19 infection with coronaviruses. Nucleic acid vaccines may include polynucleotides encoding at least one antigenic protein of SARS-CoV-2, fragments thereof, or variants thereof. The SARS-CoV-2 antigenic protein is a structural protein of SARS-CoV-2. The structural protein may be a spike protein, membrane protein, nucleocapsid phosphoprotein, or envelope protein. Non-limiting examples of the amino acid sequences of such structural proteins are shown in Table 1 (SEQ ID Nos: 1 to 6 and 15 to 19).

[0007] This article provides a COVID-19 nucleic acid vaccine for methods of vaccinating individuals against COVID-19, wherein the nucleic acid vaccine may include at least one polynucleotide encoding at least one structural protein of SARS-CoV-2 or a fragment thereof.

[0008] This document provides a method for inducing an immune response in an individual by administering an effective dose of the nucleic acid vaccine described herein. The immune response may be, but is not limited to, a T-cell response or a B-cell response. As a non-limiting example, an immune response may be generated by a single dose of the nucleic acid vaccine described herein. As another non-limiting example, an immune response may be generated by a booster dose of the nucleic acid vaccine described herein. Administering the pharmaceutical composition may generate a dose-responsive immune response in an individual. As a non-limiting example, a dose-responsive immune response may include inducing one or more of SARS-CoV-2 spike protein-specific IgG, IgG1, IgG2a, IgG2b, IgM, and IgA antibodies in the individual. As another non-limiting example, a dose-responsive immune response may include inducing one or more of IL-2+ T cells, IL-4+ T cells, and IFN-γ+ T cells. In some embodiments, administration of the pharmaceutical composition does not induce significant adverse reactions in the individual.

[0009] This article provides a method for treating and / or preventing COVID-19 in an individual by administering the nucleic acid vaccine described herein.

[0010] This article provides pharmaceutical components and formulations for the treatment and prevention of COVID-19 nucleic acid vaccines.

[0011] The nucleic acid vaccines described herein can be formulated in one or more lipid nanoparticles (LNPs).

[0012] In some embodiments, the LNP comprises (a) 50 to 85 mol% of cationic lipids; (b) 13 to 49.5 mol% of non-cationic lipids; and (c) 0.5 to 2 mol% of conjugated lipids.

[0013] In some embodiments, the LNP comprises (a) 50 to 65 mol% cationic lipids; (b) non-cationic lipids comprising 4 to 10 mol% phospholipids and 30 to 40 mol% cholesterol; and (c) 0.5 to 2 mol% conjugated lipids.

[0014] In some embodiments, the LNP comprises (a) about 30 to 60 mol% of cationic lipids; (b) about 0 to 30 mol% of non-cationic lipids; (c) about 18.5 to 48.5 mol% of sterols; and (d) about 0 to 10 mol% of polyethylene glycol-modified lipids.

[0015] In some embodiments, the LNP comprises (a) about 35 to 55 mol% of cationic lipids; (b) about 5 to 25 mol% of non-cationic lipids; (c) about 30 to 40 mol% of sterols; and (d) about 0 to 10 mol% of polyethylene glycol-modified lipids.

[0016] In some embodiments, the LNP comprises (a) about 35 to 45 mol% of cationic lipids; (b) about 25 to 35 mol% of non-cationic lipids; (c) about 20 to 30 mol% of sterols; and (d) about 0 to 10 mol% of polyethylene glycol-modified lipids.

[0017] In some embodiments, the LNP comprises (a) about 45 to 65 mol% of cationic lipids; (b) about 5 to 10 mol% of non-cationic lipids; (c) about 25 to 40 mol% of sterols; and (d) about 0.5 to 10 mol% of polyethylene glycol-modified lipids.

[0018] In some embodiments, the LNP comprises (a) about 40 to 60 mol% of cationic lipids; (b) about 5 to 15 mol% of non-cationic lipids; (c) about 35 to 45 mol% of sterols; and (d) about 0.5 to 3 mol% of polyethylene glycol-modified lipids.

[0019] In some embodiments, the LNP comprises (a) about 30 to 60 mol% of cationic lipids; (b) about 0 to 30 mol% of non-cationic lipids; (c) about 15 to 50 mol% of sterols; and (d) about 0.01 to 10 mol% of polyethylene glycol-modified lipids.

[0020] In some embodiments, the LNP comprises (a) about 10 to 75 mol% of cationic lipids; (b) about 0.5 to 50 mol% of non-cationic lipids; (c) about 5 to 60 mol% of sterols; and (d) about 0.1 to 20 mol% of polyethylene glycol-modified lipids.

[0021] In some embodiments, the LNP comprises (a) about 50 to 65 mol% of cationic lipids; (b) about 3 to 15 mol% of non-cationic lipids; (c) about 30 to 40 mol% of at least one sterol; and (d) about 0.5 to 2 mol% of polyethylene glycol-modified lipids.

[0022] In some embodiments, the LNP comprises (a) about 50 to 85 mol% of cationic lipids; (b) about 3 to 15 mol% of non-cationic lipids; (c) about 30 to 40 mol% of sterols; and (d) about 0.5 to 2 mol% of polyethylene glycol-modified lipids.

[0023] In some embodiments, the LNP comprises (a) about 25 to 75 mol% of cationic lipids; (b) about 0.1 to 15 mol% of non-cationic lipids; (c) about 5 to 50 mol% of sterols; and (d) about 0.5 to 20 mol% of polyethylene glycol-modified lipids.

[0024] In some embodiments, the LNP comprises (a) about 50 to 65 mol% of cationic lipids; (b) about 5 to 10 mol% of non-cationic lipids; (c) about 25 to 35 mol% of sterols; and (d) about 5 to 10 mol% of polyethylene glycol-modified lipids.

[0025] In some embodiments, the LNP comprises (a) about 20 to 60 mol% of cationic lipids; (b) about 5 to 25 mol% of non-cationic lipids; (c) about 25 to 55 mol% of sterols; and (d) about 0.5 to 15 mol% of polyethylene glycol-modified lipids.

[0026] In some embodiments, LNP comprises (a) 45 to 55 mol% of 3D-P-DMA; (b) 8 to 10 mol% of DSPC; (c) 36 to 42 mol% of cholesterol; and (d) 1.4 to 1.8 mol% of PEG-DMA.

[0027] In some embodiments, LNP contains (a) 49.96 mol% of 3D-P-DMA; (b) 9.97 mol% of DSPC; (c) 38.44 mol% of cholesterol; and (d) 1.61% of PEG-DMA.

[0028] This document also provides a nucleic acid vaccine for COVID-19, comprising approximately 0.2 mg / mL mRNA, wherein the mRNA contains a coding region having a nucleic acid sequence that is at least 95% identical to that of SEQ ID NO: 7. In some embodiments, the mRNA of the nucleic acid vaccine disclosed herein contains a coding region having a nucleic acid sequence as described in SEQ ID NO: 7. The nucleic acid vaccine can be prepared as a 2 mL filler in a 3 mL glass vial.

[0029] In some embodiments, administering a nucleic acid vaccine to an individual involves administering approximately 5 µg to approximately 100 µg of mRNA to the individual. For example, the method may involve administering approximately 16 µg of mRNA to the individual. Alternatively, the method may involve administering approximately 40 µg of mRNA to the individual. Alternatively, the method may involve administering approximately 100 µg of mRNA to the individual.

[0030] In some embodiments, administering a nucleic acid vaccine to an individual includes administering approximately 0.025 mL to approximately 0.5 mL of the nucleic acid vaccine to the individual. For example, the method may include administering approximately 0.025 mL of the nucleic acid vaccine to an individual, administering approximately 0.05 mL of the nucleic acid vaccine to an individual, administering approximately 0.08 mL of the nucleic acid vaccine to an individual, administering approximately 0.2 mL of the nucleic acid vaccine to an individual, or administering approximately 0.5 mL of the nucleic acid vaccine to an individual.

[0031] In some embodiments of the provided method, the administration includes intramuscular (IM) injection of a nucleic acid vaccine into an individual.

[0032] Nucleic acid vaccines can be administered to individuals in the form of a first dose of nucleic acid vaccine, followed by a second dose approximately 1 to 5 weeks later. In some implementation models, the second dose of nucleic acid vaccine is administered approximately 4 weeks after the first dose.

[0033] In some implementation scenarios, anti-spike protein IgG antibodies were detected in individuals up to day 28 after receiving the first dose of the nucleic acid vaccine.

[0034] In some implementations, anti-spike protein IgG antibodies were detected in individuals up to day 28 after receiving the first dose of the nucleic acid vaccine, and were enhanced in individuals up to day 42 after receiving the second dose of the nucleic acid vaccine.

[0035] In some implementations, the number of anti-spike protein IgG antibodies in individuals increased by up to 10 times compared to the average number of anti-spike protein IgG antibodies in serum samples from recovered SARS-CoV-2 patients.

[0036] In some implementation scenarios, SARS-CoV-2 neutralizing antibodies were detected in individuals up to day 28 after the first dose of the nucleic acid vaccine.

[0037] In some implementations, SARS-CoV-2 neutralizing antibodies were enhanced in individuals up to day 42 after the second dose of the nucleic acid vaccine.

[0038] Therefore, this disclosure provides a COVID-19 nucleic acid vaccine for a method of vaccinating an individual against COVID-19, wherein the nucleic acid vaccine comprises about 0.2 mg / mL mRNA, wherein the mRNA comprises a nucleic acid sequence having at least 95% identity with SEQ ID NO: 7, and wherein the nucleic acid vaccine is formulated for intramuscular (IM) injection and formulated in lipid nanoparticles (LNP).

[0039] Details of various embodiments are described in the following embodiments. Other features, objectives, and advantages will become apparent from the embodiments and the scope of the claims.

Implementation Method

[0066] Detailed Explanation I. Introduction

[0067] The following description illustrates exemplary components, methods, parameters, and the like. However, it should be understood that this description is not intended to limit the scope of this disclosure, but rather is provided instead as an illustrative example.

[0068] This document describes nucleic acid vaccines, specifically, nucleic acid vaccines comprising polynucleotides (e.g., mRNA), compositions, formulations, methods, and / or uses encoding one or more antigenic proteins of SARS-CoV-2, fragments thereof, or variants thereof, for the prevention, mitigation, and / or treatment of COVID-19. The antigenic protein may be a structural protein of SARS-CoV-2. The structural protein may be a spike (S) protein, a membrane (M) protein, a nucleocapsid (N) phosphoprotein, or an envelope (E) protein.

[0069] In some embodiments, at least one component of the nucleic acid vaccine is a polynucleotide encoding at least one of an antigenic protein of SARS-CoV-2 or a fragment or variant of the antigenic protein. The antigenic protein may be a structural protein of SARS-CoV-2. The polynucleotide may be an RNA polynucleotide, such as an mRNA polynucleotide.

[0070] In some embodiments, the nucleic acid vaccine includes at least one mRNA polynucleotide encoding at least one of the structural proteins or fragments or variants of the structural protein of SARS-CoV-2.

[0071] In some embodiments, the polynucleotide may be designed to encode one or more polypeptides of concern from SARS-CoV-2, or fragments or variants thereof. Such polypeptides of concern from SARS-CoV-2 may include, but are not limited to, complete polypeptides, multiple polypeptides, polypeptide fragments, or polypeptide variants, which may be independently encoded by one or more regions, or partially or entirely, of polynucleotides from SARS-CoV-2. As used herein, the term "polypeptide of concern" means any polypeptide selected to encode within the polynucleotides described herein, or whose function is influenced by the polynucleotides described herein. Any of the peptides or polypeptides described herein may be antigenic (also known as immunogenic).

[0072] As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) most commonly linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function or origin. In some embodiments, the polypeptide of interest is an antigen encoded by a polynucleotide as described herein.

[0073] In some embodiments, the encoded polypeptide is less than about 50 amino acids, and the polypeptide is subsequently referred to as a peptide. If the polypeptide is a peptide, its length will be at least about 2, 3, 4, or at least 5 amino acid residues. Therefore, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be single molecules or multi-molecule complexes, such as dimers, trimers, or tetramers. They can also contain single-chain or multi-chain polypeptides, such as antibodies or insulin, and can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide also applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids.

[0074] The term "peptide variant" refers to a molecule whose amino acid sequence differs from the native or reference sequence. Compared to the native or reference sequence, the amino acid sequence variant may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. Typically, the variant will have at least about 50% homology with the native or reference sequence, and more preferably, it will have at least about 80%, or at least about 85%, more preferably at least about 90%, or even more preferably at least about 95% homology with the native or reference sequence.

[0075] In some embodiments, a "variant mimic" is provided. As used herein, the term "variant mimic" is a mimic containing one or more amino acids that mimic the activating sequence. For example, glutamic acid esters can act as mimics of azophospho-threonine and / or azophospho-serine. Alternatively, a variant mimic can cause deactivation or deactivate a product containing the mimic, for example, phenylalanine can act as a deactivating substitution for tyrosine; or alanine can act as a deactivating substitution for serine.

[0076] When applied to amino acid sequences, "homology" is defined as the percentage of similarity between residues in a candidate amino acid sequence and residues in a second amino acid sequence after sequence alignment and, if necessary, the introduction of vacancies to achieve the maximum percentage of homology. The methods and computer programs used for alignment are well known in the art. It should be understood that homology depends on the calculation of the percentage of similarity, but its value may vary due to vacancies and penalties introduced in the calculation.

[0077] When applied to polypeptide sequences, “homology” refers to the corresponding sequence of other species that has a substantially similarity to the second sequence of the second species.

[0078] As used herein, “analyte” is intended to include peptide variants that differ in that one or more amino acid alterations, such as substitution, addition or deletion of amino acid residues that still maintain one or more characteristics of the parent or starting peptide.

[0079] In some embodiments, this disclosure covers several types of polypeptide-based compositions, including variants and derivatives. These include substitutional, insertional, deletion, and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant," but generally refers to a molecule that is modified and / or altered in any way relative to a reference molecule or a starting molecule.

[0080] For example, sequence tags or amino acids, such as one or more lysines, may be added to the peptide sequence described herein (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide purification or localization. Lysines can be used to improve peptide solubility or allow biotin labeling. Alternatively, amino acid residues in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein may be omitted as appropriate, thereby providing a truncated sequence. Alternatively, depending on the intended use of the sequence, such as when the sequence is part of a larger, soluble sequence linked to a solid carrier, certain amino acids (e.g., C-terminal or N-terminal residues) may be omitted.

[0081] When referring to a polypeptide, a “substitution variant” is a variant in which at least one amino acid residue in the native or starting sequence is removed and replaced by a different amino acid inserted at the same position. The substitution can be monosubstituted, in which only one amino acid in the molecule is substituted, or the substitution can be polysubstituted, in which two or more amino acids in the same molecule are substituted.

[0082] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include the substitution of another nonpolar residue with a nonpolar (hydrophobic) residue such as isoleucine, valine, or leucine. Similarly, examples of conservative substitution include the substitution of another polar residue with a polar (hydrophilic) residue, such as between arginine and lysine, between glutamic acid and aspartic acid, and between glycine and serine. Additionally, the substitution of another basic residue with a basic residue such as lysine, arginine, or histidine, or the substitution of another acidic residue with an acidic residue such as aspartic acid or glutamic acid, are further examples of conservative substitution. Examples of nonconservative substitutions include replacing nonpolar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, and methionine with polar (hydrophilic) residues such as cysteine, glutamic acid, glutamic acid, or lysine and / or replacing polar residues with nonpolar residues.

[0083] When referring to a polypeptide, an "insertion variant" is a variant in which one or more amino acids are inserted at a specific position adjacent to the native or starting sequence. "Immediately adjacent" amino acid means that it is connected to the α-carboxyl or α-amino functional group of the amino acid.

[0084] When referring to peptides, "deletional variants" are variants in which one or more amino acids are removed from the native or starting amino acid sequence. Typically, deletion variants will have one or more amino acid deletions in a specific region of the molecule.

[0085] When referring to polypeptides, "covalent derivatives" include modifications and / or post-translational modifications of native or starting proteins using organic or non-protein derivatizing agents. Covalent modifications are conventionally introduced by reacting the target amino acid residues of the protein with an organic derivatizing agent capable of reacting with selected side-chain or terminal residues, or by utilizing post-translational modification mechanisms that function in selected recombinant host cells. The resulting covalent derivatives are suitable for procedures targeting residues important for identifying biological activity, immunoassays, or for the immunoaffinity purification of recombinant glycoproteins to prepare anti-protein antibodies. Such modifications are performed within the capabilities of a person generally skilled in the art and without improper experimentation.

[0086] When referring to a polypeptide, "characteristic" is defined as a unique component of the molecule based on its amino acid sequence. Characteristic features of polypeptides encoded by polynucleotides as described herein include surface appearance, local conformational shape, folds, loops, hemi-loops, domains, semi-domains, sites, terminals, or any combination thereof.

[0087] As used herein, when referring to a polypeptide, the term "surface appearance" refers to the polypeptide-based component of a protein that appears on the outermost surface.

[0088] As used herein, when referring to a polypeptide, the term "local conformational shape" means the polypeptide-based protein structural representation located within a definable protein space.

[0089] As used herein, when referring to polypeptides, the term "fold" refers to the configuration of the amino acid sequence obtained at the lowest possible energy. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary folding include β-sheets and α-helices. Examples of tertiary folding include domains and regions formed by the aggregation or separation of forces with higher energy. Regions formed in this manner include hydrophobic and hydrophilic pockets, and analogues thereof.

[0090] As used herein, when relating to peptide conformation, the term “turn” means a change in the orientation of the backbone of a peptide or polypeptide and may involve bending of one, two, three, or more amino acid residues.

[0091] As used herein, when referring to a polypeptide, the term "loop" refers to a structural feature of the polypeptide that can be used to reverse the orientation of the polypeptide backbone. When a loop is found in a polypeptide and only the orientation of the backbone is changed, it may contain four or more amino acid residues. Oliva et al. have identified at least five classes of protein loops (J. Mol Bio., l266 (4): 814-830; 1997). The loop may be open or closed. A closed loop or "cyclic" loop may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids between bridging portions. Such bridging portions may be contained in the cysteine-cysteine ​​bridge (Cys-Cys) typical of polypeptides with disulfide bonds, or the bridging portions may be non-protein-based, such as the dibromoacetic acid agent used herein.

[0092] As used herein, when referring to a polypeptide, the term "semi-cyclic" means a portion of the identified ring having at least half of the amino acid residues of the ring from which that portion is derived. It should be understood that a ring may not always contain an even number of amino acid residues. Thus, in cases where a ring contains or is identified as containing an odd number of amino acids, the semi-cyclic part of the odd-numbered ring will contain an integer portion of that ring or the next integer portion (number of amino acids in the ring / 2 + / - 0.5 amino acids).

[0093] As used herein, when referring to a polypeptide, the term "domain" means a polypeptide motif having one or more identifiable structural or functional features or properties (e.g., binding force, acting as a protein-protein interaction site).

[0094] As used herein, when referring to a polypeptide, the term "half-domain" means a portion of an identified domain that has at least half of the amino acid residues of the domain from which that portion is derived. It should be understood that a domain may not always contain an even number of amino acid residues. Therefore, in cases where a domain contains or is identified as containing an odd number of amino acids, the half-domain of an odd-numbered domain will contain an integer portion of that domain or the next integer portion (the number of amino acids in the domain / 2 + / - 0.5 amino acids). For example, a domain identified as a 7-amino acid domain can produce a half-domain of 3 or 4 amino acids (7 / 2 = 3.5 + 1 - 0.5 = 3 or 4). It should also be understood that subdomains can be identified within a domain or half-domain, and these subdomains do not necessarily possess all the structural or functional characteristics identified in the domain or half-domain from which they are derived. It should also be understood that amino acids containing any of the domain types mentioned herein do not necessarily have to be linked along the polypeptide backbone (i.e., non-adjacent amino acids can fold structurally to create domains, half-domains, or subdomains).

[0095] As used herein, when referring to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when referring to an amino acid-based embodiment. A site indicates a location within a peptide or polypeptide that can be modified, manipulated, altered, derived, or changed within the molecule of the polypeptide described herein.

[0096] As used herein, when referring to a polypeptide, the term "termini" or "terminus" refers to the end of a peptide or polypeptide. Such a terminus is not limited to the first or last site of the peptide or polypeptide, but may include other amino acids in the terminal region. The polypeptide-based molecules described herein may be characterized by having an N-terminus (terminated by an amino acid having a free amino group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). In some cases, the proteins described herein consist of multiple polypeptide chains (polymers, oligomers) linked together by disulfide bonds or by non-covalent forces. Such proteins will have multiple N-termini and C-termini. Alternatively, the terminus of a polypeptide may be modified such that, depending on the specific circumstances, it begins or ends with a non-polypeptide-based portion (such as an organic conjugate).

[0097] Once any feature is identified or defined as a desired component of the polypeptide encoded by the polynucleotide described herein, any of several manipulations and / or modifications of such feature may be performed by moving, exchanging, reversing, deleting, randomizing, or copying. Furthermore, it should be understood that feature manipulation can produce the same results as the molecular modifications described herein. For example, manipulation involving domain deletion will result in a change in molecular length, just as modifying nucleic acids to encode molecules smaller than full length.

[0098] In polypeptides, the term "modification" refers to a modification relative to the standard set of 20 amino acids. Modifications can be of various kinds. In some embodiments, these regions may contain one, two, or more (as the case may vary) modifications.

[0099] Modification and manipulation can be achieved by methods known in this art, such as, but not limited to, site-directed mutagenesis or a priori incorporation during chemical synthesis. The activity of the modified molecule can then be tested using in vitro or in vivo analyses, such as those described herein or any other suitable screening analyses known in this art.

[0100] In some embodiments, the polypeptide may contain a common sequence discovered through several rounds of experiments. As used herein, a "common" sequence is a single sequence, which represents a set of sequences that allow for variation at one or more sites.

[0101] As those skilled in this art will recognize, protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of the relevant polypeptide. For example, this document provides any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical to the reference protein). The length of the protein fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids. In another example, any protein comprising an elongated segment of about 20, about 30, about 40, about 50, or about 100 or more amino acids may be used according to the nucleic acid vaccine described herein, wherein such amino acids are approximately 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any of the sequences described herein. In some embodiments, the polypeptide used according to the nucleic acid vaccine described herein includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.

[0102] Therefore, the polynucleotides disclosed herein encode peptides or polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications relative to the reference sequence, specifically the peptide or polypeptide sequence disclosed herein. Polynucleotides may also contain substitutions, insertions and / or additions, deletions and covalent modifications relative to the polynucleotide reference sequence.

[0103] A reference molecule (peptide or polynucleotide) may share a certain degree of consistency with the designed molecule (peptide or polynucleotide). As is known in this art, the term "consistency" refers to the relationship between the sequences of two or more peptides, polypeptides, or polynucleotides, as determined by comparing such sequences. In this art, consistency also means the degree of sequence correlation between them, as determined by the number of matches between strings of two or more amino acid residues or nucleosides. Consistency measures the percentage of consistent matches between the smaller of two or more sequences using gap alignments (if present), which are processed by a specific mathematical model or computer program (e.g., "algorithm"). The consistency of related peptides can be readily calculated using known methods. Such methods include, but are not limited to, those described in: *Computational Molecular Biology*, Lesk, AM, ed., Oxford University Press, NY, 1988; *Biocomputing: Informatics and Genome Projects*, Smith, DW, ed., Academic Press, NY, 1993; *Computer Analysis of Sequence Data*, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, NJ, 1994; *Sequence Analysis in Molecular Biology*, von Heinje, G., Academic Press, 1987; *Sequence Analysis Primer*, Gribskov, M. and Devereux, J., ed., M. Stockton Press, NY. 1991; and Carillo et al., SIAM J. Applied Math. 48: 1073; 1988).

[0104] In some embodiments, the encoded polypeptide variant may have the same or similar activity as the reference polypeptide. Alternatively, the variants can have altered activity (e.g., increased or decreased) relative to the reference polypeptide. In general, a variant of a particular polynucleotide or polypeptide described herein will have at least about 40%, 45%, 50% with a particular reference polynucleotide or polypeptide, as determined by resorting to sequence alignment programs and parameters known herein and by those familiar with the technology , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence consistency. Such alignment tools include the other tools of the BLAST program group (Stephen F. Altschul et al., Gapped BLAST and PSLBLAST: a new generation of protein database search programs, Nucleic Acids Res. 1997, 25:3389–3402). Other tools are described in this paper, specifically in the definition of “consistency”. II.The composition of this disclosure SARS-CoV-2

[0105] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus strain that can cause the coronavirus disease known as "COVID-19" COVID-19 affects mammals in different ways, ranging from asymptomatic individuals to individuals with a wide range of symptoms in the range from mild symptoms to severe disease or death. To date, about 80% of COVID-19 patients have mild to moderate symptoms, and about 20% can develop complications such as severe pneumonia, acute respiratory distress syndrome, sepsis, and even death. The list of symptoms associated with COVID-19 is constantly changing as doctors and scientists learn more about COVID-19 and how it affects the body, but some of the symptoms recognized to date include fever or chills, cough, shortness of breath or shortness of breath, fatigue, body aches, muscle soreness, headache, sore throat, congestion or runny nose, nausea and / or vomiting, diarrhea and a new loss of taste or smell.

[0106] The SARS-CoV-2 genome encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as non-structural proteins (named nsp1 to nsp16) and accessory proteins. The viral spike protein is typically the primary mediator for viral entry into cells. The SARS-CoV-2 spike protein binds to its receptor, human ACE2 (hACE2), via its receptor-binding domain (RBD) and is activated by human proteases via proteolytic degradation. Another characteristic of the SARS-CoV-2 spike protein is the presence of a functional furin cleavage site at the S1-S2 boundary (S1 being the receptor-binding unit and S2 the membrane fusion unit). This site can pre-activate many viruses, including SARS-CoV-2, for entry. The SARS-CoV-2 spike protein has been used as a protective antigen to elicit neutralizing antibodies in various vaccine development strategies. Membrane and envelope proteins are used for viral assembly. The envelope protein (E) forms a homopentamer cation channel that is important for viral pathogenicity (Mandala et al., Nature Structural and Molecular Bio. 2020, 27: 1202-1208). The nucleocapsid protein packages the viral genome into a helical ribonucleocapsid (RNP) and plays a role in viral self-assembly (Chang et al.; The SARS coronavirus nucleocapsid protein - Forms and functions; Antiviral Res. 2014; 103:39-50; its contents are incorporated herein by reference in their entirety). Furthermore, nucleocapsid proteins in SARS-CoV-2 can modulate host cellular mechanisms and participate in the regulation of the viral life cycle.

[0107] While not wishing to be bound by theory, it appears that SARS-CoV-2 binds to the human receptor ACE2 (hACE2). The receptor-binding domain (RBD) in the spike protein appears to be the most variable part of the coronavirus genome. Six RBD amino acids have been shown to be essential for binding to the ACE2 receptor, and the SARS-CoV-2 genome appears to possess an RBD that binds with high affinity to ACE2 in humans, ferrets, cats, and other species with high receptor homology (Anderson et al.; The Proximal Origin of SARS-CoV-2; Nature Medicine, 2020; 26(4): 450-452; the contents of which are incorporated herein by reference in their entirety).

[0108] In some embodiments, the nucleic acid vaccine described herein encodes a full-length polypeptide of a structural protein, or a fragment or variant of a structural protein of SARS-CoV-2, such as a spike protein, nucleocapsid protein, envelope protein, or membrane protein.

[0109] In some embodiments, the nucleic acid vaccines described herein encode more than one fragment or variant of the structural proteins of SARS-CoV-2, such as spike protein, nucleocapsid protein, envelope protein and / or membrane protein.

[0110] In some embodiments, the variant may be a mutant of one of the structural proteins encoded by the polynucleotides of the nucleic acid vaccine described herein, or a fragment of a mutant variant of a structural protein of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change in one of the structural proteins of SARS-CoV-2, where aspartic acid is changed to glycine.

[0111] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein encode the full-length polypeptide of the spike protein, or a fragment or variant of the spike protein of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change in the spike protein of SARS-CoV-2, where aspartic acid is changed to glycine. As a non-limiting example, the variant may be a single amino acid change in the spike protein of SARS-CoV-2, where aspartic acid at position 614 is changed to glycine (D614G) (Korber et al.; Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus; Cell; 2020, 182(4): 812-827; the contents of which are incorporated herein by reference in their entirety).

[0112] In some embodiments, the nucleic acid vaccine described herein may encode one or more proteins, peptides, fragments thereof, or variants of the structural proteins of SARS-CoV-2. Non-limiting examples of proteins, peptides, fragments thereof, or variants of the structural proteins of SARS-CoV-2 are provided in Table 1. NCBI reference numbers are also provided in the table where known. Table 1. Structural Protein Sequences of SARS-CoV-2 Serial identifier (SEQ ID NO) describe sequence type 1 Spike protein (NCBI reference number: YP_009724390.1) (“S protein”) protein 2 spike protein with D614G mutation protein 3 Envelope proteins (NCBI reference number: YP_009724392.1) protein 4 Membrane proteins (NCBI reference number: YP_009724393.1) protein 5 Nucleocapsid phosphoprotein (NCBI reference number: YP_009724397.2) protein 6 B.1.351 (South Africa) variant spike protein protein 15 B.1.17 (UK) variant spike protein protein 16 Spike protein with D614G and L452R mutations protein 17 The B.1.17 (UK) variant spike protein with the L452R mutation protein 18 The B.1.351 (South Africa) variant spike protein with the L452R mutation protein 19 P.1 (Brazil) variant spike protein protein

[0113] In some embodiments, the nucleic acid vaccine described herein may encode at least one structural protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of any sequence in Table 1 or a fragment of any sequence in Table 1 or a variant of any sequence in Table 1.

[0114] In some embodiments, the nucleic acid vaccine may be an mRNA vaccine that produces one or more structural proteins, peptides, fragments or variants of SARS-CoV-2 upon translation. Therefore, the polynucleotide of the mRNA vaccine is an mRNA polynucleotide encoding one or more structural proteins, peptides, fragments or variants of SARS-CoV-2.

[0115] In one embodiment, the coding sequence of the mRNA vaccine described herein may be based on the coding sequence of the spike (S) protein from the genome of the SARS-CoV-2 Wuhan-Hu-1 isolate (Genbank: NM908947.3, complete genome sequence). In some embodiments, a code variation of a single amino acid change from D614 to G614 is introduced to match the amino acids of currently prevalent viral strains.

[0116] Non-limiting examples of RNA sequences encoding structural proteins, peptides, fragments, or variants of SARS-CoV-2 are provided in Table 2. Table 2. Sequences of the spike protein of SARS-CoV-2 Serial identifier (SEQ ID NO) describe sequence type 7 The coding region of the spike protein with the D614G mutation RNA 20 The coding region of the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) RNA twenty one coding region of M protein RNA twenty two The coding regions of N and M proteins RNA twenty three coding region of N protein RNA twenty four Signal peptide and the coding regions of N and M proteins RNA 25 Sequence encoding the receptor-binding domain (RBD) of the S protein RNA 26 The sequence encoding the full-length S protein with a mutated furin protease site. RNA 27 Sequence encoding the spike protein with the D614G mutation RNA 28 Sequence encoding the M protein RNA 29 Sequences encoding N and M proteins RNA 30 Sequence encoding N protein RNA 31 Sequence containing a signal peptide and encoding N and M proteins RNA 32 Sequence encoding the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) RNA

[0117] In some embodiments, the mRNA sequence encoding the spike protein of SARS-CoV-2 with the D614G mutation includes the coding region of SEQ ID NO: 7 or a fragment or variant thereof.

[0118] In some embodiments, the mRNA sequence encoding the spike protein of SARS-CoV-2 with the D614G mutation includes SEQ ID NO: 27, or a fragment or variant thereof.

[0119] In some embodiments, the nucleic acid vaccine may contain a region encoding any of the sequences listed in Table 1, or a fragment or variant thereof. The nucleic acid vaccine may contain hybrid or chimeric regions, or mimics or variants. In some embodiments, the nucleic acid vaccine may contain any of the polynucleotide sequences listed in Table 3. Table 3. Illustrative sequences in nucleic acid vaccines for the treatment or prevention of COVID-19 Serial identifier (SEQ ID NO) describe sequence type 8 Sequence encoding the M protein DNA 9 Sequences encoding N and M proteins DNA 10 Sequence encoding N protein DNA 11 Sequence containing a signal peptide and encoding N and M proteins DNA 12 Sequence encoding the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) DNA 33 The coding region of the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) DNA 34 coding region of M protein DNA 35 The coding regions of N and M proteins DNA 36 coding region of N protein DNA 37 Signal peptide and the coding regions of N and M proteins DNA 38 Sequence encoding the M protein DNA 39 Sequences encoding N and M proteins DNA 40 Sequence encoding N protein DNA 41 Sequence containing a signal peptide and encoding N and M proteins DNA 42 Sequence encoding the spike protein with the D614G mutation DNA 43 Sequence encoding the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) DNA 50 Sequence encoding the receptor-binding domain (RBD) of the S protein DNA 51 Sequence encoding the S protein with a mutated furin protease site DNA

[0120] Any sequence or variant thereof mentioned in Tables 1 to 3 may also be used in the memory-enhancing vaccine described herein.

[0121] In some embodiments, the nucleic acid vaccine described herein encodes a protein, fragment thereof, or variant thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the protein provided by the amino acid sequences in Table 1. In the case of two or more polypeptide sequences, the term "identical" or percentage "identical" refers to the same two or more sequences. Percentage identity between polypeptide sequences can be determined using algorithms known in this art, such as BLAST and CLUSTAL.

[0122] The sequence of the SARS-CoV-2 protein or its fragments or variants may be obtained from any source. In some embodiments, the sequence of the SARS-CoV-2 protein or its fragments or variants comes from a strain capable of infecting or at risk of infecting human individuals.

[0123] In some embodiments, the sequence of SARS-CoV-2 protein or its fragments or variants may be modified or optimized (such as codon optimization) to be expressed in specific cells or host organisms.

[0124] In some embodiments, the nucleic acid vaccine described herein may be a multivalent vaccine. A multivalent vaccine may include polynucleotides encoding at least two different proteins, peptides, fragments thereof, or variants of SARS-CoV-2. As a non-limiting example, the polynucleotide may encode the same or different structural proteins. As a non-limiting example, the polynucleotide may encode the same structural protein, but different variants of the structural protein.

[0125] In some embodiments, the nucleic acid vaccine encodes the full-length S protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes an S protein fragment of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a receptor binding domain (RBD) fragment of the spike protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the spike protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes an S protein sequence (e.g., full-length, fragment, or variant) of SARS-CoV-2, wherein the S protein has a mutated furin cleavage site. S protein furin cleavage site mutants will remove or disable the furin cleavage site in the S protein (e.g. between the S1 and S2 borders). In some viral envelope proteins, disruption of furin cleavage sites was found to enhance performance and stability. In some embodiments, the nucleic acid vaccine encodes an S protein sequence (e.g., full length, fragment, or variant) of SARS-CoV-2, wherein the S protein includes a D614G mutation. Nucleic acid vaccines encoding the S protein of SARS-CoV-2, fragments or variants thereof may also include signal peptide and / or at least one linker (e.g., GSG linker) sequence and one or more sequences of the nucleic acid vaccine may be optimized by codons.

[0126] In some embodiments, the nucleic acid vaccine encodes the full-length M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes an M protein fragment of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the topological domain of the M protein of SARS-CoV-2 (e.g., a virion surface or intraviral region). In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of an M protein (e.g., a full-length protein or fragment) of SARS-CoV-2. Nucleic acid vaccines encoding the M protein of SARS-CoV-2, fragments or variants thereof may also include signal peptide and / or at least one linker (e.g., GSG linker) sequence and one or more sequences of the nucleic acid vaccine may be optimized by a codon.

[0127] In some embodiments, the nucleic acid vaccine encodes the full-length N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a fragment of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the RNA-binding domain of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a dimer domain of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the N protein of SARS-CoV-2 (e.g., the full-length protein or a fragment). Nucleic acid vaccines encoding the N protein of SARS-CoV-2, its fragments, or variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be codon-optimized.

[0128] In some embodiments, the nucleic acid vaccine encodes the full-length E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a fragment of the SARS-CoV-2 E protein. In some embodiments, the nucleic acid vaccine encodes a topological domain (e.g., a region on the surface or inside a viral particle) of the SARS-CoV-2 E protein. In some embodiments, the nucleic acid vaccine encodes a transmembrane domain of the SARS-CoV-2 E protein. In some embodiments, the nucleic acid vaccine encodes a variant of the SARS-CoV-2 E protein (e.g., the full-length protein or a fragment). Nucleic acid vaccines encoding the SARS-CoV-2 E protein, its fragments, or variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be codon-optimized.

[0129] In some implementations, the nucleic acid vaccine encodes two different structural proteins of SARS-CoV-2. In some implementations, the nucleic acid vaccine encodes the S protein of SARS-CoV-2, its fragments or variants, and the M protein of SARS-CoV-2, its fragments or variants. In some implementations, the nucleic acid vaccine encodes the S protein of SARS-CoV-2, its fragments or variants, and the N protein of SARS-CoV-2, its fragments or variants. In some implementations, the nucleic acid vaccine encodes the S protein of SARS-CoV-2, its fragments or variants, and the E protein of SARS-CoV-2, its fragments or variants. In some implementations, the nucleic acid vaccine encodes the M protein of SARS-CoV-2, its fragments or variants, and the N protein of SARS-CoV-2, its fragments or variants. In some implementations, the nucleic acid vaccine encodes the M protein of SARS-CoV-2, its fragments or variants, and the E protein of SARS-CoV-2, its fragments or variants. In some embodiments, the nucleic acid vaccine encodes the N protein of SARS-CoV-2, its fragments or variants, and the E protein of SARS-CoV-2, its fragments or variants. Nucleic acid vaccines encoding two different structural proteins of SARS-CoV-2, their fragments or variants, may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be codon-optimized. In some embodiments, the sequences encoding two different structural proteins of SARS-CoV-2 or their fragments or variants for the nucleic acid vaccine are constructed as a single polynucleotide.

[0130] In some embodiments, the nucleic acid vaccine encodes at least three different sequences of structural protein fragments of SARS-CoV-2 or their variants. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment, or variant sequences of SARS-CoV-2 and the M protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment, or variant sequences of SARS-CoV-2 and the N protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment, or variant sequences of SARS-CoV-2 and the E protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M protein, fragment, or variant sequences of SARS-CoV-2 and the S protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N protein, fragment, or variant sequences of SARS-CoV-2 and the S protein, fragment, or variant sequence of SARS-CoV-2. In some implementation samples, the nucleic acid vaccine encodes two different E protein, fragment, or variant sequences of SARS-CoV-2 and the S protein, fragment, or variant sequence of SARS-CoV-2. In some implementation samples, the nucleic acid vaccine encodes two different M protein, fragment, or variant sequences of SARS-CoV-2 and the N protein, fragment, or variant sequence of SARS-CoV-2. In some implementation samples, the nucleic acid vaccine encodes two different M protein, fragment, or variant sequences of SARS-CoV-2 and the E protein, fragment, or variant sequence of SARS-CoV-2. In some implementation samples, the nucleic acid vaccine encodes two different N protein, fragment, or variant sequences of SARS-CoV-2 and the M protein, fragment, or variant sequence of SARS-CoV-2. In some implementation samples, the nucleic acid vaccine encodes two different N protein, fragment, or variant sequences of SARS-CoV-2 and the E protein, fragment, or variant sequence of SARS-CoV-2. In some implementations, the nucleic acid vaccine encodes two different E protein, fragment, or variant sequences of SARS-CoV-2, and the N protein, fragment, or variant sequence of SARS-CoV-2. In some implementations, the nucleic acid vaccine encodes the S protein, fragment, or variant sequence of SARS-CoV-2, the M protein, fragment, or variant sequence of SARS-CoV-2, and the N protein, fragment, or variant sequence of SARS-CoV-2. In some implementations, the nucleic acid vaccine encodes the S protein, fragment, or variant sequence of SARS-CoV-2, the M protein, fragment, or variant sequence of SARS-CoV-2, and the E protein, fragment, or variant sequence of SARS-CoV-2.In some embodiments, the nucleic acid vaccine encodes the S protein, fragment, or variant sequence of SARS-CoV-2; the N protein, fragment, or variant sequence of SARS-CoV-2; and the E protein, fragment, or variant sequence of SARS-CoV-2. In other embodiments, the nucleic acid vaccine encodes the M protein, fragment, or variant sequence of SARS-CoV-2; the N protein, fragment, or variant sequence of SARS-CoV-2; and the E protein, fragment, or variant sequence of SARS-CoV-2. Nucleic acid vaccines encoding at least three different sequences of structural protein fragments of SARS-CoV-2 or their variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be codon-optimized. SARS-CoV-2 variants.

[0131] SARS-CoV-2 is a member of the Coronaviridae family. Multiple variants of SARS-CoV-2 (sometimes referred to as "virus strains" or "lineages") have been identified globally. The nomenclature of SARS-CoV-2 variants used in this specification is consistent with the PANGO nomenclature for new viral lineages (Rambaut, Andrew et al., A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nature microbiology, 2020, 5:: 1403-1407, the contents of which are incorporated herein by reference in their entirety). Near-real-time information related to PANGO SARS-CoV-2 lineages or variants can be accessed online using the Nextstrain SARS-CoV-2 analysis user interface (nextstrain.org / ncov / global).

[0132] To date, multiple PANGO lineage variants of SARS-CoV-2 have been identified, including the following (the numbers in parentheses represent the number of cases for each submitted PANGO lineage):

[0133] From an epidemiological perspective, variants are generally classified as variants of concern (VOC), variants of concern (VOC), and variants with serious consequences (VOC). For information on classifying specific variants as VOI, VOC, or VOHC, please refer to, for example, cdc.gov / coronavirus / 2019-ncov / cases-updates / variant-surveillance / variant-info.html.

[0134] VOIs may possess certain genetic markers associated with changes in receptor binding, reduced neutralization of antibodies against previous infections or vaccinations, decreased therapeutic efficacy, potential diagnostic impact, or increased predictive value for transmissibility or disease severity. In some cases, VOIs possess specific genetic markers that predict potential impacts on transmission, diagnosis, treatment, or immune evasion, or lead to an increased proportion of cases or unique outbreak clusters. SARS-CoV-2 VOIs include, for example, PANGO lineages B.1.1.7 (α), B.1.351 (β); B.1.427 / 429 (ε); B.1.526 (ι); B.1.525 (η); B.1.617.1 (κ); B.1.617.2 (δ); B.1.621 (μ); C37 (λ); P.1 (γ) and P.2.

[0135] VOCs may include variants that provide evidence of increased transmissibility, more severe illness (increased hospitalizations or deaths), significantly reduced neutralizing activity of antibodies produced during prior infection or vaccination, reduced effectiveness of treatment or vaccines, or failure of diagnostic tests. In some cases, VOCs may provide evidence of impact on diagnosis, treatment, and vaccines; widespread interference with diagnostic testing targets; evidence of significantly increased resistance to one or more classes of therapies; evidence of significantly reduced neutralizing activity of antibodies produced during prior infection or vaccination; evidence of reduced protection against severe illness induced by vaccines; evidence of increased transmissibility; or evidence of increased disease severity. SARS-CoV-2 VOCs may include, for example, PANGO lineages B.1.1.7 (α), P.1 (γ), B.1.351 (β), B.1.427 and B.1.429 (ε), B.1.526 (ι), B.1.525 (η), B.1.617.1 (κ), B.1.617.2 (δ), B.1.621 (μ), and C37 (λ).

[0136] VOHC may provide clear evidence that preventative measures or medical mechanisms (MCMs) have significantly reduced effectiveness relative to previously prevalent variants. In some cases, VOHC impacts medical mechanisms (MCMs) by demonstrating diagnostic failure, evidence of significantly reduced vaccine effectiveness, disproportionately high numbers of vaccine breakthrough cases, extremely low protection against vaccine-induced severe disease, significantly reduced sensitivity to multiple Emergency Use Authorization (EUA) or approved therapeutics, more severe clinical disease, and increased hospitalization rates.

[0137] The nucleic acid vaccine disclosed herein may encode one or more polypeptides of any SARS-CoV-2 variant described herein, such as one or more proteins, peptides, fragments thereof, or variants. In some embodiments, the nucleic acid vaccine disclosed herein may encode one or more polypeptides of SARS-CoV-2 VOI, VOC, and / or VOHC, such as one or more proteins, peptides, fragments thereof, or variants thereof. In some embodiments, the nucleic acid vaccine encodes a polypeptide containing a specific mutation called D614G.

[0138] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.526 SARS-CoV-2 variant (i.e., the ι variant), such as one or more of the following: spike protein substitutions for L5F, T95I, D253G, S477N, E484K, D614G and / or A701V; ORF1a substitutions for L3201P, T265I and / or Δ3675 / 3677; ORF1b substitutions for P314L and / or Q1011H; ORF3a substitutions for P42L and Q57H; ORF8 substitution for T11I; and / or 5'UTR substitution for R81C.

[0139] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.525 SARS-CoV-2 variant (i.e., the n variant), such as one or more of the following: spike protein substitutions for A67V, Δ69 / 70, Δ144, E484K, D614G, Q677H and / or F888L; ORF1b substitution for P314F; ORF1a substitution for T2007I; M protein substitution for I82T; N protein substitutions for A12G and / or T205I; and / or 5'UTR substitution for R81C.

[0140] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the P.2 SARS-CoV-2 variant, such as one or more of the following: spike protein substitution for E484K, D614G and / or V1176F; ORF1a substitution for L3468V and / or L3930F; ORF1b substitution for P314L; N protein substitution for A119S, R203K, G204R and / or M234I; 5'UTR substitution for R81C.

[0141] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.1.7 SARS-CoV-2 variant (i.e., the α variant), such as one or more of the following: spike protein substitutions Δ69 / 70, Δ144Y, E484K, S494P, N501Y, A570D, D614G and / or P681H.

[0142] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the P.1 SARS-CoV-2 variant (i.e., the γ variant), such as one or more of the following: spike protein substitutions K417N / T, E484K, N501Y and / or D614G.

[0143] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.351 SARS-CoV-2 variant (i.e., the β variant), such as one or more of the following: spike protein substitutions K417N, E484K, N501Y, and / or D614G. The B.1.351 variant is also known as the South African variant because it originated in South Africa.

[0144] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.427 SARS-CoV-2 variant, such as one or more of the following: spike protein substitution L452R and / or D614G.

[0145] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.429 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions S13I, W152C, L452R and / or D614G.

[0146] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.617.1 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions G142D, E154K, L452R, E484Q, D614G, P681R and / or Q1071H.

[0147] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant (i.e., the delta variant), such as one or more of the following: spike protein substitutions T19R, T95I, G142D, Δ156 / 157, R158G, L452R, T478K, D614G, P681R, and / or D950N. In other embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions V70F, A222V, W258L, and / or K417N.

[0148] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.617.3 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions T19R, G142D, L452R, E484Q, D614G, P681R and / or D950N.

[0149] In some implementations, the nucleic acid vaccine encoding includes one or more of the following substituted and / or deleted SARS-CoV-2 spike proteins, such as proteins, peptides, fragments or variants: A570D, A67V, A701V, D253G, D614G, E484K, F888L, K417N / T, L452R, L5F, N501Y, P681H, Q677H, S13I, S477N, S494P, T95I, V1176F, W152C, Δ144, Δ144Y and Δ69 / 70.

[0150] In some implementations, the nucleic acid vaccine code includes one or more of the following substitutions and / or deletions of SARS-CoV-2 ORF1a: L3201P, T265I, T2007I, L3468V, Δ3675-3677 and L3930F.

[0151] In some implementations, the nucleic acid vaccine code includes one or more of the following substituted SARS-CoV-2 ORF1b: P314F, P314L and Q1011H.

[0152] In some implementations, the nucleic acid vaccine code includes one or more of the following substitutions of SARS-CoV-2 ORF3a: P42L and Q57H.

[0153] In some implementations, the nucleic acid vaccine code includes SARS-CoV-2 ORF8 replaced by T11I.

[0154] In some implementations, the nucleic acid vaccine code includes the SARS-CoV-2 5'UTR replaced by R81C.

[0155] In some implementations, the nucleic acid vaccine encodes the SARS-CoV-2 M protein, which is replaced by the I82T, such as a protein, peptide, fragment, or variant.

[0156] In some implementations, the nucleic acid vaccine encodes a SARS-CoV-2 N protein containing one or more substitutions selected from the following: protein, peptide, fragment, or variant: A12G, A119S, R203K, G204R, T205I, and M234I. Components of the nucleic acid vaccine

[0157] In some embodiments, the polynucleotides described herein encode at least one polypeptide of interest, such as one or more proteins, peptides, fragments thereof, or variants of SARS-CoV-2. The SARS-CoV-2 proteins, peptides, fragments thereof, or variants disclosed herein may be wild-type, wherein they are derived from infectious agents or modified (e.g., structural proteins or fragments thereof and variants are engineered, designed, or artificial). They may have any combination of the features described herein.

[0158] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode one or more peptides or polypeptides of interest. Such peptides or polypeptides are structural proteins of SARS-CoV-2 or fragments or variants thereof, used for the prevention, mitigation, and / or treatment of COVID-19. As a non-limiting example, such peptides or polypeptides may act as antigens or antigen molecules (preferably as immunogenic molecules). The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a nucleotide polymer. Such polymers are commonly referred to as polynucleotides.

[0159] Examples of nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), diol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with aL-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-a-LNA with 2'-amino functionalization), vinyl nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) or mixtures or combinations thereof.

[0160] In some embodiments, the in vitro transcription (IVT) enzyme synthesis method can be used to prepare linear polynucleotides (referred to as "IVT polynucleotides") encoding one or more proteins, peptides, fragments or variants of SARS-CoV-2 disclosed herein.

[0161] In some embodiments, nucleic acid vaccines may include "chimeric polynucleotides" having portions or regions of different sizes and / or encoding proteins (e.g., structural proteins of SARS-CoV-2). A "chimera" is an entity having two or more incongruous or heterogeneous portions or regions. As used herein, a "portion" or "region" of a polynucleotide is defined as any part of the polynucleotide that is less than the entire length of the polynucleotide.

[0162] In some implementations, the nucleic acid vaccine comprises polynucleotides with a length of approximately 30 to approximately 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000). 00, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 To 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1, 500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).

[0163] In some embodiments, the nucleic acid vaccine includes at least one polynucleotide encoding at least one peptide or polypeptide of interest. In another embodiment, the polynucleotide may be non-coding.

[0164] In some embodiments, the length of the region encoding at least one peptide or polypeptide of interest of the polynucleotide of the nucleic acid vaccine is greater than about 30 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, 1,000, 1,100, 1,200, 1,3...). 00, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or up to and including 100,000 nucleotides). As used herein, such regions may be referred to as “coding regions” or “regional coding”.

[0165] In some embodiments, the polynucleotide of the nucleic acid vaccine is or acts as messenger RNA (mRNA). As used herein, the term "messenger RNA (mRNA)" means any polynucleotide that encodes at least one peptide or polypeptide of interest and is capable of being translated to produce, in vitro, in vivo, in situ, or ex vivo, the peptide or polypeptide of interest encoded.

[0166] The minimum length of the polynucleotide region of a nucleic acid vaccine may be a length sufficient to encode a nucleic acid sequence that is sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, this length may be sufficient to encode 2 to 30 amino acids, for example, 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. This length may be sufficient to encode a peptide with at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or not longer than 40 amino acids, for example, not longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids. Examples of dipeptides that the polynucleotide sequence may encode may include, but are not limited to, carnosine and alpha-carnosine.

[0167] The length of the polynucleotide region of a nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants of SARS-CoV-2 used for the prevention, mitigation and / or treatment of COVID-19 may be greater than about 30 nucleotides. The length may be, but is not limited to, at least or greater than about 30, 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, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,6 ...200, 1,200, 1,300, 1,400, 1,500, 1,600, 1,2 700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or up to and including 100,000 nucleotides.In some embodiments, this region comprises approximately 30 to approximately 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500). 0, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides). mRNA components.

[0168] The nucleic acid vaccine described herein may be an mRNA vaccine. An mRNA vaccine comprises at least one mRNA molecule that, upon translation, produces at least one peptide or polypeptide of interest for the prevention, mitigation, and / or treatment of COVID-19. Generally, an mRNA molecule generally includes at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. mRNA components: start codon and stop codon.

[0169] In some embodiments, the mRNA includes a region for initiating translation. This region may include any translation initiation sequence or signal, including a start codon. As a non-limiting example, the region includes a start codon. In some embodiments, the start codon may be "ATG", "ACG", "AGG", "ATA", "ATT", "CTG", "GTG", "TTG", "AUG", "AUA", "AUU", "CUG", "GUG", or "UUG".

[0170] In some embodiments, the mRNA includes a region that terminates translation. This region may include any translation termination sequence or signal, including a stop codon. As a non-limiting example, the region includes a stop codon. In some embodiments, the stop codon may be "TGA", "TAA", "TGA", "TAG", "UGA", "UAA", "UGA", or "UAG".

[0171] In some embodiments, the length of the start or stop translation region may independently be in the range of 3 to 40, for example 5 to 30, 10 to 20, 15, or at least 4, 30, or fewer nucleotides. In addition, in addition to start and / or stop codons, these regions may also contain one or more signal and / or restriction sequences.

[0172] In some embodiments, the masking agent may be used to mask the first start codon or the alternative start codon to increase the probability that translation will begin on a start codon or the alternative start codon downstream of the masked start codon or the alternative start codon.

[0173] In some embodiments, a start codon may be removed from the polynucleotide sequence so that translation of the polynucleotide begins at a codon that is not a start codon. Translation of the polynucleotide may begin at a codon following the removed start codon or at a downstream start codon or an alternative start codon. The polynucleotide sequence with the start codon removed may further contain at least one masking agent for the downstream start codon and / or alternative start codon to control or attempt to control the initiation of translation, the length of the polynucleotide, and / or the structure of the polynucleotide. mRNA component: coding region

[0174] In some embodiments, the coding region of the polynucleotide of the nucleic acid vaccine may encode at least one peptide or polypeptide of interest. Non-limiting examples of peptides or polypeptides of interest include one or more proteins, peptides, fragments thereof, or variants of SARS-CoV-2, for the prevention, mitigation, and / or treatment of COVID-19. mRNA component: non-translated region

[0175] The polynucleotides of the nucleic acid vaccines described herein may contain one or more regions or portions that act as or are used as untranslated regions (UTRs). The wild-type UTR of a gene is transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start point 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. Although not wishing to be bound by theory, UTRs may play a role in the stability of nucleic acid molecules and in translation. Variants of the UTR may be utilized in which one or more nucleotides are added to or removed from the end, including A, T, C, or G.

[0176] In some embodiments, the length of the UTR of the polynucleotide of the nucleic acid vaccine may independently range from 15 to 1,000 nucleotides (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[0177] Wild-type 5' UTRs include features that play a role in translation initiation because such 5' UTRs include sequences such as the Kozak sequence, which are known to be involved in how ribosomes initiate the translation of many genes. 5' UTR formation is also known to involve secondary structures for elongation factor binding. Other non-UTR sequences (e.g., introns or portions of intron sequences) can also be used as regions or subregions that can increase protein yield and polynucleotide content.

[0178] It is known that natural or wild-type 3' UTRs contain embedded adenosine and uridine elongations. These AU-rich tags are particularly prevalent in genes with high turnover rates. The introduction, removal, or modification of 3' UTR-rich AU elements (AREs) can be used to regulate the stability of polynucleotides in nucleic acid vaccines.

[0179] UTRs from any gene may be incorporated into the polynucleotide region of a nucleic acid vaccine. Alternatively, artificial UTRs of variants that are not wild-type regions may also be used in the polynucleotide of a nucleic acid vaccine. The placement orientation of such UTRs or portions thereof may be the same as in the transcripts from which they are selected, or their orientation or position may be varied. As used herein, the term "alteration" in relation to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. As a non-limiting example, 5' or 3' UTRs may be inverted, shortened, or elongated from one or more other 5' or 3' UTRs from different parental sequences.

[0180] In some embodiments, the flanking regions are selected from transcript families of proteins that share common functions, structures, and characteristics. For example, the polypeptide of interest may belong to a protein family that is expressed in a particular cell, tissue, or at a specific time during development. A UTR from any such gene may be exchanged with any other UTR from the same or different protein family to produce a new polynucleotide. As used herein, "protein family" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, characteristic, location, origin, or mode of expression.

[0181] The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having the sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). In some embodiments, the 5' UTR of the polynucleotide of the nucleic acid vaccine disclosed herein consists of the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). In some embodiments, the 5' UTR is directly at the 5' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. In some embodiments, the 5' UTR is separated from the 5' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine by 1, 2, 3, 4, 5, 6, or more nucleotides; for example, a spacer sequence of 1, 2, 3, 4, 5, 6, or more nucleotides separates the 5' UTR from the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 91% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 94% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 96% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR, which has a sequence that is at least 99% sequence identical to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR, which has a sequence that is at least 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).

[0182] The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having the sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). In some embodiments, the 3' UTR of the polynucleotide of the nucleic acid vaccine disclosed herein consists of the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). In some embodiments, the 3'UTR is directly at the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. In some embodiments, the 3'UTR is separated from the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine by 1, 2, 3, 4, 5, 6, or more nucleotides; for example, a spacer sequence of 1, 2, 3, 4, 5, 6, or more nucleotides separates the 3'UTR from the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 91% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 94% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 96% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 99% sequence identical to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).

[0183] The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having the sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). In some embodiments, the 3' UTR of the polynucleotide of the nucleic acid vaccine disclosed herein consists of the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). In some embodiments, the 3'UTR is directly at the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. In some embodiments, the 3'UTR is separated from the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine by 1, 2, 3, 4, 5, 6, or more nucleotides; for example, a spacer sequence of 1, 2, 3, 4, 5, 6, or more nucleotides separates the 3'UTR from the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 91% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 92% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 93% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 94% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 96% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 99% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). mRNA components: Cap and IRES sequences.

[0184] In some embodiments, the polynucleotides of the nucleic acid vaccines disclosed herein may include a 5' cap structure. The 5' cap structure of natural mRNA participates in nuclear export, increases mRNA stability, and binds to the mRNA cap-binding protein (CBP). This protein associates with the poly(A)-binding protein via the CBP to form a mature circular mRNA species responsible for the stability and translation capacity of the mRNA in the cell. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

[0185] In some embodiments, the 5' end cap region of the polynucleotide of the nucleic acid vaccine may contain a single cap or a series of nucleotides forming that cap. The length of the cap region may be 1 to 10, for example 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2, or 10 or fewer nucleotides. In some instances, the cap region may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the cap is absent.

[0186] In some embodiments, end-cap analogs, also referred to herein as synthetic end-cap analogs, chemical end-caps, chemical end-cap analogs, or structural or functional end-cap analogs, can be used in nucleic acid vaccines. End-cap analogs may be chemically (e.g., non-enzymatic) or enzymatically synthesized, and their chemical structure may differ from that of natural (e.g., endogenous, wild-type, or physiological) 5'-caps, but they retain end-cap function.

[0187] In some embodiments, the 5' end cap of the polynucleotide of the nucleic acid vaccine may include an endogenous end cap or an end cap analog. As a non-limiting example, the 5' end cap may contain a guanine analog. Suitable guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine (m1G), 2'-fluoro-guanosine, 7-dezo-guanosine, 8-side-oxy-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0188] Those familiar with this technique should understand that the 5' cap can be generated via enzymes or other synthetic methods. Endogenous mRNA molecules are 5'-capped, with a 5'-ppp-5'-triphosphate bond formed between the terminal guanosine cap residue and the sense nucleotide transcribed from the 5' end of the mRNA molecule. This 5'-guanosine cap can then be methylated to produce an N7-methyl-guanosine residue. The ribose of the nucleotide transcribed from the 5' end and / or proximal end of the mRNA may also be 2'-O-methylated, depending on the case. The 5'-uncapping, achieved through hydrolysis and cleavage of the guanosine cap structure, can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0189] The polynucleotides of the nucleic acid vaccines described herein, such as mRNA, can be modified to include a non-hydrolyzable end-cap structure, thereby preventing end-cap removal and thus increasing the mRNA half-life. Since end-cap hydrolysis requires cleavage of the 5'-ppp-5' phosphodiester bond, modified nucleotides can be used during the end-capping reaction. For example, a vaccinia virus end-capping enzyme, available from companies such as New England Biolabs (Ipswich, MA), can be used with α-thioguanosine nucleotides according to the manufacturer's instructions to generate a thiophosphate bond in the 5'-ppp-5' end cap. Additional modified guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, can be used.

[0190] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminus and / or the 5'-preterminal nucleotide of the mRNA at the 2'-hydroxyl group of the sugar ring (as described above). Multiple unique 5'-cap structures can be used to generate nucleic acid molecules, such as the 5'-cap of mRNA molecules.

[0191] End-cap analogs, also referred to herein as synthetic end-cap analogs, chemical end-caps, or structural or functional end-cap analogs, differ in chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5'-caps while retaining end-cap function. End-cap analogs can be chemically (e.g., non-enzymatically) or enzymatically synthesized and linked to nucleic acid molecules, such as mRNA molecules.

[0192] For example, the anti-reverse cap analog (ARCA) cap contains two guanines linked by 5'-5'-triphosphate groups, one of which contains an N7 methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which can be equivalently designated as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA). The N7- and 3'-O-methylated guanine provides the terminal portion of the capped nucleic acid molecule (e.g., mRNA).

[0193] Another example is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0194] Although cap analogs allow for the addition of caps to nucleic acid molecules during in vitro transcription reactions, up to 20% of transcripts remain capless. This, along with the structural differences between cap analogs and endogenous 5'-cap structures, can lead to reduced translational capacity and decreased cell stability.

[0195] In the exemplary embodiments disclosed herein, polynucleotides (e.g., mRNA) can be capped post-transcriptionally using an enzyme. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can create a typical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of mRNA and the guanine capping nucleotide, wherein the capping guanine contains N7 methylation and the 5'-terminal nucleotide of mRNA contains 2'-O-methyl. Such structures are referred to as Cap 1 structures. In some embodiments, Cap 1 structures provide higher translational efficiency and cellular stability and reduced activation of pro-inflammatory cytokines compared to other 5'-cap analog structures known in this art, for example. Cap structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')N1mpNp (Cap 1), and 7mG(5')-ppp(5')N1mpN2mp (Cap 2).

[0196] In one embodiment, the polynucleotide of the nucleic acid vaccine described herein contains a Cap 1 structure.

[0197] Because polynucleotides, such as mRNA, can be capped post-transcriptionally, and because this process is more efficient, up to 100% of polynucleotides, such as mRNA, can be capped. This contrasts with approximately 80% of capped analogues ligated to mRNA during in vitro transcription reactions.

[0198] In some embodiments, the polynucleotides of nucleic acid vaccines may contain an internal ribosome entry site (IRES) sequence. While not wishing to be bound by theory, the IRES plays an important role in initiating protein synthesis in the absence of a 5' cap structure. The IRES can serve as a single ribosome binding site or as one of multiple ribosome binding sites for mRNA. mRNA component: Tailing region

[0199] In some embodiments, the polynucleotides of the nucleic acid vaccine, such as mRNA, include a tailing region. Non-restrictive examples of tailing regions include poly-A sequences, poly-C sequences, and / or polyA-G tetrads.

[0200] In some embodiments, the mRNA includes a chain-terminating nucleoside. Non-limiting examples of chain-terminating nucleosides include 2'-O methyl, F, and locked nucleosides (LNA).

[0201] In some embodiments, the length of the polynucleotide tailing region of the nucleic acid vaccine can range from non-existent to 500 nucleotides (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). If the tailing region is a poly-A tail, the length can be described in units of poly-A binding protein binding or as a function of poly-A binding protein binding.

[0202] In some implementations, the poly-A tail can also be added after the kernel output of the construct.

[0203] In some embodiments, a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide (such as an mRNA molecule) during RNA processing to increase stability. Immediately following transcription, the 3' end of the transcript can be cleaved to release a 3' hydroxyl group. Then, a poly-A polymerase adds the adenine nucleotide chain to the RNA. This process, called polyadenylation, adds the poly-A tail, which can be, for example, about 80 to about 250 residues in length, including lengths of about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues.

[0204] In some embodiments, the length of the poly-A tail (when present) is greater than 30 nucleotides (e.g., at least or greater than about 30, 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, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A tail region comprises approximately 30 to approximately 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500). 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,500, and 2,500 to 3,000 nucleotides).

[0205] In some embodiments, the poly-A tail is about 99 nucleotides long (SEQ ID NO: 44).

[0206] In some embodiments, the poly-A tail is designed relative to the length of the entire polynucleotide or the length of a specific region of the polynucleotide. This design may be based on the length of the coding region, the length of a specific feature or region, or the length of the final product expressed from the polynucleotide.

[0207] In this case, the length of the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the polynucleotide or its characteristic. The poly-A tail can also be designed as part of its constituent polynucleotide. In this case, the poly-A tail can be the total length of the construct, the construct region, or the total length of the construct minus 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or greater, of the poly-A tail. Furthermore, engineered binding sites and polynucleotide binding of poly-A binding proteins can enhance expression. Signal sequence

[0208] In some embodiments, the polynucleotides of the nucleic acid vaccine may also encode additional features that facilitate the transport of the polypeptide to therapeutically relevant sites. One such feature that facilitates protein transport is a signal sequence. As used herein, a "signal sequence" or "signal peptide" is a polynucleotide or polypeptide of about 9 to 200 nucleotides (3 to 60 amino acids) incorporating into the 5' end of the coding region or the N-terminal polypeptide it encodes. In some embodiments, the addition of such sequences enables the transport of the encoded polypeptide to the endoplasmic reticulum via one or more secretory pathways. After protein transport, some signal peptides are cleaved from the protein by signal peptidase.

[0209] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein include a signal sequence comprising SEQ ID NO: 45 (DNA) or SEQ ID NO: 49 (RNA). Codon optimization

[0210] Polynucleotides, regions, portions, or subregions of nucleic acid vaccines may be codon-optimized. Codon optimization methods are known in this art and can be applied to achieve one or more of a number of objectives. These objectives include, but are not limited to, matching codon frequencies in the target organism and the host organism to ensure correct folding; altering GC content to increase mRNA stability or reduce secondary structures; minimizing the movement of tandem repeat codons or bases that may impair gene construction or expression; customizing transcription and translation control regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins; adding, removing, or reorganizing protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; regulating translation rates to allow proper folding of protein domains; or reducing or eliminating problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in this art, and non-limiting examples include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), and / or proprietary methods. In some implementations, the ORF sequence is optimized using an optimization algorithm. The codon selections for each amino acid are given in Table 4. Table 4. Codon Selection single letter Nomenclature Amino acid name Codon selection A alanine GCT, GCC, GCA, GCG C Cysteine TGT, TGC D Aspartic acid GAT, GAC E glutamic acid GAA, GAG F Phenylan TTT, TTC G Glycine GGT, GGC, GGA, GGG H histidine CAT, CAC I Isoleucine ATT, ATC, ATA K lysine AAA, AAG L Leucine CTT, CTC, CTA, CTG, TTA, TTG M Methionine ATG N Aspartic acid AAT, AAC P proline CCT, CCC, CCA, CCG Q glutamic acid CAA, CAG R Arginine CGT, CGC, CGA, CGG, AGA, AGG s serine TCT, TCC, TCA, TCG, AGT, AGC Sec selenocysteine UGA in mRNA in the presence of selenocysteine ​​insertion elements (SECTS) termination stop codon TAA, TAG, TGA T threonine ACT, ACC, AC A, ACG V Valine GTT, GTC, GTA, GTG w tryptophan TGG γ Tyrosine TAT, TAC

[0211] In some embodiments, the nucleic acid vaccine is vectorized after codon optimization. Non-limiting examples of vectors include, but are not limited to, plasmids, viruses, myxosomes, and artificial chromosomes. Modification

[0212] The nucleic acid vaccines (including mRNA vaccines) disclosed herein may include one or more modifications. The term "modification" or, where appropriate, "modified" refers to a modification of A, G, U, or C ribonucleotides. Generally, these terms are not intended to refer to ribonucleotide modifications in the 5'-terminal cap portion of naturally occurring mRNA. In peptides, the term "modification" refers to a modification relative to the standard set of 20 amino acids.

[0213] As described herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof ("nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside that includes a phosphate group or other backbone bond (nucleoside inter-bond).

[0214] Modifications can be of various kinds. In some embodiments, the coding region, non-translated region, flanking region, and / or terminal or tailed region may contain one, two, or more (as the case may vary) nucleoside or nucleotide modifications. In some embodiments, the nucleic acid vaccine disclosed herein contains one or more modifications that, compared to unmodified polynucleotides, make the nucleic acid molecule more resistant to degradation in the cell and / or more stable in the cell upon introduction.

[0215] The polynucleotides of the nucleic acid vaccines described herein may include any useful modifications, such as modifications to sugars, nucleobases, or nucleoside bonds (e.g., modifications to the phosphate ester / phosphodiester bond / phosphodiester backbone). One or more pyrimidine nucleobase atoms may be substituted or replaced, for example, by substitution of an amino group, a thiol group, an alkyl group (e.g., methyl or ethyl), a halogen group (e.g., chlorine or fluorine) atom or group, depending on the situation. In some embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and nucleoside bonds. Modifications according to this disclosure may be used to modify ribonucleic acid (RNA) into deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or mixtures thereof. Additional modifications are described herein.

[0216] In some embodiments, the modification includes nucleotides modified with 2'-O-methyl or 2'-O-methoxyethyl (2'-OMe and 2'-MOE, respectively).

[0217] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may contain at least one of the modifications described herein.

[0218] The polynucleotides of the nucleic acid vaccines described herein may include combinations of modifications to sugars, nucleobases and / or nucleoside bonds.

[0219] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that can be used in the vaccines disclosed herein include, but are not limited to, any modifications as described in PCT Publication WO2017070626, the contents of which are incorporated herein by reference in their entirety, including, for example, modifications or deletions of nucleotides (or codons) encoding one or more N-linked glycosylation sites of the translated polypeptide. Modifications that can be used in the vaccines disclosed herein may also include any modifications as described in PCT Publication WO2018200892, the contents of which are incorporated herein by reference in their entirety. The vaccine disclosed herein may further include features or modifications as described in PCT patent applications published in WO2020255063, WO2020182869, WO2016011222, WO2016011226, WO2016005004, WO2016000792, WO2015176737, WO2015085318, WO2015048744 and WO2015034925, and U.S. patent applications published in US20200254086, US20200206362, US20180311336 and US20180303929; each of these contents is incorporated herein by reference in its entirety.

[0220] For example, the polynucleotide of the mRNA molecule of the nucleic acid vaccine described herein may include the following modifications: The nucleoside internucleotide bond of the polynucleotide may be partially or completely modified. The polynucleotide may contain modifications to one or more nucleobases. The polynucleotide may contain 5-methylcytosine replacing all cytosine nucleobases / cytidine nucleotides. In addition, the polynucleotide may have one or more modifications to one or more sugar subunits of the nucleoside. The sugar modification may be one or more locked nucleic acids (LNA) or 2'-O-methoxyethyl modifications ("2'-MOE"). The polynucleotide may be designed as a patterned array with sugar, nucleobase, or bond modifications. In some embodiments, the polynucleotide may contain modifications that maximize stability. In some embodiments, the polynucleotide may be completely modified with 2'-MOE-sugar. Modified nucleobases

[0221] Modified nucleosides and nucleotides may include modified nucleobases. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine.

[0222] In some embodiments, the modified nucleobase is modified uracil. Exemplary nucleobases and nucleosides containing modified uracil include pseudouridine (ψ), pyridin-4-one nucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (S 2U), 4-thio-uridine (S 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (Ho 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (M 3U), 5-methoxy-uridine (Mo 5U), uridine 5-oxyacetic acid (CMo 5U), methyl uridine 5-oxyacetic acid (MCMo 5U), 5-carboxymethyl-uridine (CM 5U), 1-carboxymethyl-pseudouridine, and 5-carboxymethylhydroxymethyl-uridine (CHM 5U), 5-carboxymethylhydroxymethyluridine methyl ester (mchm 5U), 5-methoxycarbonylmethyluridine (mcm 5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5s 2U), 5-aminomethyl-2-thiouridine (nm 5s 2U), 5-methylaminomethyluridine (mnm 5U), 5-methylaminomethyl-2-thiouridine (mnm 5s 2U), 5-methylaminomethyl-2-selenouridine (mnm 5se 2U), 5-aminomethylmethyluridine (ncm 5U), 5-carboxymethylaminomethyluridine (cmnm 5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5s 2U), 5-propynyluridine, 1-propynyl-pseudouridine, 5-tauronic acid methyluridine (τm 5U), 1-Taurate methyl-pseuuridine, 5-Taurate methyl-2-thio-uridine (τm 5s 2U), 1-Taurate methyl-4-thio-pseuuridine, 5-Methyl-uridine (m 5U, i.e., with nucleobase deoxythymidine), 1-Methylpseuuridine (m 1ψ), 5-Methyl-2-thio-uridine (m 5s 2U), 1-Methyl-4-thio-pseuuridine (m 1s 4ψ), 4-Thio-1-methylpseuuridine, 3-Methylpseuuridine (m 3ψ), 2-Thio-1-methylpseuuridine, 1-Methyl-1-deazo-pseuuridine, 2-Thio-1-methyl-1-deazo-pseuuridine, dihydrouridine (D), dihydropseuuridine, 5,6-Dihydrouridine, 5-Methyl-Dihydrouridine (m 5D), 2-Thio-Dihydrouridine, 2-Thio-Dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-Thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-Thio-pseudouridine, N1-Methyl-pseudouridine (also known as 1-methylpseudouridine (m 1ψ)), 3-(3-amino-3-carboxypropyl)uridine (acp 3U), 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3ψ), 5-(isopentenylaminomethyl)uridine (inm 5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5s 2U), α-Thio-uridine, 2'-O-Methyl-uridine (Um), 5,2'-O-Dimethyl-uridine (m 5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5Um), 5-aminomethoxymethyl-2'-O-methyl-uridine (ncm 5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5Um), 3,2'-O-dimethyl-uridine (m 3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5Um), 1-thio-uridine, deoxythymidine, 2'-F-arasu-uridine, 2'-F-uridine, 2'-OH-arasu-uridine, 5-(2-methoxycarbonylvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0223] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudocytidine, 3-methylcytidine (m 3C), N4-acetylated cytidine (ac 4C), 5-methylated cytidine (f 5C), N4-methylcytidine (m 4C), 5-methylcytidine (m 5C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm 5C), 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, and 2-thio-cytidine (s 2C), 2-thio-5-methyl-cytidine, 4-thio-pseudo-cytidine, 4-thio-1-methyl-pseudo-cytidine, 4-thio-1-methyl-1-de-nitro-pseudo-cytidine, 1-methyl-1-de-nitro-pseudo-cytidine, 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-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysidine (k 2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4Cm), N4,2'-O-dimethyl-cytidine (m 4Cm), 5-methacryl-2'-O-methyl-cytidine (f 5Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-arasacchar-cytidine, 2'-F-cytidine, and 2'-OH-arasacchar-cytidine.

[0224] In some embodiments, the modified nucleobase is modified adenine. Exemplary nucleobases and nucleosides containing modified adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deazo-adenosine, 7-deazo-8-aza-adenosine, 7-deazo-2-amino-purine, 7-deazo-8-aza-2-amino-purine, 7-deazo-2,6-diamino-purine, 7-deazo-8-aza-2,6-diamino-purine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A), and 2-methylthio-N6-methyl-adenosine (ms2m). 6A), N6-isopentenyl-adenosine (i 6A), 2-methylthio-N6-isopentenyl-adenosine (ms 2i 6A), N6-(cis-hydroxyisopentenyl)adenosine (io 6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2io 6A), N6-glycineaminomethyladenosine (g 6A), N6-threonineaminomethyladenosine (t 6A), N6-methyl-N6-threonineaminomethyladenosine (m 6t 6A), 2-methylthio-N6-threonineaminomethyladenosine (ms 2g 6A), N6,N6-dimethyl-adenosine (m 6A) 2A), N6-hydroxyn-valine acetoaminomethyladenosine (hn 6A), 2-methylthio-N6-hydroxyn-valine acetoaminomethyladenosine (ms 2hn 6A), N6-acetyladenosine (ac 6A), 7-methyladenosine, 2-methylthioadenosine, 2-methoxyadenosine, α-thioadenosine, 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m 6Am), N6,N6,2'-O-trimethyladenosine (m 6 2Am), 1,2'-O-dimethyladenosine (m 1Am), 2'-O-ribosyl adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arose-adenosine, 2'-F-adenosine, 2'-OH-arose-adenosine, and N6-(19-amino-pentazo-nonadecanyl ester)-adenosine.

[0225] In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 4-demethyl wyoside (imG-14), isowyoside (imG2), wyoside (yW), peroxywyoside (o2yW), hydroxywyoside (OHyW), undermodified hydroxywyoside (OHyW*), 7-deazo-guanosine, Q nucleoside (Q), epoxy-Q nucleoside (oQ), galactosyl-Q nucleoside (galQ), mannosyl-Q nucleoside (manQ), 7-cyano-7-deazo-guanosine (preQ0), 7-aminomethyl-7-deazo-guanosine (preQ1), and archaenoside (G). +), 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine (m 7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1G), N2-methyl-guanosine (m 2G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7G), N2,N2,7-dimethyl-guanosine (m 2,2,7G), 8-side-oxy-guanosine, 7-methyl-8-side-oxy-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1Im), and 2'-O-ribo-guanosine (phosphate) (Gr(p)).

[0226] The nucleobases of a nucleotide may be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. For example, each nucleobase may be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase may also include, for example, naturally occurring and synthetic derivatives of the base, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen (e.g., 8-bromo), 8-amino, 8-thiol, 8-thiouracil, etc. Alkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen (specifically 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deadenine, 7-deadenine, 3-deadenine, deadenine, 7-deadenine, 3-deadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazine, 9-deadenine, imidazo[4,5-d]pyrazol, thiazo[4,5-d]pyrimidine, pyrazol-2-one, 1,2,4-triazine, tazo; and 1,3,5-triazine.

[0227] Various sugar modifications, nucleotide modifications, and / or nucleotide bonds (e.g., backbone structure) can be introduced at various positions in the polynucleotides described herein. Those skilled in the art will understand that nucleotide analogs or other modifications can be located at any position on the polynucleotide without substantially reducing its function. The polynucleotides disclosed herein may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any or more of A, G, T / U, or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10%). (up to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%).

[0228] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may be modified into circular nucleic acids. The ends of the polynucleotides may be linked by chemical reagents or enzymes to produce circular polynucleotides without free ends. Circular polynucleotides are expected to be more stable than their linear counterparts and resistant to exonuclease digestion. Circular polynucleotides may further include other structural and / or chemical modifications relating to A, G, T / U, or C ribonucleotides / deoxyribonucleotides.

[0229] In some embodiments, at least 50% of the polynucleotide is modified, for example, at least 50% of the nucleotides are modified. In some embodiments, at least 75% of the polynucleotide is modified, for example, at least 75% of the nucleotides are modified. It should be understood that since nucleotides (sugar, base, and phosphate groups, such as bonds) can be modified individually, any modification to any part of a nucleotide or nucleoside will constitute a modification.

[0230] In some embodiments, the polynucleotide is modified in at least 10% of only one component of the nucleotide, wherein such component is a nucleobase, sugar, or bond between nucleosides. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleobase, sugar, or bond of the polynucleotide described herein may be modified.

[0231] As a non-limiting example, all uracil nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all guanine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all guanine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all cytosine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all adenine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different.

[0232] In one embodiment of this disclosure, the polynucleotide of the nucleic acid vaccine is modified to include N1-methyl-pseudouridine nucleotide. Sugar modification.

[0233] Modified nucleosides and nucleotides that can be incorporated into polynucleotides (e.g., RNA or mRNA, as described herein) may be modified at the sugars of ribonucleic acid. For example, the 2' hydroxyl group (OH) may be modified or substituted with several different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halogen, substituted C1-6 alkyl, substituted C1-6 alkoxy, substituted C6-10 aryloxy, substituted C3-8 cycloalkyl, substituted C3-8 cycloalkoxy, substituted C6-10 aryloxy, substituted C6-10 aryl-C1-6 alkoxy, substituted C1-12 (heterocyclic)oxy; sugars (e.g., ribose, pentose, or any sugar described herein); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR, wherein R is H or substituted alkyl, and n is 0 to 20. (e.g., integers of 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20); "locked" nucleic acid (LNA), wherein the 2'-hydroxyl group is connected to the 4'-carbon of the same ribose by a C1-6 alkyl or C1-6 heteroalkyl bridge, wherein exemplary bridges include methylene, propyl, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0234] In some embodiments, the polynucleotide, such as the mRNA of the nucleic acid vaccine described herein, contains at least one sugar modification. Generally, RNA comprises a glycosylribose as a 5-membered ring with oxygen. Exemplary non-restrictive modified nucleotides include the substitution of oxygen in the ribose (e.g., with S, Se, or an alkyl group, such as methylene or ethyl); the addition of a double bond (e.g., by substituting the ribose with a cyclopentenyl or cyclohexenyl group); ring condensation of the ribose (e.g., to form a 4-membered ring with cyclobutane or oxygen); ring expansion of the ribose (e.g., to form a 6- or 7-membered ring with an additional carbon or heteroatom, such as anhydrous hexitol, altritol, mannitol, cyclohexyl, cyclohexenyl, and N-hydroxyl (also having an aminophosphate backbone)); and polycyclic forms (e.g., tricyclic and "unlocked" forms, such as glycol nucleic acid (GNA)). (e.g., R-GNA or S-GNA, where the ribose is replaced by an ethylene glycol unit linked to a phosphodiester bond), threononucleotides (TNA, where the ribose is replaced by an α-L-threofuranosyl-(3'→2') group), and peptide nucleotides (PNA, where the ribose and phosphodiester backbone are replaced by a 2-aminoethyl-glycine bond). The glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in the ribose. Therefore, polynucleotide molecules (including mRNA) as described herein may include nucleotides containing, for example, arabinose as a sugar.

[0235] Non-limiting examples of sugar modifications may include the modifications provided in Table 5. The polynucleotides disclosed herein may have one or more nucleotides carrying the modifications provided in Table 5. In some embodiments, each of the polynucleotides described herein carries any of the modifications provided in Table 5, or does not carry any of the modifications provided in Table 5. Table 5. Nucleotide Sugar Modifications

[0236] In some embodiments, at least one 2' position (OH in RNA or H in DNA) of the nucleotide sugar of the polynucleotide is substituted with -OMe, referred to as 2'-OMe. In some embodiments, at least one 2' position (OH in RNA or H in DNA) of the nucleotide sugar of the polynucleotide is substituted with -F, referred to as 2'-F. Nucleoside internucleotide bond

[0237] The polynucleotides disclosed herein may include any modification to the nucleoside internucleotide bond (e.g., to the phosphate ester / phosphodiester bond / phosphodiester backbone). In the context of the polynucleotide backbone, the terms "phosphate ester" and "phosphodiester" are used interchangeably. The backbone phosphate ester group may be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include the complete replacement of the unmodified phosphate ester portion with another nucleoside internucleotide bond as described herein. Examples of modified phosphate ester groups include, but are not limited to, thiophosphates, methylphosphates, selenophosphates, boranophosphates, boranophosphate esters, hydrophosphonates, aminophosphates, diaminophosphates, alkyl or arylphosphonates, and triphosphates. In dithiophosphates, both non-linked oxygen atoms are replaced with sulfur. Phosphate linkers can also be modified by replacing oxygen with nitrogen (bridging amino phosphates), sulfur (bridging thiophosphates), and carbon (bridging methylene-phosphonates).

[0238] An α-thio-substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides via non-natural thiophosphate backbone bonding. Thiophosphate DNA and RNA exhibit increased nuclease resistance, followed by a longer half-life in the cellular environment. It is anticipated that thiophosphate-linked polynucleotide molecules will also reduce innate immune responses through weaker binding / activation to cellular innate immune molecules.

[0239] In a specific embodiment, the modified nucleoside includes α-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine or 5'-O-(1-thiophosphate)-pseuuridine).

[0240] In some embodiments, the polynucleotide contains at least one thiophosphate bond or methylphosphonate bond between nucleotides.

[0241] In some embodiments, the polynucleotide contains at least one 5'-(E)-phosphonovinyl ester (5'-E-VP), a phosphate ester mimic as a modification.

[0242] In one embodiment of this disclosure, the polynucleotides (e.g., mRNA) of the COVID-19 nucleic acid vaccine may be modified. (Variance)

[0243] The valence of the nucleic acid vaccine disclosed herein can vary. "Valence" refers to the number of antigenic components in the nucleic acid vaccine or its polynucleotides. The antigenic components of the nucleic acid vaccine may be on the same polynucleotide or on different polynucleotides. In some embodiments, the nucleic acid vaccine may be monovalent. In some embodiments, the nucleic acid vaccine may be bivalent. In some embodiments, the nucleic acid vaccine may be trivalent. In some embodiments, the nucleic acid vaccine may be multivalent, containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 antigens or antigenic portions, such as, but not limited to, antigenic peptides. As a non-limiting example, the antigenic peptide may be one or more fragments or variants of the structural protein of SARS-CoV-2. Synthetic enzyme method: in vitro transcriptase synthesis.

[0244] The cDNA encoding the polynucleotides of the nucleic acid vaccine described herein can be transcribed using an in vitro transcription (IVT) system. This system typically includes a transcription buffer, nucleotide triphosphates (NTPs), a ribonuclease inhibitor, and a polymerase. The NTPs may be manufactured in-house, optionally from a supplier, or synthesized as described herein. The NTPs may be selected from, but are not limited to, the NTPs described herein, including natural and non-natural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, and polymerase variants.

[0245] In some embodiments, the DNA template is removed from the IVT reaction using DNase I. The digested DNA and nucleotides are then removed during oligo dT purification of the mRNA. This purification method is based on the affinity of the poly-A tail of the mRNA for the poly-dT column bed. Centrifugation can be used, but may not be necessary to remove the digested DNA and nucleotides. After purification using a reverse-phase column (e.g., SDVB) to remove double-stranded RNA from the mRNA, ultrafiltration followed by one or more filtration steps can be used. After purification, residual DNA can be measured using PCR to confirm that DNA in plastid regions outside the region transcribed into mRNA has been removed. In some embodiments, where product concentration is required, permeation followed by one or more filtration steps can be used to remove any biological load (e.g., biomolecules or other biological material).

[0246] Any number of RNA polymerases or variants can be used for the synthesis of polynucleotides in the nucleic acid vaccines described herein. RNA polymerases can be modified by inserting or deleting amino acids from the RNA polymerase sequence.

[0247] Polynucleotide or nucleic acid synthesis reactions can be performed using enzymatic methods employing polymerases. Polymerases catalyze the formation of phosphodiester bonds between nucleotides in polynucleotide or nucleic acid chains. Currently known DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis. The DNA polymerase I (pol I) or A polymerase family, including the Klenow fragment of *E. coli*, Bacillus DNA polymerase I, *Taq* DNA polymerase, and T7 RNA and DNA polymerase, is one of the most thoroughly studied families. Another large family is the DNA polymerase a (pol a) or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from bacteriophages T4 and RB69. Although these polymerase families employ similar catalytic mechanisms, they differ in terms of acceptor specificity, acceptor analog binding efficiency, primer elongation extent and rate, DNA synthesis mode, exonuclease activity, and sensitivity to inhibitors. Solid-phase chemical synthesis

[0248] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be manufactured entirely or partially using solid-phase technology. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a reactant solution. Impurities and excess reagents are washed away, and purification is not required after each step. This process can be automated on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows for the rapid production of polynucleotides or nucleic acids on a relatively large scale, making some polynucleotides or nucleic acids commercially available.

[0249] In some embodiments, automated solid-phase synthesis is used, wherein the chain is synthesized along the 3' to 5' direction. The hydroxyl group at the 3' end of the nucleoside is plugged into the solid support via a chemically or photo-cleavable linker. Activated nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG, and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides are sequentially added to the nucleosides bound to the support. At the end of the synthesis, a cleaving agent such as ammonia or ammonium hydroxide is added to remove all protecting groups and release the polynucleotide chain from the solid support. Light can also be applied to cleave the polynucleotide chain. The product can then be further purified by high-performance liquid chromatography (HPLC) or electrophoresis. Liquid-phase chemical synthesis

[0250] The synthesis of polynucleotides for the nucleic acid vaccines described herein by sequentially adding monomer building blocks can be carried out in the liquid phase. Covalent bonds are formed between monomers or between the terminal functional groups of the growing chain and the entering monomer. Functional groups that do not participate in the reaction must be temporarily protected. After each monomer building block is added, the reaction mixture must be purified before the next monomer building block is added. Functional groups at one end of the chain must be deprotected before reacting with the next monomer building block. Liquid-phase synthesis is time-consuming, labor-intensive, and cannot be automated. Despite its limitations, liquid-phase synthesis can still be used for the large-scale preparation of short polynucleotides. Because the system is homogeneous, it does not require large amounts of excess reagents and is cost-effective in this respect. Quantification and purification

[0251] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may be quantified in exosomes or when derived from one or more body fluids. As used herein, "body fluids" include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or preejaculate fluid, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual blood, pus, sebum, vomitus, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood. Alternatively, exosomes can be taken from organs selected from the following groups: lungs, heart, pancreas, stomach, intestines, bladder, kidneys, ovaries, testes, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0252] In exosome quantification methods, no more than 2 mL of sample is obtained from an individual, and exosomes are separated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanofiltration, immunoadsorption capture, affinity purification, microfluidic separation, or combinations thereof. In the analysis, the level or concentration of polynucleotides can be the expression level, presence, absence, truncation, or alteration of the administered construct. This level has a favorable correlation with one or more clinical phenotypes or with the analysis of biomarkers for human diseases. The analysis can be performed using construct-specific probes, cell counting, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof, while exosomes can be separated using immunohistochemical methods, such as enzyme-linked immunosorbent assay (ELISA). Exosomes can also be separated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanofiltration, immunoadsorption capture, affinity purification, microfluidic separation, or combinations thereof.

[0253] These methods enable researchers to monitor the levels of remaining or delivered polynucleotides in real time. This is possible because the polynucleotides described herein differ from their endogenous forms due to structural modifications.

[0254] In some embodiments, polynucleotides can be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). Quantitative polynucleotides can be analyzed to determine whether the polynucleotides are of appropriate size and to check for degradation. Polynucleotide degradation can be checked by methods such as, but not limited to, agarose gel electrophoresis; HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography-mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE).

[0255] The purification of polynucleotides in the nucleic acid vaccines described herein may include, but is not limited to, polynucleotide cleanup, quality assurance, and quality control. Cleanup may be performed by methods known in this art, such as, but not limited to, AAGEN-COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes (EX-IQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). When the term "purified" is used in connection with polynucleotides, such as "purified polynucleotides" refers to polynucleotides isolated from at least one contaminant. As used herein, "contaminant" is any substance that makes another unsuitable, impure, or inferior. Thus, purified polynucleotides (e.g., DNA and RNA) exist in a form or setting different from their natural occurrence or setting, or in a form or setting different from their state or setting prior to the treatment or purification method.

[0256] Quality assurance and / or quality control checks may be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. III. Pharmaceutical Compositions and Delivery

[0257] The nucleic acid vaccines described herein can be used as therapeutic or preventative agents. In some embodiments, this disclosure provides pharmaceutical compositions comprising at least one medically acceptable carrier and a nucleic acid vaccine, i.e., a nucleic acid vaccine for COVID-19. Therefore, pharmaceutical compositions comprising the nucleic acid vaccines described herein can be used to prevent, alleviate, and / or treat COVID-19.

[0258] This document provides nucleic acid vaccines and their pharmaceutical compositions that can be used in combination with one or more pharmaceutically acceptable excipients. The pharmaceutical compositions may, where appropriate, contain one or more additional active substances, such as therapeutic and / or prophylactic active substances. The pharmaceutical compositions of the nucleic acid vaccines described herein may be sterile and / or pyrogen-free.

[0259] In some embodiments, the composition is administered to humans, human patients, or individuals. For the purposes of this disclosure, the phrase "active ingredient" generally refers to a nucleic acid vaccine delivered as described herein, or a polynucleotide contained therein, such as a polynucleotide encoding one or more proteins, peptides, fragments or variants of SARS-CoV-2, for the prevention, mitigation and / or treatment of COVID-19.

[0260] Although the descriptions of pharmaceutical compositions provided herein are primarily directed at those intended for human administration, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, including non-human mammals. It should be fully understood that modifications to pharmaceutical compositions intended for human administration are necessary to make them suitable for administration to a variety of animals, and that a generally skilled veterinary pharmacologist can design and / or make such modifications using only standard experimental design (if applicable). The individuals covered by the administration of pharmaceutical compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or poultry, including commercially relevant poultry such as poultry, chickens, ducks, geese, and / or turkeys. Formulations

[0261] Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, as used herein, including, but not limited to, any and all solvents, dispersion media, diluents or other liquid media, dispersants or suspenders, surfactants, isotonants, thickeners or emulsifiers, preservatives and the like, suitable for the desired particular dosage form. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare such compositions are known in this art (see Remington: The Science and Practice of Pharmacy, 21st edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient media may be covered within the scope of this disclosure unless any known carrier medium is incompatible with the substance or its derivatives, such as producing any adverse biological effects or otherwise interacting harmfully with any other component of the pharmaceutical composition.

[0262] Formulations of pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacology. Generally, such preparation methods include the following steps: combining the active ingredient with excipients and / or one or more other adjuncts, and subsequently, as needed and / or desired, dividing, shaping and / or encapsulating the product into the desired single-dose or multi-dose units.

[0263] Pharmaceutical compositions according to this disclosure can be prepared, packaged, and / or sold in bulk, in single unit doses, and / or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to an individual and / or an appropriate fraction of that dose, such as half or one-third of such a dose.

[0264] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition disclosed herein will vary depending on the individual's identity, body type, and / or condition and further on the route of administration of the composition. For example, the composition may contain between 0.1% and 100%, such as between 0.5% and 50%, between 1% and 30%, between 5% and 80%, or at least 80% (w / w) of the active ingredient.

[0265] In some embodiments, the formulation described herein may contain at least one nucleic acid vaccine component, such as a nucleic acid vaccine for COVID-19, or an mRNA vaccine for COVID-19. As a non-limiting example, the formulation may contain 1, 2, 3, 4, or 5 nucleic acid vaccine components with different sequences, such as 1, 2, 3, 4, or 5 mRNA vaccine components with different sequences. In some embodiments, the formulation contains at least two nucleic acid vaccine (e.g., mRNA vaccine) components with different sequences. In some embodiments, the formulation contains at least three nucleic acid vaccine (e.g., mRNA vaccine) components with different sequences. In some embodiments, the formulation contains at least four nucleic acid vaccine (e.g., mRNA vaccine) components with different sequences. In some embodiments, the formulation contains at least five nucleic acid vaccine (e.g., mRNA vaccine) components with different sequences.

[0266] The nucleic acid vaccine composition disclosed herein may be formulated with one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g., from a reservoir of nucleic acid vaccine compositions); (4) alter biodistribution (e.g., target the nucleic acid vaccine composition to a specific tissue or cell type); (5) increase the translation of protein-encoded proteins in vivo; and / or (6) alter the release characteristics of protein-encoded proteins in vivo.

[0267] In addition to conventional excipients (such as any and all solvents, dispersion media, diluents, or other liquid media), the dispersants or suspending agents, surfactants, isotonics, thickeners or emulsifiers, preservatives, and excipients disclosed herein may include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with nucleic acid vaccine compositions (e.g., for transplantation into individuals), hyaluronidase, nanoparticle mimics, and combinations thereof. Therefore, the formulations disclosed herein may include one or more excipients, each present together to increase the stability of the nucleic acid vaccine composition and / or increase the amount of cell transfection of the nucleic acid vaccine composition. Furthermore, the nucleic acid vaccine compositions disclosed herein may be formulated using self-assembled nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients, and delivery agents for use in formulating nucleic acids of the nucleic acid vaccine compositions disclosed herein are disclosed in PCT Patent Application Publication WO 2013 / 090648, the contents of which are incorporated herein by reference in their entirety. lipids

[0268] The nucleic acid vaccine components disclosed herein can be formulated using one or more lipids.

[0269] The synthesis of lipids has been extensively described, and formulations containing such compounds are particularly suitable for delivering oligonucleotides or nucleic acids (see Mahon et al., Bioconjug Chem. 2010, 21:1448-1454; Schroeder et al., J Intern Med. 2010, 267:9-21; Akinc et al., Nat Biotechnol. 200, 8 26:561-569; Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869; Siegwart et al., Proc Natl Acad Sci US A. 2011, 108:12996-3001; all of which are incorporated herein by reference in their entirety).

[0270] Although these lipids have been used to efficiently deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci US A. 2008, 105:11915-11920; Akinc et al., Mol Ther. 2009, 17:872-879; Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; the contents of these documents are incorporated herein by reference in their entirety), this disclosure covers formulations thereof and their use in delivering at least one pharmaceutically acceptable carrier, including nucleic acid vaccines. Complexes, micelles, liposomes, or particles containing such lipids can be prepared, and therefore can be used to effectively deliver nucleic acid vaccine components after injection of lipid formulations via local and / or systemic routes. Lipid complexes containing nucleic acid vaccine components can be administered by various routes, including but not limited to intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), or intraventricular (ICV) administration.

[0271] In vivo delivery of nucleic acids can be affected by many parameters, including but not limited to formulation composition, the nature of particle PEGylation, loading level, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are incorporated herein by reference in their entirety). For example, small variations in the anchor chain length of poly(ethylene glycol) (PEG) lipids can have a significant impact on in vivo efficacy. In vivo activity can be tested for formulations with different lipid classes, including but not limited to penta[3-(1-lauroaminopropyl)]-triethylenetetramine hydrochloride (TETA-5LAP; also known as 98N12-5, see Murugaiah et al., Analytical Biochemistry, 2010, 401:61; the contents of which are incorporated herein by reference in their entirety), C12-200 (including derivatives and variants), and MD1.

[0272] The lipid class referred to in this paper as "98N12-5" was revealed by Akinc et al., Mol Ther. 2009, 17:872-879, and the contents of that literature are incorporated herein by reference in their entirety.

[0273] The lipid referred to herein as "C12-200" is disclosed by Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869 and Liu and Huang, Molecular Therapy. 2010, 669-670; the contents of these references are incorporated herein by reference in their entirety. Lipid formulations may include particles containing three or four or more components in addition to nucleic acid vaccine components. For example, formulations containing certain lipids include, but are not limited to, 98N12-5 and may contain 42% lipids, 48% cholesterol, and 10% PEG (C14 alkyl chain length). As another example, formulations containing certain lipids include, but are not limited to, C12-200 and may contain 50% lipids, 10% distearate phosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

[0274] In some embodiments, a nucleic acid vaccine composition formulated with lipids is used for systemic intravenous administration. For example, the final optimized intravenous formulation, which allows greater than 90% of the formulation to be distributed to the liver, uses a nucleic acid vaccine composition and contains a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipids, having a final weight ratio of total lipids to nucleic acid vaccine composition of approximately 7.5:1 and C14 alkyl chain length on the PEG lipids with an average particle size of approximately 50 to 60 nm. (See Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are incorporated herein by reference in their entirety). In another example, intravenous formulations using C12-200 lipids (see PCT patent application publication WO2010129709, the contents of which are incorporated herein by reference in their entirety) can have a C12-200 / distearate phosphatidylcholine / cholesterol / PEG-DMG molar ratio of 50 / 10 / 38.5 / 1.5, wherein a total lipid:nucleic acid weight ratio of 7:1 and an average particle size of 80 nm can effectively deliver nucleic acid vaccine components (see Love et al., Proc Natl Acad Sci US A.2010, 107:1864-1869, the contents of which are incorporated herein by reference in their entirety).

[0275] In some embodiments, formulations containing MD1 lipids can be used to efficiently deliver nucleic acid vaccine components to hepatocytes in vivo. The properties of optimized lipid formulations for intramuscular or subcutaneous routes can vary significantly depending on the target cell type and the ability of the formulation to diffuse into the bloodstream via the extracellular matrix. While particle sizes smaller than 150 nm may be required for efficient hepatocyte delivery due to the size of endothelial pores (see Akinc et al., Mol Ther. 2009, 17:872-879, the contents of which are incorporated herein by reference in their entirety), similar size limitations may not apply to the delivery of formulations to other cell types (including but not limited to endothelial cells, bone marrow cells, and muscle cells) using lipid-formed nucleic acid vaccine components.

[0276] The use of lipid modulators for in vivo delivery of siRNA to other non-hepatic cells, such as bone marrow cells and endothelial cells, has been reported (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009, 19:3112-3118; 8th International Judah Folkman Conference, Cambridge, MA, October 8-9, 2010; each of these entries is incorporated herein by reference in its entirety). For efficient delivery to bone marrow cells, such as monocytes, lipid modulators may have a similar compositional molar ratio. Different ratios of lipids to other components (including but not limited to distearate, phosphatidylcholine, cholesterol, and PEG-DMG) can be used to optimize the formulation of nucleic acid vaccine components for delivery to different cell types, including but not limited to hepatocytes, bone marrow cells, and muscle cells. For example, component molar ratios may include, but are not limited to, 50% C12-200, 10% distearate, phosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG (see Leuschner et al., Nat Biotechnol 2011, 29:1005-1010; the contents of which are incorporated herein by reference in their entirety). Local delivery of nucleic acids to cells via subcutaneous or intramuscular delivery using lipid formulations may not require all the formulation components needed for systemic delivery, and therefore may consist only of lipids and nucleic acid vaccine components. Liposomes

[0277] The nucleic acid vaccine components disclosed herein can be formulated using one or more liposomes.

[0278] In some embodiments, the pharmaceutical components of the nucleic acid vaccine composition include liposomes. Liposomes are artificially prepared vesicles that may consist primarily of a lipid bilayer and can be used as delivery media for nutrients and pharmaceutical formulations. Liposomes can have different sizes, such as, but not limited to, multilayer vesicles (MLVs) with diameters of hundreds of nanometers and containing a series of concentric bilayers separated by narrow aqueous compartments, small single-cell vesicles (SUVs) with diameters less than 50 nm, and large monolayer vesicles (LUVs) with diameters between 50 nm and 500 nm. Liposome design may include, but is not limited to, opsonins or ligands, to improve liposome attachment or activation events with unhealthy tissues, such as, but not limited to, endocytosis. Liposomes may contain low or high pH to improve the delivery of pharmaceutical formulations.

[0279] The formation of liposomes may depend on physicochemical characteristics, such as, but not limited to, the encapsulated pharmaceutical formulation and liposome components; the nature of the medium in which the liposomes are dispersed; the effective concentration of the encapsulated substance and its potential toxicity; any other processes involved in the application and / or delivery of the vesicles; the optimal size, polydispersity and shelf life of the vesicles for the intended application; and batch-to-batch reproducibility and the possibility of large-scale production of safe and efficient liposome products.

[0280] In some embodiments, the pharmaceutical composition comprising the nucleic acid vaccine described herein may include, but is not limited to, liposomes, such as liposomes formed from 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), SMARTICLES® / NOV340 (Marina Biotech, Bothell), 1,2-dilinolenoyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinolenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (DLin-KC2-DMA), and MC3 (US Patent Application Publication US20100324120; the contents of which are incorporated herein by reference in their entirety), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006, 5(12): 1708-1713); the contents of which are incorporated herein by reference in their entirety), hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel), and liposomes capable of delivering small molecule drugs, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[0281] In some embodiments, pharmaceutical components comprising the nucleic acid vaccines described herein may include, but are not limited to, liposomes, such as liposomes formed from synthetic stable plastid-lipid particles (SPLP) or stable nucleic acid-lipid particles (SNALP), which have been previously described and demonstrated to be suitable for in vitro and in vivo oligonucleotide delivery (see Wheeler et al. Gene Therapy. 1999, 6:271-281; ​​Zhang et al. Gene Therapy. 1999, 6:1438-1447; Jeffs et al. Pharm Res. 2005, 22:362-372; Morrissey et al., Nat Biotechnol. 2005, 2:1002-1007; Zimmermann et al., Nature. 2006, 441:111-114; Heyes et al. J Contr Rel. 2005, 107:276-287; Semple et al. Nature Biotech. 2010, 28:172-176; Judge et al. J Clin Invest. 2009, 119:661-673; de Fougerolles Hum Gene Ther. 2008, 19:125-132; each of these contents is incorporated herein by reference in its entirety. Wheeler et al.'s original manufacturing method was a detergent dialysis method, which was subsequently improved by Jeffs et al. and termed the spontaneous vesicle formation method. In addition to nucleic acid vaccine components, liposome formulations can consist of 3 to 4 lipid components. As a non-limiting example, liposomes may contain, but are not limited to, 55% cholesterol, 20% distearate phosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. In another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipids, wherein the cationic lipids may be 1,2-distearate phosphatidyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilininoxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.In another example, the nucleic acid-lipid particle may comprise about 50 mol% to about 85 mol% of the total lipids present in the particle; about 13 mol% to about 49.5 mol% of the total lipids present in the particle; and about 0.5 mol% to about 2 mol% of the total lipids present in the particle, as described in WO2009127060 issued to Maclachlan et al., the contents of which are incorporated herein by reference in their entirety. In another example, the nucleic acid-lipid particle may be any nucleic acid-lipid particle disclosed in US2006008910 issued to Maclachlan et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the nucleic acid-lipid particle may comprise the cationic lipids of Formula I, the noncationic lipids, and the conjugated lipids that inhibit particle aggregation.

[0282] In some embodiments, the nucleic acid vaccine composition disclosed herein can be formulated in lipid vesicles that are cross-linked between functionalized lipid bilayers.

[0283] In some embodiments, the liposomes may contain the glycomodified lipids disclosed in U.S. Patent No. US5,595,756 to Bally et al., the contents of which are incorporated herein by reference in their entirety. The lipids may be gangliosides and cerebrosides in an amount of about 10 moles.

[0284] In some embodiments, the nucleic acid vaccine composition disclosed herein can be formulated in liposomes containing cationic lipids. The liposomes may have a molar ratio (N:P ratio) between 1:1 and 20:1 for nitrogen atoms in the cationic lipids and phosphate in the nucleic acid vaccine composition, as described in PCT Patent Application Publication No. WO2013006825, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the liposomes may have an N:P ratio greater than 20:1 or less than 1:1.

[0285] In some embodiments, the nucleic acid vaccine composition disclosed herein may be formulated in a lipid-polycationic complex. The formation of the lipid-polycationic complex may be achieved by methods known in the art and / or as described in U.S. Patent Publication No. 20120178702, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the polycation may include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyguanine, and / or polyarginine, and cationic peptides as described in PCT Patent Application Publication No. WO2012013326; the contents of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid vaccine composition may be formulated in a lipid-polycationic complex, which may further include neutral lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0286] Liposome formulations can be influenced by factors including, but not limited to, the selection of cationic lipid components, cationic lipid saturation, the nature of PEGylation, the ratio of all components, and biophysical parameters such as size. In one example by Semple et al. (Semple et al., Nature Biotech. 2010, 28:172-176; the contents of which are incorporated herein by reference in their entirety), the liposome formulation consisted of 57.1% cationic lipids, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.

[0287] In some embodiments, the pharmaceutical composition may be formulated using any of the amphoteric liposomes disclosed in: PCT Patent Application Publication No. WO 2008043575 issued to Panzner and U.S. Patent No. US 8,580,297 issued to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety. Amphoteric liposomes may comprise mixtures of lipids, including cationic amphiphilic molecules, anionic amphiphilic molecules, and, where appropriate, one or more neutral amphiphilic molecules. Amphoteric liposomes may comprise amphoteric compounds based on amphiphilic molecules, wherein the head group of such amphiphilic molecules is substituted with one or more amphoteric groups. In some embodiments, the pharmaceutical composition may be formulated with amphoteric lipids comprising one or more amphoteric groups having isoelectric points of 4 to 9, as disclosed in U.S. Patent Application Publication No. 20140227345 to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated with liposomes comprising sterol derivatives as disclosed in U.S. Patent No. 7312206 to Panzner et al. (Novosom), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated using amphoteric liposomes comprising at least one amphiphilic cationic lipid, at least one amphiphilic anionic lipid, and at least one neutral lipid, or liposomes comprising at least one amphiphilic lipid having both positive and negative charges and at least one neutral lipid, wherein the liposomes are stable at pH 4.2 and pH 7.5, as disclosed in U.S. Patent No. 7,780,983 to Panzner et al., the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated using liposomes capable of encapsulating the nucleic acid vaccine composition disclosed herein, such liposomes comprising a serum-stable mixture of lipids taught in U.S. Patent Application Publication No. 20110076322 to Panzner et al., the contents of which are incorporated herein by reference in their entirety. The lipid mixture comprises phosphatidylcholine and phosphatidylethanolamine in a ratio ranging from about 0.5 to about 8. The lipid mixture may also include pH-sensitive anionic and cationic amphiphilic molecules, making the mixture amphoteric, negatively or neutral at pH 7.4 and positively charged at pH 4. The drug / lipid ratio can be adjusted to target the liposomes to specific organs or other sites in the body. In some embodiments, lipid systems loaded with the nucleic acid vaccine components disclosed herein are prepared by the method disclosed in U.S. Patent Application Publication No. US 20120021042 to Panzner et al., the contents of which are incorporated herein by reference in their entirety.The method includes mixing an aqueous solution of a polyanionic surfactant with an alcoholic solution of one or more amphiphilic molecules and buffering the mixture to an acidic pH, wherein one or more amphiphilic molecules readily form amphoteric liposomes at the acidic pH, thereby forming an amphoteric liposome complex in a suspension of the encapsulating surfactant.

[0288] The nucleic acid vaccine composition disclosed herein can be formulated using one or more lipid complexes.

[0289] In some implementations, the nucleic acid vaccine components can be formulated as lipid complexes, such as, but not limited to, the ATUPLEX™ system, the DACC system, the DBTC system, and other siRNA-lipid complex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, MA), and targeted and non-targeted nucleic acid delivery based on polyethyleneimine (PEI) or protamine (Aleku et al. Cancer Res. 2008, 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther, 2012, 50:76-78; Santel et al., Gene Ther, 2006, 13:1222-1234; Santel et al., Gene Ther., 2006, 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010, 23:334-344; Kaufmann et al. Microvasc Res., 2010, 80:286-293; Weide et al. J Immunother., 2009, 32:498-507; Weide et al. J Immunother., 2008, 31:180-188; Pascolo., Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., J. Immunother., 2011, 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci US A. 2007, 6;104:4095-4100; deFougerolles Hum Gene Ther.2008, 19:125-132; each of these contents is incorporated herein by reference in its entirety. Lipid nanoparticles (LNP)

[0290] In some embodiments, the nucleic acid vaccine composition disclosed herein can be formulated in lipid nanoparticles (LNPs). Generally, LNPs are characterized as small solid or semi-solid particles having an outer lipid layer with a hydrophilic outer surface exposed to a non-LNP environment; an internal space that can be aqueous (vesicle-like) or non-aqueous (micelle-like); and at least one hydrophobic intermembrane space. The LNP membrane can be layered or non-layered and can consist of 1, 2, 3, 4, 5, or more layers. In some embodiments, the LNP may contain a load or effective payload entering its internal space, entering its intermembrane space, entering its outer surface, or any combination thereof.

[0291] The LNPs used herein are known in this art and generally contain cholesterol (which contributes to stability and promotes membrane fusion), phospholipids (which provide structure for the LNP bilayer and also help endosome escape), polyethylene glycol (PEG) derivatives (which reduce LNP aggregation and "protect" LNPs from nonspecific endocytosis by immune cells) and ionizable lipids (which complex with negatively charged RNA and enhance endosome escape), forming LNP-forming components.

[0292] The components of the LNP can be selected based on the desired target, orientation, load, size or other desired characteristics or properties.

[0293] LNP may be the lipid nanoparticles described in PCT Patent Application Publication No. WO2012170930, the contents of which are incorporated herein by reference in their entirety.

[0294] In some embodiments, the nucleic acid vaccine composition disclosed herein may be formulated in an LNP containing at least one cationic lipid.

[0295] In some embodiments, the cationic lipids that can be used in the formulations disclosed herein may be selected from, but are not limited to, the cationic lipids described in the following: PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, and WO2 U.S. Patent Nos. 012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276, U.S. Patent Nos. 7,893,302, 7,404,969 and 8,283,333, and U.S. Patent Publication Nos. US20100036115 and US20120202871; each of these contents is incorporated herein by reference in its entirety. Cationic lipids may also be selected from, but are not limited to, Formula A described in the following: PCT Patent Application Publications Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of these contents is incorporated herein by reference in its entirety. Alternatively, the cationic lipid may be selected from, but not limited to, the formula CLI-CLXXIX of PCT Patent Application No. WO2008103276, the formula CLI-CLXXIX of U.S. Patent No. 7,893,302, the formula CLI-CLXXXXII of U.S. Patent No. 7,404,969, and the formulas I-VI of U.S. Patent Publication No. US20100036115; each of these contents is incorporated herein by reference in its entirety. The cationic lipid may be a multivalent cationic lipid, such as the cationic lipid disclosed in U.S. Patent No. 7,223,887 to Gaucheron et al., the contents of which are incorporated herein by reference in their entirety. The cationic lipid may have a positively charged head group comprising two quaternary amino groups and a hydrophobic portion comprising four hydrocarbon chains, as described in U.S. Patent No. 7,223,887 to Gaucheron et al. Cationic lipids may be biodegradable, such as those disclosed in U.S. Patent Application Publication No. 20130195920 to Maier et al., the contents of which are incorporated herein by reference in their entirety. Cationic lipids may have one or more biodegradable groups located in the lipid portion of the cationic lipid, as described in Formulas I-IV of US20130195920 to Maier et al.In some embodiments, the cationic lipid may also be the cationic lipid disclosed in US20130156845 and US 20130129785 issued to Manoharan et al., WO 2012047656 issued to Wasan et al., WO 2010144740 issued to Chen et al., WO 2013086322 issued to Ansell et al., or WO 2012016184 issued to Manoharan et al., each of which is incorporated herein by reference in its entirety.

[0296] As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonadecano-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethylhexadecano-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentadecano-16,19-diene-8-amine, (13Z,16Z)-N,N-dimethyltetradecano-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethyltetradecano-12,15-diene-4-amine, (14Z,17Z)-N,N-dimethyltetradecano-14,17-diene-6-amine, (15Z,18Z)-N,N-di Methyltetracos-15,18-diene-7-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltetracos-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethyloctadec-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexadec-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethyldiene-7-amine Pentadecyl-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethyltridecyl-22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyltridecyl-21,24-diene-9-amine, (18Z)-N,N-dimethylheptadecyl-18-en-10-amine, (17Z)-N,N-dimethylhexadecyl-17-en-9-amine, (19Z,22Z)-N,N-dimethylhexadecadecyl-19,22-diene-7-amine, N,N-dimethylheptadecane-10-amine, (20Z,23Z)-N-ethyl-N-methylhexadecadecyl-20,23-diene-10-amine, 1-[(11Z,14Z)-l-nonyl] [Eicosene-11,14-dien-l-yl]pyrrolidine, (20Z)-N,N-dimethylheptadec-20-en-l0-amine, (15Z)-N,N-dimethylheptadec-15-en-l0-amine, (14Z)-N,N-dimethylheptadec-14-en-l0-amine, (17Z)-N,N-dimethylheptadec-17-en-l0-amine, (24Z)-N,N-dimethyltridec-24-en-l0-amine, (20Z)-N,N-dimethylheptadec-20-en-l0-amine, (22Z)-N,N-dimethyltridec-22-en-l0-amine, (16Z)-N,N-dimethylpentadecan-16-en-8-amine, (12Z,(15Z)-N,N-dimethyl-2-nonyldocosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]tecosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R, 2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, RN,N -Dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)prop-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-octyl-5-en-1-yloxy]prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-octyl-5-en-1-yloxy]prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)methyl} (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-(nonoxy)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-dien-1-yloxy]-3-(octyloxy)prop-2-amine, (2S)-N,N-Dimethyl-1-[(6Z,9Z,12Z)-Octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)prop-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentoxy)prop-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylprop-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylprop-2-amine, [(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octoxy)prop-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octoxy)prop-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexoxy)-N,N-dimethylprop-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexoxy)-N,N-dimethylprop-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, 1-[(9Z)-hexadecane-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-decane-1-yloxy]-prop-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-decane-1-yloxy]-prop-2-amine [Octocarbon-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-(octoxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)prop-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octoxy)prop-2-amine, and (11E,20Z,23Z)-N,N-dimethyl-nonadecanocarbon-11,20,2-trien-10-amine, or a pharmaceutically acceptable salt or stereoisomer thereof. Lipid nanoparticle (LNP) composition.

[0297] In some embodiments, the lipid nanoparticles may consist of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, at least one additional LNP functional component, or any combination thereof. In some embodiments, the lipid nanoparticles may consist of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP may consist of at least one cationic lipid, at least one non-cationic lipid, and at least one sterol. In some embodiments, the LNP may consist of at least one cationic lipid, at least one non-cationic lipid, and at least one additional LNP functional component. In some embodiments, the LNP may consist of at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP may consist of at least one cationic lipid and at least one non-cationic lipid. In some embodiments, the LNP may consist of at least one cationic lipid and at least one sterol. In some embodiments, the LNP may consist of at least one cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP may consist of at least one non-cationic lipid and at least one sterol. In some embodiments, the LNP may consist of at least one non-cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP may consist of at least one sterol and at least one additional LNP functional component. In some embodiments, the LNP may consist of at least one cationic lipid. In some embodiments, the LNP may consist of at least one non-cationic lipid. In some embodiments, the LNP may consist of sterol. In some embodiments, the LNP may consist of additional LNP functional components.

[0298] In some embodiments, the at least one cationic lipid may comprise at least one ionizable cationic lipid, at least one amino lipid, at least one saturated cationic lipid, at least one unsaturated cationic lipid, at least one zwitterionic lipid, at least one polyvalent cationic lipid, or any combination thereof. In some embodiments, the LNP may substantially contain no at least one cationic lipid. In some embodiments, the LNP may contain no amount of at least one cationic lipid.

[0299] In some embodiments, at least one cationic lipid may be selected from, but is not limited to, at least one of the following: 1,3-bis-(1,2-bis-tetradecoxypropyl-3-dimethylethoxyammonium bromide)-prop-2-ol ((R)-PLC-2)), 2-(dinonylamino)ethanol-1-ol (17-10), 2-(di-dodecylamino)ethanol-1-ol (17-11), 3-(di-dodecylamino)prop-1-ol (17-12), 4-(di-dodecylamino)but-1-ol (17-13), 2-(hexyl((9Z,12Z)-octadec-9,12-dien-1-yl)amino)ethanol-1-ol (17-2), 2-(nonylamino)prop-1-ol (17-13), 2-(di-dodecylamino)but-1-ol (17-14), 2-(di-dodecylamino)but-1-ol (17-15), 2-(di-dodecylamino)but-1-ol (17-16 ... 2-(dodecyl(9Z,12Z)-octadecyl-9,12-dien-1-yl)amino) ethyl-1-ol (17-3), 2-(dodecyl(9Z,12Z)-octadecyl-9,12-dien-1-yl)(tetradecyl)amino) ethyl-1-ol (17-4), 2-((9Z,12Z)-octadecyl-9,12-dien-1-yl)(tetradecyl)amino) ethyl-1-ol (17-5), 2-((9Z,12Z)-octadecyl-9,12-dien-1-yl)(tetradecyl)amino) ethyl-1-ol (17-6), 2-(di-tetradecylamino) ethyl-1-ol (17-7), 2-(di((Z)-octadecyl-9-dien-1-yl)amino) ethyl- 1-Alcohol (17-8), (9Z,12Z)-N-(2-methoxyethyl)-N-((9Z,12Z)-octadec-9,12-dien-1-yl)octadec-9,12-dien-1-amine (17-9), N-nonyl-N-(2-(piperazine-1-yl)ethyl)non-1-amine (19-1), N-dodecyl-N-(2-(piperazine-1-yl)ethyl)dodecyl-1-amine (19-2), (9Z,12Z)-N-((9Z,12Z)-octadec-9,12-dien-1-yl)-N-(2-(piperazine-1-yl)ethyl)octadec-9,12-dien-1-amine (19-3), N-dodecyl-N-(2-( 4-Methylpiperazine-1-yl)ethyl)dodecane-1-amine intermediate 1: 2-(di-dodecylamino)ethanol-1-ol (19-4), N-dodecyl-N-(2-(4-(4-methoxybenzyl)piperazine-1-yl)ethyl)dodecane-1-amine (19-5), (9Z,12Z)-N-(2-(4-dodecylpiperazine-1-yl)ethyl)-N-((9Z,12Z)-octadec-9,12-dien-1-yl)octadec-9,12-dien-1-amine (19-6), (3-((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropyl-1-amine) (1-Bl 1),N-(2-(di-dodecylamino)ethyl)-N-dodecylglycine (20-1), 8,8'-((2-(dodecyl(2-hydroxyethyl)amino)ethyl)ureidyl)dioctanoate dinonyl ester (20-10), 3-((2-(di-tetradecylamino)ethyl)(dodecyl)amino)prop-1-ol (20-11), 2-((2-(di-tetradecylamino)ethyl)(tetradecyl)amino)ethanol-1-ol (20-12), 2-((2-(di((9Z,12Z)-octadec-9,12-dien-1-yl)amino)ethyl)(dodecyl)amino)ethanol-1-ol (20-13 ... ,12-dien-1-yl)amino)ethyl)((9Z,12Z)-octadec-9,12-dien-1-yl)amino)ethyl-1-ol (20-14), 2-((2-(di-dodecylamino)ethyl)(hexyl)amino)ethyl-1-ol (20-15), 2-((2-(di-nonylamino)ethyl)(nonyl)amino)ethyl-1-ol (20-16), 2-((2-(di-dodecylamino)ethyl)(nonyl)amino)ethyl-1-ol (20-17), 2-((2-(di-nonylamino)ethyl)(dodecyl)amino)ethyl-1-ol (20-18), 2-((2-(di-dodecylamino)ethyl)amino)ethyl-1-ol (20-19), 6 -(dodecyl(2-(dodecyl(2-hydroxyethyl)amino)ethyl)amino)pentyl hexanoate (20-2), 2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl-1-ol (20-20), 3-((2-(di-dodecylamino)ethyl)(dodecyl)amino)prop-1-ol (20-21), 4-((2-(di-dodecylamino)ethyl)(dodecyl)amino)but-1-ol (20-22), (Z)-2-((2-(di-dodecylamino)ethyl)dodec-6-en-1-yl)amino)ethyl-1-ol (20-23), 2-((2-(di-dodecylamino)ethyl)(tetradecyl)amino)ethyl-1- Alcohols (20-24), 2-((2-(di-dodecylamino)ethyl)((9Z,12Z)-octadec-9,12-dien-1-yl)amino)ethyl-1-ol (20-25), 6-((2-(di-dodecylamino)ethyl)(2-hydroxyethyl)amino)pentyl hexanoate (20-3), 6,6'-((2-(dodecyl(2-hydroxyethyl)amino)ethyl)ureidyl)dipentyl hexanoate (20-4), 6,6'-((2-((6-(heptoxy)-6-sideoxyhexyl)(2-hydroxyethyl)amino)ethyl)ureidyl)diheptyl hexanoate (20-5), 6-((2-(2-(dinonylamino)ethyl)(2-hydroxyethyl)amino)pentyl hexanoate (20-6)6-(dodecyl(2-(dodecyl(2-hydroxyethyl)amino)ethyl)amino)heptaate (20-7), 8-((2-(di-dodecylamino)ethyl)(2-hydroxyethyl)amino)nonyl octanoate (20-8), 18-((2-(di-dodecylamino)ethyl)(2-hydroxyethyl)amino)heptadecane-9-ester of octanoate (20-9), 1-(2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)cyclopropyl)-N,N-dimethylmethylamine) 21-1), 3,3-bis((9Z,12Z)-octadec-9,12-dien-1-yl)cyclobutyl 4-(dimethylamino)butyric acid (21-2), 3,3-bis((9Z,12Z)-octadec-9,12-dien-1-yl)cyclopentyl 3-(dimethylamino)propionic acid (21-3), 3,3-bis((9Z,12Z)-octadec-9,12-dien-1-yl)cyclopentyl 4-(dimethylamino)butyric acid (21-4), 1-(2,3-bis((8Z,11)-cyclopentyl 4-(dimethylamino)butyric acid (21-5), 1-(2,3-bis((8Z,11)-cyclopentyl 4-(dimethylamino)butyric acid (21-6), 1-(2,3-bis((8Z,11)-cyclopentyl 4-(dimethylamino)butyric acid (21-7), 1-(2,3-bis((8Z,11)-cyclopentyl 4-(dimethylamino)butyric acid (21-8), 1-(2,3-bis((8Z,11)-cyclopentyl 4-(dimethylamino)butyric acid (21-9), 1-(2,3-bis((8Z,11)-cyclopentyl 4-(dimethylamino)butyric acid (21-2 ... Z)-heptadec-8,11-dien-1-yl)cyclopropyl)-N,N-dimethylmethylamine (21-6), unknown (75-016B), poly{4-((2-(dimethylamino)ethyl)thio)tetrahydro-2H-pyran-2-one}-r-poly{4-(octylthio)tetrahydro-2H-pyran-2-one} (A7), (3aR5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopentan-1, 3-Dioxo-5-amine (ALN100), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopentane[d][l,3]dioxo-5-amine (ALN1001), ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopentane[d][l,3]dioxo-5-amine)) (ALNY-100), Dimyroxytrimethylammonium propane (aminolipid 6), Benzamidiπ-dialkyl-formic acid (BADACA), N,N-dihydroxyethylmethyl-N-2-(cholesteroloxycarbonylamino)ethylammonium bromide (BHEM-Chol), N,N-bis-(2-hydroxyethyl)-N-methyl-N-(2-cholesteroloxycarbonylamino-ethyl)ammonium bromide (BHEM-Chol1), 2-{4-[(3β)-cholesterol-5-en-3-yloxy]butoxy}-iV?N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propyl-1-amine (butyl-CLinDMA)(2JR)-2-{4-[(3β)-cholesterol-5-en-3-yloxy]butoxy}-Λr^dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxyprop-1-amine (butyl-CLinDMA (2R)), (25)-2-{4-[(3β)-cholesterol-5-en-3-yloxy]butoxy}-iVy / V-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (butyl-CLinDMA (2S)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylureadiyl)di-dodecane-2-ol (C 12-200), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)ureidyl)bis(dodecane-2-ol) (C12-200), cholesterolyl-succinylsilane (C2), bis(octadecano-9,12-dienoic acid) (9Z,9'Z,12Z,12'Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy Propyl-1,3-diester (cationic lipid A2), octadecyl-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (cationic lipid A3), l-(3-cholesterolyl)-oxycarbonyl-aminomethylimidazolium (CHIM), [(2-morpholino-4-yl-ethylaminomethyl)methyl]-carbamate cholesterol ester (Chol-C3N-Mo2 ... [l-methyl-2-(2-morpholin-4-yl-ethylaminomethyl)-propyl]-carbamate cholesterol ester (Chol-DMC3N-Mo2), 1,17-bis(2-octylcyclopropyl)heptadecane-9-ester of 4-(dimethylamino)butyrate (CL), 3-(dimethylamino)butyrate heptadecane-6,9,28,31-tetraen-19-ester of heptadecane (CL01), 3-(dimethylamino) Cholesterol propionate (CL06), 2-(dimethylamino)acetic acid cholesterol ester (CL08), N,N-dimethyl-2,3-bis(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)propyl-1-amine (CL-1), N-methyl-2-(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)-N-(2-((((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)ethyl)ethyl-1-amine (CL-11)(3R,4R)-3,4-bis(((Z)-hexadec-9-en-1-yl)oxy)-1-methylpyrrolidone (compound CL-12) (CL-12), 2-(dimethylamino)-N-((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl)acetamide (CL-13), (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid)-3-(dimethylamino)prop-1,2-diester (CL-14), (9Z,12Z)-di((9Z,12Z)-octadec-9,12-dien-1-yl)amine (CL-15), di-dodecanoic acid 7-hydroxy7-(4-((1-methylpiperidin-4-carbonyl)oxy)butyl)tridecane-1,13-diester (C L15B6), ditetradecanoic acid 7-hydroxy7-(4-((1-methylpiperidin-4-carbonyl)oxy)butyl)tridecane-1,13-diester (CL15C6), dipalmitoic acid 7-hydroxy7-(4-((1-methylpiperidin-4-carbonyl)oxy)butyl)tridecane-1,13-diester (CL15D6), dioleoic acid 7-hydroxy7-(4-((1-methylpiperidin-4-carbonyl)oxy)butyl)tridecane-1,13-diester (CL15H6), bis(2-(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)ethyl)amine (CL-16), (9Z,12Z)-N -Methyl-N-(2-(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)ethyl)octadec-9,12-dien-1-amine (CL-17), (9Z,12Z)-N-(3-(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)propyl)octadec-9,12-dien-1-amine (CL-18), di((11Z,14Z)-eicosano-11,14-dien-1-yl)carbamate (1-methylpiperidin-3-yl)methyl ester (CL-19), N-methyl-N,N-bis(2-(((Z)-hexadec-9-enyloxy)ethyl)amine ( CL-2), (13Z,16Z)-N,N-dimethyl-4-((9Z,12Z)-octadec-9,12-dien-1-yl)teicosto-3,13,16-trien-1-amine (CL-20), (S)-2-amino-3-hydroxy-N,N-bis(2-(((Z)-octadec-9-en-1-yl)oxy)ethyl)propionic acid (CL-21), C2:N,N-di-hexadecyl-N'-(3-triethoxysilylpropyl)succinidine (CL3), trans-1-methyl-3,4-bis((((Z)-octadec-9-en-1-yl)oxy)methyl)pyrrolidine (CL-3)Bis(methylene)(9Z,9'Z,12Z,12'Z)-bis(octadecanoic-9,12-dienoic acid) trans-1-methylpyrrolidone-3,4-diester (CL-4), 7-(4-(diisopropylamino)butyl)-7-hydroxytetrazane-1,13-diester of di-tetradecanoic acid (CL4C6), 7-(4-(diisopropylamino)butyl)-7-hydroxytetrazane-1,13-diester of dipalmitoic acid (CL4D6), 11-(4-(diisopropylamino)butyl)-11-hydroxytetradecane-1,21-diester of dioleoic acid (CL4H10), 7-(4-(diisopropylamino)butyl)-7-hydroxytetrazane-1,13-diester of dioleoic acid (CL4H6), di Oleic acid 9-(4-(diisopropylamino)butyl)-7-hydroxyheptadecane-1,17-diester (CL4H8), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-ester (CL-5), 2-(dimethylamino)-N-(2-(((Z)-octadecane-9-en-1-yl)oxy)ethyl)-N-((9Z,12Z)-octadecane-9,12-dien-1-yl)acetamide (CL-53), 3-((2-(((Z)-octadecane-9-en-1-yl)oxy)ethyl)((9Z,12Z)-octadecane-9,12-dien-1-yl)amino)propane-1-All (CL-54), 1-Methyl-3,3-bis((((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)methyl)azicyclic butane (CL-55), 1-Methyl-3,3-bis(2-(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)ethyl)azicyclic butane (CL-56), 1-Methyl-3,3-bis(2-(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)propyl)azicyclic butane (CL-57), 2-(3,3-di((9Z,12Z)-octadec-9,12-dien-1-yl)azicyclic butane-1-yl)ethanol (CL-58) ), 2-(3,3-di((9Z,12Z)-octadec-9,12-dien-1-yl)azacyclobutan-1-yl)prop-1-ol (CL-59), 3-(di((9Z,12Z)-octadec-9,12-dien-1-yl)amino)prop-1-ol (CL-6), 3,3-di((9Z,12Z)-octadec-9,12-dien-1-yl)azacyclobutan-1-carboxylic acid 3-(dimethylamino)propyl ester (CL-60), 2-(di((Z)-octadec-9-en-1-yl)amino)ethanol-1-ol (CL-61), 3-(di((Z)-octadec-9-en-1-yl)amino)prop-1-ol (CL-62)(11Z,14Z)-2-((dimethylamino)methyl)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)eicos-11,14-dien-1-ol (CL-63), (11Z,14Z)-2-(dimethylamino)-2-((9Z,12Z)-octadec-9,12-dien-1-yl)eicos-11,14-dien-1-ol (CL-64), 3-(dimethylamino)-2,2-bis((((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)methyl)prop-1-ol (CL-65), (9Z,12Z)-N-(2 -(((Z)-octadec-9-en-1-yl)oxy)ethyl)octadec-9,12-dien-1-amine (CL-7), 1-methyl-3,3-di((9Z,12Z)-octadec-9,12-dien-1-yl)azacyclobutane (CL-8), N,2-dimethyl-1,3-bis(((9Z,12Z)-octadec-9,12-dien-1-yl)oxy)prop-2-amine (CL-9), 3-dimethylamino-2-(cholesterol-5-en-3B-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholesterol-5-en-3B-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholesterol-5-en-3B-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA) (c-5-en-3-oxy)-3′-oxaproloxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), acetyltrimethylammonium bromide (CTAB), 1-diarachidonicyloxy-TV-dimethyl-propyl-S-amine (DAraDMA), O,O'-di-tetradecyl-N-(α-trimethylammonium acetyl)diethanolamine chloride (DC-6-14), 3β-[N-(N′,N′-dimethylaminoethane)aminomethyl]cholesterol (DC-Chol), dimethyl di-octadecylammonium (DDA ... Alkyl ammonium bromide (DDA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-di-docohexenyloxy-(7V,N-dimethyl)propyl-3-amine (DDocDMA), N-(2-(dimethylamino)ethyl)-4,5-bis(dodecylthio)pentanylamine (DEDPA), 3-dimethylamino-2-(cholesterol-5-en-3β-oxypentan-3-oxa-an-5-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (DEG-CLinDMA), 1,6-dioleyltriethylenetetramine (dio-TETA)Nl,N19-bis((S,23E,25E,27E,29E)-16-((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexyl-1-en-1-yl)non-2,4,6,8-tetraenenylamino)-24,28-dimethyl-15,22-disiderol-30-(2,6,6-trimethylcyclohexyl-1-en-1-yl)-4,7,10-trioxa-14,21-diazatriacontane-23,2 5,27,29-Tetraen-1-yl)-4,7,10,13,16-pentaenodecane-1,19-diaminoamine (diVA-PEG-diVA), DiLin-N-methylpiperazine (DL-033), DiLin-N,N-dimethylglycine (DL-036), dioleyl-N,N-dimethylglycine (DL-048), 3-((1,3-bis(((9Z,12Z)-octadec-9,12-dienyl)oxy)propane-2 1,2-Linoleoylaminopropionic acid (DLAPA), 1,2-di-linolenic acid-3-dimethylaminopropane (DLenDMA), 1-linolenic acid-2-linolenic acid-3-dimethylaminopropane (DLin-2-DMAP), 3-(N,N-dilinolenic acid)-1,2-propanediol (DLinAP), 1,2-N,N′-dilinolenic acid-methyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Linolenic acid methyl-3-dimethylaminopropane (DLinCDAP), 1,2-linolenic acid methyl-3-dimethylaminopropane (DLin-C-DAP), 1,2-linolenic acid-3-(dimethylamino)acetylated propane (DLin-DAC), 1,2-linolenic acid-3-dimethylaminopropane (DLinDAP), 1,2-linolenic acid-N,N-dimethylaminopropane (DLinDMA) ), 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLinDMA 1), 1,2-Dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), linoleyl-4-aminobutyric acid (DLinFAB), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxacyclopentane (DLin-K-DMA), 1,2-Dilinoleyloxy-3-morpholinylpropane (DLin-MA), 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-ester (DLin-MC3-DMA)1,2-Dilinolenic acid-3-(N-methylpiperidinyl)propane (DLinMPZ), 1,2-Dilinolenic acid-3-(N-methylpiperidinyl)propane (DLin-MPZ), 1,2-Dilinolenic acid-3-piperidinylpropylamine (DLinPip), 1,2-Dilinolenic acid-3-(3'-hydroxypiperidinyl)-propylamine (DLinPip-3OH), 1,2-Dilinolenic acid-3-(4'-hydroxypiperidinyl)-propylamine (DLinPip-4OH), 1,2-Dilinolenic acid-3-hydroxypropane (DLinPO), 1,2-Dilinolenic acid-thio-3-dimethylaminopropane (DLin-S-DMA), 1,2-Dilinolenic acid-3-hydroxypropane (DLinPO), 1,2-Dilinolenic acid-thio-3-dimethylaminopropane (DLin-S-DMA), 1,2-Dilinolenic acid-3-hydroxypropane (DLinPO), 1,2-Dilinolenic acid-3-dimethylaminopropane (DLin-S-DMA), 1,2-Dilinolenic acid-3-hydroxypropane (DLinPO), 1,2-Dilinolenic acid-3-hydroxy ... Linolenic acid-3-trimethylaminopropane (DLinTAP), 1,2-dilinolenic acid-3-trimethylaminopropane hydrochloride (DLin-TAP.Cl), 1,2-dilinolenic acid-3-trimethylaminopropane (DLinTMA), 1,2-dilinolenic acid-3-trimethylaminopropane hydrochloride (DLin-TMA.Cl), 3-((1,3-bis(((9Z,12Z,15Z)-octadecano-9,12,15-trienyl)oxy)prop-2-yl)amino)propionic acid (DLLAPA), 1,2-dilinolenic acid-3-(N,N-dimethyl-propylamine) (DLmDEA), 1,2-dilauryl-sn-glycolic acid DL-3-phosphoethanolamine (DLPE), 1,2-dilauryl-sn-glycerol-3-glycerol (DLPG), N,N-dimethyl-3,4-dioleoxybenzamine (DMOBA), dimyristylphosphatidylserine (DMPS), N-[l-(2,3-dimyristoxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 1,2-dimyristoxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE1), 1,2-dimyristyl-3-trimethylammonium propane (DMTAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 3-((1,3-bis(oleyloxy) (DOAPA)-propyl-2-amino-3-propionic acid, 1,2-N,N′-dioleoylaminomethyl-3-dimethylaminopropane (DOcarbDAP), 1,2-dioleoylaminomethyl-3-dimethylammonium-propane (DOCDAP), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-dihydroxyethylN,N-di-octadecylammonium chloride (DODEAC), N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), dioleoyl-4-aminobutyric acid (DOFAB), di-octadecylaminoglycinylspermine (DOGS)1,2-Dioleoyl-3-methyl-(methoxycarbonyl-ethyl)ammonium-propane (DOMCAP), 1,2-dioleoyl-3-N-pyrrolidone-propane (DOP5P), 1,2-dioleoyl-3-N-pyridinium-propane bromide (DOP6P), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxybutylammonium bromide (DORIE-HB), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxypropylammonium bromide (DORIE-HP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxypentyl bromide Ammonium (DORIE-Hpe), 2,3-dioleoxy-N-[2(spermine-methamido)ethyl]-N,N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA), 1,3-dioleoxy-2-(6-carboxy-spermine)-propylamine (DOSPER), N-(1-(2,3-dioleoxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoxy-3-trimethylammonium-propane (DOTAP1), N-[5'-(2',3'-dioleoxy)uridine]-N',N',N'-trimethylammonium toluenesulfonate (DOTAU), 1-[2-(9(Z)-octadecenoxy)ethyl]-2-(8(Z)-decanoyl)-ethyl Heptacarben-3-(2-hydroxyethyl)imidazolium chloride (DOTIM), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylinuridine phosphatidylcholine (DOUPC), 1,2-diphytanyloxy)-(WN-dimethyl)-but-4-amine (DPan-C2-DMA), 1,2-diphytanyloxy)-3-(iV,7V-dimethyl)propylamine (DPanDMA), (2-(dimethylamino)ethyl)carbamate 2,3-bis(dodecylthio)propyl ester (DPDEC), dipalmitoyl-4-aminobutyric acid (DPFA) B) 1,2-Dipalmitoxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE), 1,2-dipalmitoxypropyl-3-trimethylammonium propane (DPTAP), 1-[2-(hexadecyloxy)ethyl]-2-pentadecanyl-3-(2-hydroxyethyl)imidazolium chloride (DPTIM), 3-((1,3-bis(stearyloxy)propyl-2-yl)amino)propionic acid (DSAPA), distearyldimethylammonium (DSDMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA1), 1,2-distearyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE), 1,2-distearyloxy-3-trimethylammonium propane (DSTAP)Ditetradecyltrimethylammonium (DTDTMA), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (EDOPC), N2-[N2,N5-bis(3-aminopropyl)-L-ormithyl]-N,N-di-octadecyl-L-glutamine tetrahydrotrifluoroacetate (GC33), cholesty-5-en-3-ol (3P)-,3-[(3-aminopropyl)[4-[(3-aminopropyl)amino]butyl]amine Formate esters (GL67), glyceryl monooleate (GMO), guanidino-dialkyl-formic acid (GUADACA), 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-sideoxyethane ammonium bromide (HEDC), 2,2'-(tert-butoxycarbonylureadiyl)bis(ethyl-2,1-diyl) ester of di-tetradecanoic acid (HEDC-BOC-TN), 1-(2-(((3S) ,10R,13R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentan[a]phenanthrene-3-yldithioalkyl)ethyl)guanidine (HGT4002), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadec-9,12-dien-l-yl)tetradecano Tetracarbon-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadec-9,12-dien-1-yl)tetracarbon-4,15,18-trien-1-amine (HGT5001), histamine-cholesterol hemisuccinate (HisChol), histamine-cholesterol hemisuccinate (Hist-Chol), HydroSoyPC (HSPC), Imidazole Cholesterol Ester (ICE), 3-(di-dodecylamino)-N1,N1,4-tri-dodecyl-1-piperazine (KL10), N1-[2-(di-dodecylamino)ethyl]-N1,N4,N4-tri-dodecyl-1,4-piperazine diethylamine (KL22), 14,25-di-tridecyl-15,18,21,24-tetraaza-octacosandecane (KL25) ), N,N-di-n-tetradecyl,N-methyl-N-(2-guanidinyl)ethylammonium (lipoprotein 1), N,N-di-n-octadecyl,N-methyl-N-(2-guanidinyl)ethylammonium chloride (lipoprotein 2), (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (lipoprotein A),(9Z,12Z)-Octadecano-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (lipoprotein A1), 2,2-dilinolenico-4-dimethylaminoethyl-[l,3]-dioxacyclopentane (lipoprotein A2), bis(decanoic acid)((5-(((dimethylamino)methyl)-l,3-epoxyphenyl)bis(oxy))bis(octyl-8,l-diyl) ester (lipoprotein B), 9Z,9'Z,12Z,12'Z)-bis(octadecano-9,12-dienoic acid)2-((4-(((3-(dimethylamino)propoxy)carbonyl)) (Oxy)hexadecyl)oxy)propane-1,3-diester (lipid C), 3-octylundecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octyloxy)tridecane ester (lipid D), 5-(dimethylamino)valerate (6Z,16Z)-12-((Z)-dec-4-en-1-yl)teicos-6,16-dien-11-ester (lipid I), di-octadecyl-(2-hydroxy-3-propylamino)amino polylysine (lipid T), (3-((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropyl-1-amine (M) C3 ether), as described in U.S. Provisional Application No. 61 / 384,050 (MC3 thioester), (4-((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbut-1-amine (MC4 ether), 3-((2-(((9Z,12Z)-octadec-9,12-dienyl)oxy)ethyl)amino)propionic acid (MLAPA), 3-((2-(((9Z,12Z,15Z)-octadec-9,12,15-trienyl)oxy)ethylamino)propionic acid (MLLAPA), mono-omycolyl glycerol (MM) G), 3-((2-(oleyloxy)ethyl)amino)propionic acid (MOAPA), 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MoChol), 1,2-dioleyl-3-N-morpholino-propane (MoDO), methylpyridinyl-dialkyl-carboxylic acid (MPDACA), monopalmitoylphosphatidylcholine (MPPC), 3-((2-(stearoyloxy)ethyl)amino)propionic acid (MSAPA), N1-[2-((lS)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylmethoxy)ethyl]-3,4-di[oleyloxy]benzylamine (MVL5)2-({8-[(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA) (2R)), phosphatidylcholine (PC), 1,3-bis-(1,2-bis-tetradecoxypropyl-3-dimethylethoxyammonium bromide)-prop-2-ol (PCL-2), palmityl-oleyl-n-arginine (PONA), stearylamine (STA), 2-(((tri-butyldimethylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol (Synthesis Example 1 (A)), (9Z)-tetradec-9-enoic acid 3-((tri-butyl(dimethyl)silyl)oxy)-2,2-bis((((9Z)-tetradec-9-enoxy)methyl)propyl ester (Synthesis Example 1 (B)), (9Z)-tetradec-9-enoic acid 3-hydroxy-2,2-bis((((9Z)-tetradec-9-enoxy)methyl)propyl ester (Synthesis Example 1) (C)), (9Z)-tetradec-9-enoic acid 3-((4-(dimethylamino)butyryl)oxy)-2,2-bis((((9Z)-tetradec-9-enyloxy)methyl)propyl ester (Synthetic Example 1 (D)), 3-(5-(bis(2-hydroxydodecyl)amino)pent-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pent-2-yl)-1,4-dialkyl-2,5-dione) (Target 24), molasses 6'6'-disorbate (TDB), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylureadiyl)di-dodecane-2-ol (Tech) G1), 3-((1,3-bis(((9Z,12Z)-octadec-9,12-dienyl)oxy)-2-((((9Z,12Z)-octadec-9,12-dienyl)oxy)methyl)propyl-2-yl)amino)propionic acid (TLAPA), (l-(2,3-linolenic acid propoxy)-2-(linolenic acid)-(7V,Λ / -dimethyl)-propyl-3-amine) (TLinDMA), 3-((1,3-bis(((9Z.12Z.15Z)-octadec-9,12,15-trienyl)oxy)-2-((((9Z.12Z.15E)-octadec-9,12,15-trienyl)oxy)methyl)propyl-2-yl)amino)propionic acid (TLLAPA),N-(α-trimethylammonium acetylated)-di-dodecyl-D-glutamic acid (TMAG), 3-((1,3-bis(((Z)-octadec-9-enyl)oxy)-2-((((Z)-octadec-9-enyl)oxy)methyl)prop-2-yl)amino)propionic acid (TOAPA), 3-((1,3-bis(stearyloxy)-2-((stearyloxy)methyl)prop-2-yl)amino)propionic acid (TSAPA), 1,N19-bis((16E,18E,20E,22E)-17,21-dimethyl-15-sideoxy-23-(2,6,6-trimethylcyclohex-1-en-1-yl)-4,7,10-trioxa-14-azatrioctane -16,18,20,22-tetraen-1-yl)-4,7,10,13,16-pentaenodecane-1,19-diaminoamine (VA-PEG-VA), 2,2-dilinolenoyl-4-dimethylaminoethyl-[1,3]-dioxane (XTC), disclosed in Non-Patent Literature 11 (YSK05), 1,2-di-γ-linolenoyloxy-N,N-dimethylaminopropane (γ-DLenDMA), aD-tocopherol hemisuccinyl, bis(octadecanoic-9,12-dienoic acid)(9Z,9,Z,12Z,12,Z)-2-((2-(((3-(dimethylamino)propoxy)carbonyl)oxy)tetradecyloxy)oxy)prop-1,3 Dioctanoic acid 2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienyl)oxy)prop-1,3-diester, dioctanoic acid 2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dienyl)oxy)prop-1,3-diester, dioctanoic acid 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester, bis(decanoic acid) 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester, bis(decanoic acid) 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester, dioctanoic acid 2-(10-dodecyl-3-ethyl-8,14-di-side-oxy-7,9,13-trioxa-3-azaeicosano-20-yl)prop-1,3-diester, (9Z,9′Z)bis-tetradec-9-enoic acid 2-(((4-(dimethylamino)butyryl)oxy)methyl)-2-((octyloxy)methyl)prop-1,3-diester, bis(octadec-9,12-dienoic acid)(9Z,9′Z,12Z,12′Z)-2-(((1-(cyclopropylmethyl)piperidin-4-carbonyl)oxy)methyl)prop-1,3-diester,bis(decanoic acid)((2-(((1-isopropylpiperidin-4-carbonyl)oxy)methyl)-1,4-epoxyphenyl)bis(oxy))bis(octyl-8,1-diyl) ester, 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester of dodecanoic acid, 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester of dodecanoic acid, 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester of dodecanoic acid, 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)prop-1,3-diester of di-tetradecanoic acid, 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl) Di(dimethylamino)propoxy)prop-1,3-diester, di-tetradecanoic acid 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecoxy)prop-1,3-diester, di-tetradecanoic acid 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecoxy)prop-1,3-diester, dioleoic acid (Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecoxy)prop-1,3-diester, bis(octadec-9,12,15-trienoic acid)(9Z,9,Z,12Z,12,Z,15Z,15,Z)-2-((4-(((3-(dimethylamino)propoxy) propyl-1,3-diester, bis(octadecyl-9,12-dienoic acid)(9Z,9,Z,12Z,12,Z)-2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propyl-1,3-diester, bis(octadecyl-9,12-dienoic acid)(9Z,9,Z,12Z,12,Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propyl-1,3-diester, N,N,N-trimethyl-5-sideoxy-5-(3-((3-pentyloctyl)oxy)-2,2-bis(((3-pentyloctyl)oxy)methyl)propoxy )Pentane-1-ammonium iodide, 3-pentyloctanoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis(((3-pentyloctyl)oxy)methyl)propyl ester, 3-dimethylaminopropyl carbonate (9Z,12Z)-octadec-19,22-dien-11-ester, (9Z,9′Z)bis-tetradec-9-enoic acid 2-(((N,N-dimethyl-β-propylamino)oxy]methyl}-2-[(octyloxy)methyl)propyl-1,3-diester, 8-dimethyldioctanoic acid 0,O1-(2-(7-dodecyl-14-methyl-3,9-disidel-2,4,8,10-tetraoxa-14-azapentadecanyl)propyl-1,3-diyl)ester,Dioctanoic acid 8-dimethylO,O1-(2-((((1-methylpyrrolidin-3-carbonyl)oxy)methyl)prop-1,3-diyl) ester, 8-methyloctanoic acid 1-(3-(((6,6-bis((2-propylpentyl)oxy)hexyl)oxy)-2-(((1,4-dimethylpiperidin-4-carbonyl)oxy)methyl)propyl) ester, octadecanoic acid (9Z,12Z)-5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecanyl ester, octanoic acid 5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecanyl ester, 10-octyl sebacate 1-(3-((6,6-bis((2-propylpentyl)) 1,4-Dimethylpiperidin-4-carbonyl)oxy)methyl)propyl) ester, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyl ester, 8-methyloctanoic acid 1-(16-(((4,4-bis(octyloxy)butyryl)oxy)methyl)-9-dodecyl-2-methyl-7,13-disideoxy-6,8,12,14-tetraoxa-2-azaheptadecane-17-yl) ester, (9Z,12Z)-octadec-9,12-dienoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis(((9Z)-tetradec-9-enyloxy)methyl)propyl ester, 3 -Pentyloctanoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis(((3-pentyloctyl)oxy)methyl)propyl ester, bis(octadecanoic acid)(9Z,9'Z,12Z,12'Z)-2-(((3-(diethylamino)propionic)oxy)methyl)propyl-1,3-diester, bis(decanoic acid)((2-(((4-(dimethylamino)butyryl)oxy)methyl)-1,4-epoxyphenyl)bis(oxy))bis(octyl-8,1-diyl) ester, 8-methyloctanoic acid 1-(3-((4,4-bis(octyl)butyryl)oxy)-2-(((1-methylpyrrolidone-3-carbonyl)oxy)methyl)propyl) ester, 1- Methylpyrrolidone-3-carboxylic acid 3-((4,4-bis(octoxy)butyryl)oxy)-2-((palmitoyloxy)methyl)propyl ester, 1-methylpyrrolidone-3-carboxylic acid 3-((4,4-bis(octoxy)butyryl)oxy)-2-((tetradecanoyloxy)methyl)propyl ester, 9-pentyltetradecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octoxy)tridecyl ester, 1-methylpyrrolidone-3-carboxylic acid 3-((4,4-bis(octoxy)butyryl)oxy)-2-((dodecanoyloxy)methyl)propyl ester, 9-pentyltetradecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl ester,7-Hexyltridecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octyloxy)tridecanoic acid 2-(5-(3-((1-methylpyrrolidin-3-carbonyl)oxy)-2-((tetradecanoyloxy)methyl)propoxy)-5-sideoxypentyl)prop-1,3-diester, 5-heptyldodecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octyloxy)tridecanoic acid 2-(5-(3-((1-methylpyrrolidin-3-carbonyl)oxy)-2-((palmitoyloxy)methyl)propoxy)-5-sideoxypentyl)prop-1,3-diester, 5-heptyldodecanoic acid 3 -(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl ester, bis(6,6-bis(octyloxy)hexanoic acid)2-(((1-methylpyrrolidin-3-carbonyl)oxy)methyl)prop-1,3-diester, octadecanoic acid (9Z,12Z)-3-(((3-dimethylamino)propoxy)carbonyl)oxy)-13-(octyloxy)tridecyl ester, (9Z)-octadecanoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis(((9Z)-tetradecano-9-enyloxy)methyl)propyl ester, dioctanoic acid 2-(10-dodecyl-3-ethyl-8,14-disidel-7, 9,13-trioxa-3-azanonadecan-19-yl)prop-1,3-diester, bis(decanoic acid)((2-(((1-methylpiperidin-4-carbonyl)oxy)methyl)-1,4-epoxyphenyl)bis(oxy))bis(octyl-8,1-diyl)ester, bis(4,4-bis(octyloxy)butanoic acid)2-(((3-(dimethylamino)propionic)oxy)methyl)prop-1,3-diester, octadecanoic acid (9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propionic)oxy)eicosano-11,14-dien-1-yl)oxy)ethyl ester, bis(4,4-bis(octyloxy)butanoic acid)2-(( (1,3-Dimethylpyrrolidone-3-carbonyl)oxy)methyl)prop-1,3-diester, succinic acid (13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-ester heptadecane-9-ester, 3-((3-ethyl-10-((9Z,12Z)-octadec-9,12-dien-1-yl)-8,15-disideoxy-7,9,14-trioxa-3-azaheptadecane-17-yl)dithioalkyl)propionate 2,2-bis(heptyloxy)ethyl ester, bis(4,4-bis(octyloxy)butyric acid)2-(((1-methylpyrrolidone-3-carbonyl)oxy)methyl)prop-1,3-diester,10-Octyl sebacate 1-(3-((1,3-dimethylpyrrolidin-3-carbonyl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienoxy)methyl)propyl) ester, 2,2-bis(heptoxy)acetic acid (13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-ester, 2,2-bis(heptoxy)acetic acid (13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa-13,16-dien-1-ester, Acetic acid (20,23R)-2-methyl-9-[(9Z,12Z)-octadec-9,12-dien] -1-yl]-7-sideoxy-6,8,11-trioxa-2-aza-nonadecan-20-en-23-ester, 3-(dimethylamino)propyl carbonate (11Z,14Z)-1-{[(9Z,12R)-12-hydroxyoctadec-9-en-1-yl] ester, 3-(dimethylamino)propionic acid (12Z,15Z)-1-((((9Z,12Z)-octadec-9,12-dien-1-yloxy)carbonyl)oxy)tetradec-12,15-dien-3-ester, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(dimethylamino)propyl)aminomethyl) propyl ester, 9-pentyltetradecanoic acid (12Z,15Z)-3-((4-(dimethylamino)butyryl)oxy)tetradec-12,15-diene-1-ester, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((((1,2,2,6,6-pentamethylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl ester, 7-hexyltridecanoic acid (12Z,15Z)-3-((4-(dimethylamino)butyryl)oxy)tetradec-12,15-diene-1-ester, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)tetradec-12,15-diene-1-ester, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)tetradec-12,15-diene-1-ester (Octooxy)butyryl)oxy)-2-(((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)methyl)propyl ester, 5-heptyldodecanoic acid (12Z,15Z)-3-((4-(dimethylamino)butyryl)oxy)tetradec-12,15-dien-1-ester, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octooxy)butyryl)oxy)-2-(((((1-ethylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl ester, 3-octylundecanoic acid (12Z,15Z)-3-((4-(dimethylamino)butyryl)oxy)tetradec-12,15-dien-1-ester, formate,(9Z)-Hexadec-9-enoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis((((9Z)-tetradec-9-enyloxy)methyl)propyl ester, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((((1-methylazacyclobutane-3-yl)oxy)carbonyl)oxy)methyl)propyl ester, octadec-9,12-dienoic acid (9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propyryl)oxy)tetradec-12,15-dien-1-ester, 4,4-bis((2-ethylhexyl)oxy)butyric acid 2-(((3 -(diethylamino)propoxy)carbonyl)oxy)tetradecane ester, octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl ester, octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((((1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)methyl)propyl ester, octadecano-9,12-dienoic acid (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecanyl ester, octadecano-9,12-di Acenoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(4-methylpiperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl ester, 3-(dimethylamino)propyl triacontadecane-11-yl carbonate triacontadecane-11-ol, octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl ester, octadec-9,12-dienoic acid (9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecanyl ester, 4-((diethyl) 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, 3-((dimethylamino)methyl)benzoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester, (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl ester,1-Methylpiperidin-3-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester, 1-methylpiperidin-4-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester, 1,4-dimethylpiperidin-4-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester, 1,4-dimethylpiperidin-4-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester (9Z)-Octadecano-9,12-dienoxy)methyl)propyl ester, (9Z)-hexadecano-9-enoic acid 3-((4-(dimethylamino)butyryl)oxy)-2,2-bis((((9Z)-tetradecano-9-dienoxy)methyl)propyl ester, dioctanoic acid 2-(10-dodecyl-3-ethyl-8,14-disidel-7,9,13-trioxa-3-azahexadecano-16-yl)propyl-1,3-diester, bis(octadecano-9,12-dienoic acid)(9Z,9'Z, 12Z,12'Z)-2-(((4-(piperidin-1-yl)butyryl)oxy)methyl)propyl-1,3-diester, 4-methylmorpholino-2-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)propyl ester, 1-methylpyrrolidone-2-carboxylic acid (2R)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z) )-Octadecano-9,12-dienoxy)methyl)propyl ester, 1-methylpyrrolidone-2-carboxylic acid (2S)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z)-octadecano-9,12-dienoxy)methyl)propyl ester, bis(octadecano-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(( (9Z,12Z)-Octadecano-9,12-dienoxy)methyl)propyl-1,3-diester, Octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester, 1-(cyclopropylmethyl)piperidin-4-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((9Z,12Z) (9Z,12Z)-Octadecano-9,12-dienoxy)methyl)propyl ester, 1-isopropylpiperidin-4-carboxylic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((3-(dimethylamino)propyl)oxy)methyl)propyl ester, Octadecano-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((3-(dimethylamino)propyl)oxy)methyl)propyl ester,4-(dimethylamino)butyl carbonate (6Z,9Z,26Z,29Z)-pentadecano-6,9,26,29-tetraen-18-ester, (9Z)-tetradecano-9-enoic acid 3-((6-(dimethylamino)hexyl)oxy)-2,2-bis(((9Z)-tetradecano-9-enoxy)methyl)propyl ester, 3-(dimethylamino)propyl carbonate 2,5-bis(((9Z,12Z)-octadecano-9,12-dienoxy)benzyl ester, bis(octadecano-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((4-(pyrrolidin-1-yl)butyryl)oxy)methyl)propyl-1,3-diester, 5-heptyldodecanoic acid 3-(((3-(dimethyl) Pentadecyl acetate (7R,9Z)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)octadecano-9-en-7-ester, 9-pentyltetradecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl acetate, octadecano-9,12-dienoic acid (9Z,12Z)-3-((6,6-bis(octyloxy)hexyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, 7-hexyltridecano-6-enoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl acetate, octadecano-9,12-dienoic acid (9Z,12Z)-3-(2,2- Bis(heptyl)ethoxy)-2-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)propyl ester, 3-octyldodecano-2-enoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, octadecano-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((5-heptyldodecyl)oxy)methyl)propyl ester, 3-octylundecanoic acid 3-(((3-dimethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, octadecano-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)oxy)-2-(((9-pentyltetradecanoic acid) Alkyl)oxy)methyl)propyl ester, diacetic acid (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl)oxy)tripentadecano-9,26-diene-7,29-diester, 8,8-bis((2-propylpentyl)oxy)octanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanoyl ester, octadecanoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)oxy)2-(((7-hexyltridecyl)oxy)methyl)propyl ester, 8,8-bis((2-propylpentyl)oxy)octanoic acid 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecanoyl ester,Octadecto-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-octylundecyloxy)oxy)methyl)propyl ester, 8,8-bis((2-propylpentyl)oxy)octanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, 8,8-dibutoxyoctanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, (9Z)-tetradec-9-enoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis(((9Z)-tetradec-9-enyloxy)methyl)propyl ester, 3-(dimethylamino)propyl carbonate (6Z,9Z, 26Z,29Z)-Tridecano-6,9,26,29-tetraen-18-ester, 2,5-bis(((9Z,12Z)-octadecano-9,12-dien-1-yloxy)benzyl ester, bis(octadecano-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((3-(4-methylpiperyl-1-yl)propoxy)methyl)prop-1,3-diester, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, 3-(dimethylamino)propyloctadecano-11-ester of carbonate, 2,4-bis(((9Z,12Z)butanoic acid) -Octadecano-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((2-heptyloylundecyl)oxy)methyl)propyl ester, 6,6-bis((2-ethylhexyl)oxy)hexanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanoyl ester, bis(2-heptyloylundecanoic acid)2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propyl-1,3-diester, 6,6-bis(hexyl)hexanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanoyl ester, 4-(dimethylamino)butyric acid 4-methyl 2,5-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)benzyl ester, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester of 6,6-bis(octyloxy)hexanoic acid, 4-methyl-2,5-bis((9Z,12Z)-octadec-9,12-dienoxy)benzylmethyl carbonate 4-(dimethylamino)butyl ester, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl ester of 4,4-bis((2-propylpentyl)oxy)butyric acid, 2-(12-dodecyl-3-ethyl-8,14-di-side-oxy-7,9,13-trioxa-3-azaoctadecane-18-yl)prop-1,3-diester of dioctanoic acid,Dioctanoic acid 2-(5-sidekto-5-((3-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecanyl)prop-1,3-diester, 4-methyl-2,5-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)benzylmethylcarbonate 3-(dimethylamino)propyl, 4,4-bis((2-propylpentyl)oxy)butyrate 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecanyl, dioctanoic acid 2-(11-dodecyl-3-ethyl-9,15-sidekto-8,10,14-trioxa-3-azanonadecan-19-yl)prop-1,3-diester, dioctanoic acid 2-(10-dodecyl)prop-1,3-diester Alkyl-3-ethyl-8,15-di-side-oxy-7,9,14-trioxa-3-azanonadecan-19-yl)prop-1,3-diester, dioctanoic acid 2-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-side-oxypentyl)prop-1,3-diester, dioctanoic acid 2-(5-((4-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)hexadecyl)oxy)-5-side-oxypentyl)prop-1,3-diester, dioctanoic acid 2-(5-((4-(((((R)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-side-oxypentyl)prop-1,3-diester Dioctanoic acid 2-(5-((4-(((((S)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-sideoxypentyl)prop-1,3-diester, dioctanoic acid 2-(5-sideoxy-5-((4-((((S)-pyrrolidin-2-carbonyl)oxy)hexadecyl)oxy)pentyl)prop-1,3-diester, dioctanoic acid 2-(5-((4-((1,3-dimethylpyrrolidin-3-carbonyl)oxy)hexadecyl)oxy)-5-sideoxypentyl)prop-1,3-diester, dioctanoic acid 2-(5-((4-((1,4-dimethylpiperidin-4-carbonyl)oxy)hexadecyl)oxy)-5-sideoxypentyl)prop-1,3-diester, 4,4-bis(octyloxy)butyl(3-(diethylamino)propyl)pentadecano-1,3-diester, 4,4-bis((2-propylpentyl)oxy)butyric acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanoate, bis(decanoic acid)((2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-1,4-epenylphenyl)bis(oxy))bis(octyl-8,1-diyl)ester, 5-(((3-(diethylamino)propoxy)carbonyl)oxy)heptadecanoic acid 4,4-bis(octyloxy)butyl ester, octanoic acid 6-((6,6-bis(octyloxy)hexyl)oxy)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexyl ester,6,6-Di(octyloxy)hexanoic acid (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecano-12,15-dien-1-ester, 6,6-Di(octyloxy)hexanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl ester, 6,6-Di(octyloxy)hexanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl ester, 5-(4,6-diheptyl-1,3-dialkyl-2-yl)valerate 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, 6,6-Di(octyloxy)hexanoic acid 3-((5-(diethylamino)pentyl)oxy)pentadecanyl ester, 1,4-dimethyl Piperidin-4-carboxylic acid 1-((6,6-bis(octyloxy)hexyl)oxy)pentadecanyl-3-ester, 6,6-bis(octyloxy)hexanoic acid 3-((3-(1-methylpiperidin-4-yl)propoxy)oxy)pentadecanyl ester, 1,3-dimethylpyrrolidin-3-carboxylic acid 1-((6,6-bis(octyloxy)hexyl)oxy)pentadecanyl-3-ester, 4,4-bis((2-ethylhexyl)oxy)butyric acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, bis(8-(octyloxy)octanoic acid)2-(((1,3-dimethylpyrrolidin-3-carbonyl)oxy)methyl)prop-1,3-diester, bis(decanoic acid)((2-((((3-(dimethylamino)propoxy)propoxy) 1,4-Phenyl(oxy)-1,4-dimethyl ... (((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, 6,6-bis((2-propylpentyl)oxy)hexanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, 6,6-bis(octyl)hexanoic acid 3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecanyl ester, 6,6-bis(octyl)hexanoic acid 3-(((3-morpholinylpropoxy)carbonyl)oxy)pentadecanyl ester, 6,6-bis(octyl)hexanoic acid 3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecanyl ester, 6,6-bis(octyl)hexanoic acid 3-(((3-(4-methylpiperidin-1-yl)propoxy)carbonyl)oxy)pentadecanyl ester,4,4-Di(octyloxy)butyric acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, (9Z,9′Z)bis-tetradec-9-enoic acid 2-(((4-(dimethylamino)butyryl)oxy)methyl)-2-((dodecyloxy)methyl)prop-1,3-diester, bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((4-(dimethylamino)butyryl)oxy)methyl)prop-1,3-diester, 6,6-Di(octyloxy)hexanoic acid 3-(((4-(diethylamino)butoxy)carbonyl)oxy)pentadecanyl ester, 6,6-Di(octyloxy)hexanoic acid 3-(((3-(piperyl-1-yl)propoxy)carbonyl)oxy) Pentadecyl ester, 6,6-bis(octyloxy)hexanoic acid 3-(((3-piperidin-1-yl)propoxy)carbonyl)oxy)pentadecanyl ester, 4,4-bis(octyloxy)butyric acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecanyl ester, bis(octadecyl-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(9-dodecyl-2-methyl-7,12-disideloxy-6,8,13-trioxa-2-azatetradecane-14-yl)prop-1,3-diester, octadecyl-9,12-dienoic acid(9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadecyl-9,12-dienyloxy)ethyl)-8, 13-Dioxa-7,9-dioxa-3,14-diazahexadecane-16-ester, dioctanoic acid 2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl)oxy)prop-1,3-diester, dioctanoic acid 2-(9-dodecyl-2-methyl-7,13-dioxa-6,8,12-trioxa-2-azanonadecan-19-yl)prop-1,3-diester, (9Z,9′Z)bis-tetradecanoic acid 2-((decyloxy)methyl)-2-(((4-(dimethylamino)butyryl)oxy)methyl)prop-1,3-diester, bis(octadecanoic acid) (9Z,9′Z,12Z,12′Z)-2-(((3-morpholino) Propyl(oxy)methyl)prop-1,3-diester, 3-(dimethylamino)propyl carbonate (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-ester, 4-(dimethylamino)butyric acid 2,5-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)benzyl ester, dioctanoic acid 2-(10-dodecyl-3-ethyl-8,14-disideoxy-7,9,13-trioxa-3-azaoctadecane-18-yl)prop-1,3-diester, bis(octadec-9,12-dienoic acid) (9Z,9'Z,12Z,12'Z)-2-(((1,3-dimethylpyrrolidin-3-carbonyl)oxy)methyl)prop-1,3-diester,Trioctanoic acid ((5-((dimethylamino)methyl)phenyl-1,2,3-triyl)tri(oxy))tri(dec-10,1-diyl) ester, 9-dioctyldiadenosine O',O-(((5-(((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(prop-3,1-diyl)) ester, Octadecto-9,12-dienoic acid (9Z,12Z)-3-(3-((dimethylamino)methyl)-5-(3-(((3-octylundecylacryl)oxy)propoxy)phenoxy)propyl ester, bis(decanoic acid)((((5-(((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(prop-3,1-diyl))bis(oxy))bis(4-sideoxybut-4,1-diyl) ester Dioctanoic acid (R)-4-(3-((R)-3,4-bis(octyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)but-1,2-diester, dioctanoic acid (S)-4-(3-((S)-3,4-bis(octyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)but-1,2-diester, dioctanoic acid (R)-4-(3-((S)-3,4-bis(octyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)but-1,2-diester, tetraoctanoic acid 4,4'-((5-((dimethylamino)methyl)-1,3-epoxy)bis(but-1,2-diyl)ester, 6,6'-((5-((dimethylamino)methyl)) -1,3-Einylphenyl)bis(oxy))dihexanoic acid di-dodecyl ester, 5,5'-((5-(((dimethylamino)methyl)-1,3-Einylphenyl)bis(oxy))divalerate di((9Z,12Z)-octadec-9,12-dien-1-yl) ester, bis(decanoic acid)(((5-((dimethylamino)methyl)-1,3-Einylphenyl)bis(methylene))bis(oxy))bis(6-sideoxyhex-6,1-diyl) ester, bis(methylene)bis(8-(octyloxy)octanoic acid)(5-((dimethylamino)methyl)-1,3-Einylphenyl) ester, bis(methylene)bis(10-(octyloxy)decanoic acid)(5-((dimethylamino)methyl)-1,3-Einylphenyl) ester, dioctanoic acid (((5 -((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene))bis(oxy))bis(6-sideoxyhexyl-6,1-diyl) ester, bis(decanoic acid)(((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene))bis(oxy))bis(8-sideoxyoctyl-8,1-diyl) ester, bis(octadecanoic acid)(9Z,9'Z,12Z,12'Z)-(((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene))bis(oxy))bis(4-sideoxybut-4,1-diyl) ester, 8-dinonyldioctanoic acid O',O-((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene)) ester,bis(10-(octyloxy)decyl)disuccinic acid 0,0'-((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene)) ester, bis((9Z,12Z)-octadec-9,12-dien-1-yl)disuccinic acid 0,0'-((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene)) ester, bis(methylene)bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-(5-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-1,3-epoxyphenyl) ester, octadec-9,12-dienoic acid(9Z,12Z)-4-(3-((dimethylamino)propoxy)carbonyl)oxy)methyl)-1,3-epoxyphenyl) ester, octadec-9,12-dienoic acid(9Z,12Z)-4-(3-((dimethylamino)propoxy)carbonyl)propoxyphenyl)methyl)-1,3-epoxyphenyl) ester, octadec-9,12-dienoic acid(9Z,12Z)-4-(3-((dimethylamino)propoxyphenyl ... Dioleoyl (Z)-5-(4-(dimethylamino)methyl)butoxy)phenoxy)butyl ester, bis(octadecano-9,12,15-trienoic acid)(9Z,9'Z,12Z,12'Z,15Z,15'Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, ditetradecanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, dioleoic acid (Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, dodecanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)-1 3-Ethylphenyl)bis(oxy))bis(hexyl-6,1-diyl) ester, bis(octadecano-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((((5-((diethylamino)methyl)-1,3-epylphenyl)bis(oxy))bis(ethyl-2,1-diyl))bis(oxy))bis(ethyl-2,1-diyl) ester, 8,8'-((5-(((dimethylamino)methyl)-1,3-epylphenyl)bis(oxy))dioctanoic acid didecyl ester, bis(3-octylundecanoic acid)((5-(((dimethylamino)methyl)-1,3-epylphenyl)bis(oxy))bis(prop-3,1-diyl) ester, bis(octadecano-9,12-dienoic acid)(9Z .9'Z.12Z.12'Z)-((5-((diethylamino)methyl-2-methyl-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, dodecanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(octyl-8,1-diyl) ester, bis(decanoic acid)((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(octyl-8,1-diyl) ester, bis(octadecanoic acid)(9Z.9'Z.12Z.12'Z)-((5-((dimethylamino)methyl-2-methyl-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester,bis(dodecano-8-enoic acid) (8Z,8'Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(hexyl-6,1-diyl) ester, bis(octadecano-9,12-dienoic acid) (9Z,9'Z,12Z, 12'Z)-((5-((3-hydroxyazyrazine-1-yl)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, dioctanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(hexyl-6,1-diyl) ester, bis(decanoic acid)((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(hexyl-6,1-diyl) ester, bis(octadecanoic-9,12-dienoic acid)(9Z.9'Z.12Z.12'Z)-((5-((dimethylamino)methyl-1,3-epoxy)bis(oxy))bis(octanoic-8 ,1-diyl) ester, bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(hexane-6,1-diyl) ester, dihexanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(decane-10,1-diyl) ester, dioctanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(decane-10,1-diyl) ester, dioctanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(octane-8,1) -diyl) ester, dihexanoic acid ((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(octyl-8,1-diyl) ester, bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(ethyl-2,1-diyl) ester, bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(propyl-3,1-diyl) ester, bis(octadec-9,12-dienoic acid) (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(methylene)di-tetrazanoic acid (5-((dimethylamino)methyl)-1,3-epoxy) ester, bis(methylene)bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-(5-((dimethylamino)methyl)-1,3-epoxy) ester, 3-(dimethylamino)propionic acid (2,6-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)pyridin-4-yl) methyl ester,bis(octadecanoic acid)(9Z,9'Z,12Z,12'Z)-5-(((3-(dimethylamino)propionic acid)oxy)methyl)-1,3-benzene ester, 1-(3,5-bis((9Z,12Z)-octadecano-9,12-dien-1-yloxy)phenyl)-N,N-dimethylmethylamine, 3-(dimethylamino)propionic acid 3,5-bis((9Z,12Z)-octadecano-9,12-dien-1-yloxy)benzene ester, 1-(3,5-bis(4,4-bis(octyloxy)butoxy)phenyl)-N,N-dimethylmethylamine, tetraoctanoic acid ( (((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl))bis(oxy))bis(propane-3,2,1-triyl) ester, bis(decanoic acid)((5-(((4-(dimethylamino)butyryl)oxy)methyl)-1,3-epoxy)bis(oxy))bis(octyl-8,1-diyl) ester, bis(decanoic acid)((5-(((3-(dimethylamino)propane)oxy)methyl)-1,3-epoxy)bis(oxy))bis(octyl-8,1-diyl) ester, bis(octadecanoic acid))(9Z,9'Z, 12Z,12'Z)-((5-(3-morpholinylpropyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(octadec-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-(3-(dimethylamino)propyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(octadec-9, 12-Dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-(3-(piperidin-1-yl)propyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(methylene)bis(9-pentyltetradecanoic acid)(5-((dimethylamino)methyl)-1,3-epoxy) ester, bis(methylene)bis(7-hexyltridecanoic acid)(5-((dimethyl) (amino)methyl)-1,3-epoxy) ester, bis(methylene)bis(5-heptyldodecanoic acid)(5-((dimethylamino)methyl)-1,3-epoxy) ester, bis(3-octylundecanoic acid)((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(5-heptyldodecanoic acid)((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(9-pentyltetradecanoic acid)((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester, bis(7-hexyltridecanoic acid)((5-((dimethylamino)methyl)-1,3-epoxy)bis(oxy))bis(but-4,1-diyl) ester,bis(octadecano-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-(pyrrolidin-1-ylmethyl)-1,3-epoxyphenyl)bis(oxy))bis(but-4,1-diyl) ester, tetraoctanoic acid(((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(oxy))bis(methylene))bis(propyl-3,2,1-triyl) ester, tetraoctanoic acid(((5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(oxy))bis(but-4,1-diyl))bis(propyl-3,2,1-triyl) ester, octadecano-9,12-dienoic acid(9Z.12Z)-4-(3-((dimethylamino)methyl-5-(4-((3-octyldecyl) 1,0'-(5-((dimethylamino)methyl)-1,3-epoxyphenyl)bis(methylene))disuccinate bis(1,3-bis(octyloxy)prop-2-yl) ester, bis(methylene)bis(6-(((nonoxy)carbonyl)oxy)hexanoic acid)(5-((dimethylamino)methyl)-1,3-epoxyphenyl) ester, 2-(3-(4-(5-((dimethylamino)methyl)-2-methyl-3-((9Z,12Z)-octadec-9,12-dien-1-yloxy)phenoxy)butoxy)-3-epoxypropyl)prop-1,3-diester of dihexanoic acid, 3-octylundecanoic acid 3-((dimethylamino)methyl)-5- (((8-(octyloxy)octyl)oxy)methyl)benzene, trioctanoic acid ((5-((diethylamino)methyl)phenyl-1,2,3-triyl)tri(oxy))tri(dec-10,1-diyl) ester, 1-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-N,N-dimethylmethylamine, N'-methyl-N',N”,N”-tris((2E.6E)-3.7.11-trimethyldodec-2.6.10-trien-1-ylpropyl-1,3-diamine, 4-(dimethylamino)butyric acid 1,17-bis(2-((2-pentylcyclopropyl)methyl)cyclopropyl)heptadecane-9-ester, (7Z)-17-{[4-(dimethylamino)butyric acid [Z]-6-(2-(dimethylamino)-3-(octadec-9-en-1-yloxy)propoxy)methyl hexanoate, 2-(di-dodecylamino)-1-(4-(N-(2-(dinonylamino)ethyl)-N-dodecylglycyl)piperidin-1-yl)aceto-1-one, hexanoate 3-((3-(1-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propoxy)piperidin-4-yl)propyl)(nonyl)amino)propyl hexanoate, hexanoate 3-((3-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propoxy)piperidin-1-yl)-3-sideoxypropyl)(nonyl)amino)propyl hexanoate,3-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4-(3-(dinonylamino)propyl)piperidin-1-yl)prop-1-one, 4-((3-(1-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propionyl)piperidin-4-yl)propyl)(nonyl)amino)pentyl butyrate, 4-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)pentyl butyrate, 4-(((1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)methyl)(nonyl)amino)pentyl butyrate, 4-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)methyl)(nonyl)amino)pentyl butyrate, 4-((2-(1-(N-(2-(dinonylamino)ethyl) 4-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl(nonyl)amino)butyrate pentyl ester, 2-(di-dodecylamino)-1-(4-(N-(2-(dinonylamino)) 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(3-(2-(dinonylamino)ethyl)piperidin-1-yl)ethyl-1-one, 4,4'-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-1-) Dipentyl(2-(4-(N-Nonyl-N-(2-(nonyl(4-sideoxy-4-(pentoxy)butyl)amino)ethyl)glycine)piperyl-1-yl)-2-sideoxyethyl)amino)pentyl)butyrate, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1 -(3-((dinonylamino)methyl)pyrrolidin-1-yl)acet-1-one, 2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)-1-(4-(dinonylglycyl)piperazine-1-yl)acet-1-one, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(3-(2-(dinonylamino)ethyl) 4-((3-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propionic)piperidin-1-yl)-3-sideoxypropyl)(nonyl)amino)pentyl butyrate, hexanoic acid 3-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl) 5-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl(nonyl)amino)valerate butyl ester, 2-((2-(di-dodecylamino)ethyl(nonyl)amino)-1-(4-(dinonylglycyl)piperidin-1-yl)acetoone,6-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)propyl hexanoate, 7-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)heptarate, 8-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)octanoate, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-1-yl)-2-ethyroxyl)(nonyl)amino)propyl hexanoate, 5-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-1-yl)-2-sideoxyethyl)(nonyl)amino)butyl valerate, 6-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-2-sideoxyethyl)(nonyl)amino)hexyl) Acetic acid ester, ethyl heptanoate of 7-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-1-yl)2-sideoxyethyl)(nonyl)amino)heptanoate, ethyl heptanoate of 3-(dinonylamino)-1-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propionic acid)piperyl-1-yl)propionic acid -1-one, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4-(di-tetradecylglycyl)piperidin-1-yl)ethyl-1-one, 2-(dinonylamino)-1-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl)piperidin-1-yl)ethyl-1-one, 2-(dinonylamino)-1-(4-(N-(2-(dinonylamino)ethyl)-N-dodecylglycyl)piperidin-1-yl)ethyl-1-one, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4-(2-(dinonylamino)ethyl)piperidin-1-yl)ethyl-1-one, 8-((2-(4- Methyl octanoate of (dinonylglycyl)piperyl-1-yl)-2-electro-ethyl)(2-((8-methoxy-8-electro-octyl)(nonyl)amino)ethyl)amino)octanoate, methyl octanoate of 8-((2-(dinonylamino)ethyl)(2-(4-(dinonylglycyl)piperyl-1-yl)-2-electro-ethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoate, methyl octanoate of 4-((2-(4-(dinonylglycyl)piperyl-1-yl)-2-electro-ethyl)(nonyl)amino)octanoate,8-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-1-yl)-2-sideoxyethyl)(nonyl)amino)methyl octanoate, 2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)-1-(5-(dinonylglycyl)-2,5-diazabiscyclic[2.2]. 1] Hept-2-yl)ethyl-1-one, 2-(dinonylamino)-1-(5-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)ethyl-1-one, N1,N1,N2-tris((9Z,12Z)-octadec-9,12-dien-1-yl)- N2-(2-(piperazine-1-yl)ethyl)ethyl-1,2-diamine, N1,N1,N2-tris((Z)-octadec-9-en-1-yl)-N2-(2-(piperazine-1-yl)ethyl)ethyl-1,2-diamine, 2-(dinonylamino)-l-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl) Piperazine-1-yl)ethyl-1-one, N1,N1,N2-tri-dodecyl-N2-(2-(piperazine-1-yl)ethyl)ethyl-1,2-diamine, N1,N1,N2-trinonyl-N2-(2-(piperazine-1-yl)ethyl)ethyl-1,2-diamine, N1,N1,N2-trihexyl-N2-(2-(piperazine-1-yl)ethyl)ethyl N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl)-N1,N2,N2-tris((9Z,12Z)-octadec-9,12-dien-1-yl)ethyl-1,2-diamine, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl)-N1,N2,N2-tris((Z)-octadec-9-dien-1-yl)ethyl-1,2-diamine, N1-(2-(4-(2-(di-tetradecylamino)ethyl)piperazine-1-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, N1-(2-(4-(2-(di-tetradecylamino)ethyl)piperazine-1-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl) N1,N2,N2-tri-tetradecylethyl-1,2-diamine, N1-(2-(4-(2-(dinonylamino)ethyl)piperyl-1-ethyl)N1,N2,N2-tri-tetradecylethyl-1,2-diamine, 2-(di-dodecylamino)-l-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperyl-1-ethyl)1-one, N1-(2-(4-(2-(di(9Z,12Z)-octadec-9,12-dien-1-yl)amino)ethyl)piperyl-1-ethyl)N1,N2,N2-tri-dodecylethyl-1,2-diamine,N1-(2-(4-(2-(di((Z)-octadec-9-en-1-yl)amino)ethyl)piperyl-1-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1,N1,N2-tri-dodecyl-N2-(2-(4-(2-(dodecyl((9Z,12Z)-octadec-9,12-dien-1-yl)amino)ethyl)piperyl-1-yl)ethyl-1,2-diamine, N1-(2-(4-(2-(di-tetradecylamino)ethyl)piperyl-1-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(di((Z)-dodecyl- 6-en-l-yl)amino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N-tri-dodecylethyl-1,2-diamine, (Z)-N1-(2-(4-(2-dodec-6-en-l-yl(dodecyl)amino)ethyl)piperazine-l-yl)ethyl)-N,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(dinonylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(dioctylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-( 2-(4-(2-(dihexylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(di-tetradecylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-nonylethyl-1,2-diamine, 2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)-l-(4-(2-(di-dodecylamino)ethyl)piperazine-l-yl)ethyl-1-one, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-nonylethyl-1,2-diamine, N1-(2-(4-) (2-(dinonylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-trinonylethyl-1,2-diamine, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-trihexylethyl-1,2-diamine, 12,12'-((2-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperazine-l-yl)ethyl)ureidyl)di-dodecanoate dimethyl ester, 12-((2-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperazine-l-yl)ethyl)(dodecyl)amino)methyl dodecanoate,6,6'-((2-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperazine-1-yl)ethyl)ureadiyl)dipentyl hexanoate, 6-((2-(4-(2-((2-(di-tetradecylamino)ethyl)(tetradecyl)amino)ethyl)piperazine-1-yl)ethyl)(dodecyl)amino)pentyl hexanoate, 6-((2-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperazine-1-yl)ethyl)(dodecyl)amino)pentyl hexanoate, 2-(di-dodecylamino)-1-(4-(N-(2-(di-dodecylamino)ethyl)-N-dodecylglycine) 2-(di-dodecylamino)-1-(4-(N-(2-(di-dodecylamino)ethyl)-N-nonylglycyl)piperyl-1-(ethyl)-1-one, 2-(di-dodecylamino)-N-(2-(4-(2-(di-dodecylamino)ethyl)piperyl-1-(ethyl) )-N-dodecylacetamide, (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid)((2-((3,S',4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)ureidyl)bis(ethyl-2,1-diyl) ester, 2-amino-N,N-di-hexadecyl-3-(1H-imidazol-5-yl) (2-amino-N,N-di-hexadecyl-3-(1H-imidazol-5-yl)propionic acid, (9Z)-19-[2-(dimethylamino)ethyl]heptadec-9-enoic acid methyl ester, 8-(2-{9-[2-(dimethylamino)ethyl]octadecyl}cyclopropyl)octanoic acid methyl ester, (9Z)-19-[2-(dimethylamino)ethyl]heptadecyl-9-enoic acid methyl ester, 8-(2-{ll-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid ethyl ester, 8-(2-{ll-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid ethyl ester, 3-(((2-(dimethylamino)ethoxy)carbonyl)amino)glutaric acid di((9Z) ,12Z)-octadec-9,12-dien-l-yl) ester, 6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)heptaate, 8-(2-{ll-[(dimethylamino)methyl]nonadecanyl}cyclopropyl)octanoate, 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)pentyl)octanoate, 8-(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)octanoate, 8-(2-{9-[(dimethylamino)methyl]pentadecanyl}cyclopropyl)octanoate,3-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(tetradecyl)amino)propyl decanoate, 6-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-l-yl)-2-ephthylethyl)(tetradecyl)amino)heptyl hexanoate, 8-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, 8-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-2-ephthylethyl)(tetradecyl)amino)pentyl octanoate, 8-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate Ethyl heptadecanoyl (cyclopropyl)octanoate, methyl 6-(2-(8-(2-(dimethylamino)-3-(nonoxy)propoxy)octyl)cyclopropyl)hexanoate, methyl (9Z)-21-(dimethylamino)heptadec-9-enoate, methyl (9Z)-21-{[4-(dimethylamino)butyryl]oxy}heptadec-9-enoate, methyl (2R)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]dodecane-2-amine, (15Z,18Z)-N,N-dimethyltetradec-15,18-dien-5-amine, 8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)octyl) Ethyl hexanoate, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-1-yl)-2-sideoxyethyl)(tetradecyl)amino)propyl ester, ethyl 4-(2-{11-[(dimethylamino)methyl]eicosyl}cyclopropyl)butyrate, ethyl 8-(2-{7-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, ethyl hexanoate, 3-((3-(1-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propyl)piperidin-4-yl)propyl)(nonyl)amino)propyl ester, ethyl 6-(2-{9-[(dimethylamino)methyl]pentadecanyl}cyclopropyl)hexanoate, 3-((3- (4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propionyl)piperidin-1-yl)-3-sideoxypropyl)(nonyl)amino)propyl ester, 6-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)hexanoate, 3-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4-(3-(dinonylamino)propyl)piperidin-1-yl)prop-1-one, 4-((3-(l-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propionyl)piperidin-4-yl)propyl)(nonyl)amino)pentyl butyrate, 6-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)hexanoate,4-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)pentyl butyrate, 6-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)hexanoate, 4-(((l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)methyl)(nonyl)amino)pentyl butyrate, (9Z)-21-[(dimethylamino)methyl]heptadec-9-enoate, 4-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)pentyl butyrate, (9Z)-21-[(di-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)pentyl butyrate, (9Z)-21-[(di-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)pentyl butyrate, (9Z)-21-[(di-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)pentyl butyrate, (9Z)-21-[(di-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)pentyl butyrate, [Methylamino]methyl[2-octadec-9-enoic acid ethyl ester, (9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoic acid)((2-((3,S',4R)-3,4-dihydroxypyrrolidin-1-yl)acetylated)ureidindiyl)bis(ethyl-2,1-diyl)ester, 4-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl)(nonyl)amino)pentyl butyrate ester, (9Z)-21-[(dimethylamino)methyl]nonadecano-9-enoic acid ethyl ester, 6-(2-(8-(2-(dimethylamino)-3-(heptoxy)propoxy)octyl)cyclopropyl)hexanoate methyl ester, (9Z)-21-{[4-( Methyl dimethylamino)butyryl]oxy}octadec-9-enoate, (9Z)-21-(dimethylamino)octadec-9-enoate, 2-(di-dodecylamino)-1-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-1-yl)ethyl-1-, (2S)-NN-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]non-2-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-dien-10-amine, (9Z)-21-[(dimethylamino)methyl]tridec-9-enoate, (9Z)-19-[(dimethylamino)methyl]pentadecan-9 1-Acenoic acid ethyl ester, (9Z)-19-[(dimethylamino)methyl]hexadec-9-acenoic acid ethyl ester, (9Z)-19-[(dimethylamino)methyl]heptadec-9-acenoic acid ethyl ester, (9Z)-19-[(dimethylamino)methyl]hexadec-9-acenoic acid ethyl ester, (5Z)-17-[(dimethylamino)methyl]hexadec-5-acenoic acid ethyl ester, (9Z)-17-[(dimethylamino)methyl]hexadec-9-acenoic acid ethyl ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(3-(2-(dinonylamino)ethyl)piperidin-l-yl)ethyl-1-one, (7Z)-17-[(dimethylamino)methyl]tridec-7-acenoic acid ethyl ester4,4'-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-2-sideoxyethyl)ureidyl)dipentyl dibutyrate, 4-(nonyl(2-(4-(N-nonyl-N-(2-(nonyl(4-sideoxy-4-(pentoxy)butyl)amino)ethyl)glycyl)piperazine-l-yl) (7Z)-17-[(dimethylamino)methyl]tetraco-7-enoic acid ethyl ester, (7Z)-17-[(dimethylamino)methyl]pentaco-7-enoic acid ethyl ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(3-((dinonylamino)methyl)pyrrolidine) -1-yl)ethyl-1-one, trans-3-[(3}7-dimethyloctyl)oxy]-1-methyl-4~[(9Z,12Z)-octadec-9512-dien-1-oxypyrrolidine, methyl 6-(2-(8-(2-(dimethylamino)-3-(hexoxy)propoxy)octyl)cyclopropyl)hexanoate, methyl (9Z)-21-{[4-(dimethylamino)butyryl]oxy}nonadecano-9-enoic acid, methyl (9Z)-21-(dimethylamino)nonadecano-9-enoic acid, methyl (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-oxy]tridecane-2-amine, (15Z,18Z)-N,N-dimethyltetracosyl C15,18-diene-7-amine, (7Z)-17-[(dimethylamino)methyl]hexadecano-7-enoate ethyl ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(3-(2-(dinonylamino)ethyl)pyrrolidin-1-yl)acetoone, 6-(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)hexanoate methyl 10-(2-{7-[(dimethylamino)methyl]hexadecyl}cyclopropyl)decanoate methyl 8-(2-{ll-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoate methyl 8-(2-{ll-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate methyl 8-(2-{ll-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate methyl 8- Methyl octanoate ([(dimethylamino)methyl]nonadecanyl}cyclopropyl)octanoate, methyl octanoate (8-(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)octanoate, methyl 4-((3-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propionyl)piperidin-l-yl)-3-sideoxypropyl)(nonyl)amino)pentadecanate, methyl octanoate (8-(2-{9-[(dimethylamino)methyl]pentadecanyl}cyclopropyl)octanoate, methyl 8-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, methyl hexanoate (3-(2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)propyl)8-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-(dimethylamino)-3-((6-(((2-octylcyclopropyl)methoxy)-6-sideoxyhexyl)oxy)propoxy)octanoic acid methyl ester, 5-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)valerate butyl ester, trans-1-methyl-3-[(12Z)-octadec-12-en-1-yloxy]-4-(octoxy)pyrrolidine, (9Z)-21-{[4-(dimethylamino)butyryl]oxy}tridec-9-enoic acid methyl ester, (9Z)-21-(dimethylamino)tridec- 9-Ocenoic acid methyl ester, 2-((2-(di-dodecylamino)ethyl)(nonyl)amino)-1-(4-(dinonylglycyl)piperazine-1-yl)ethyl-1-one Step 1: N-(2-(di-dodecylamino)ethyl)-N-nonylglycine methyl ester, 1-((2R,3S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-disideloxy-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran methanesulfonate, (Z)-16-(3-(decyloxy)-2-(dimethylamino)propoxy)hexadec-7-enoic acid methyl ester, (2S)-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]non-2 -Amine, (14Z,17Z)-N,N-dimethyltetracarbo-14,17-diene-6-amine, 6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)propyl hexanoate, methyl heptaate of 7-(2-(dimethylamino)-3-((6-(((2-octylcyclopropyl)methoxy)-6-sideoxyhexyl)oxy)propoxy)heptanoate, methyl heptaate of (7Z)-19-[(dimethylamino)methyl]octadec-7-enoate, methyl heptadec-11-enoate of (HZ)-19-[(dimethylamino)methyl]octadec-11-enoate, methyl heptadecyl)7-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl) Ethyl heptahydrate (2-octylcyclopropyl) hexanoate, methyl 6-(2-(dimethylamino)-3-((5-methoxy-5-sideoxypentyl)oxy)propoxy)hexanoate, methyl 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl(nonyl)amino)octanoate, methyl (9Z)-21-[(dimethylamino)methyl]heptadec-9-enoate, methyl 6-(2-(dimethylamino)-3-(4-methoxy-4-sideoxybutoxy)propoxy)hexanoate, methyl (9Z)-21-[(dimethylamino)methyl]heptadec-9-enoate,3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-2-sideoxyethyl)(nonyl)amino)propyl hexanoate, (Z)-8-(2-(dimethylamino)-3-((6-sideoxy-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)octanoate methyl ester, (9Z)-21-[(dimethylamino)methyl]nonadecano-9-enoate methyl ester, 5-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-2-sideoxyethyl)(nonyl)amino)valerate butyl ester, (Z)-7-(2-(dimethylamino)-3-( Methyl heptanoate (6-side-oxy-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)heptaate, propyl hexanoate ((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-l-yl)-2-side-oxyethyl)(nonyl)amino)hexanoate, methyl (9Z)-21-[(dimethylamino)methyl]tetrate-9-enoate, methyl (Z)-6-(2-(dimethylamino)-3-((5-methoxy-5-side-oxypentyl)oxy)propoxy)hexanoate undec-2-en-1-ester, methyl (9Z)-19-[(dimethylamino)methyl]pentadecanoate-9-enoate, methyl (7-(2-(4-(N-(2-(di-4-ylamino)ethyl)-N-nonylglycyl)piperyl-l-yl)-2-side-oxyethyl)(nonyl)amino)hexanoate, methyl (9Z)-21-[(dimethylamino)methyl]tetrate-9-enoate, methyl (7-(2-(4-(N-(2-(di-4-ylamino)methyl)-N-nonylglycyl)propoxy)hexanoate, methyl (9Z)-21-[(dimethylamino)methyl]tetrate-9-enoate, methyl (7-(2-(4-(N-(2-(di-4-ylamino)methyl)-N-nonylglycyl)proptaate, methyl (9Z)-21-[(dimethylamino)methyl)-N-nonylamino)-hexanoate, methyl (9Z)-21-[(dimethylamino)methyl]tetrate-9-enoate, methyl (9Z)-21-[(dimethylamino)methyl]tetrate-9-enoate, methyl (9Z)-2 (Nonylamino)ethyl)-N-nonylglycyl)piperazine-1-yl)-2-sideoxyethyl)(nonyl)amino)heptanate ethyl ester, (Z)-6-(2-(dimethylamino)-3-(4-methoxy-4-sideoxybutoxy)propoxy)hexanoate undec-2-en-1-ester, 6-(2-(dimethylamino)-3-((6-((2-octylcyclopropyl)methoxy)-6-sideoxyhexyl)oxy)propoxy)hexanoate methyl ester, (9Z)-19-[(dimethylamino)methyl]hexadec-9-enoate methyl ester, 3-(dinonylamino)-1-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propoxy)piperazine-1-yl)prop- 1-keto, (9Z)-19-[(dimethylamino)methyl]heptadec-9-enoic acid methyl ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(4-(di-tetradecylglycyl)piperazine-1-yl)ethyl-1-keto, (Z)-6-(2-(dimethylamino)-3-((6-sideoxy-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)methyl hexanoate, 8-(2-(dimethylamino)-3-((8-(2-(6-methoxy-6-sideoxyhexyl)cyclopropyl)octyl)oxy)propoxy)methyl octanoate, 8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)methyl octanoate,2-(dinonylamino)-l-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl)piperidin-1-yl)ethyl-1-one, trans-l-methyl-3-[(9Z)-octadec-9-en-l-yloxy]-4-(octoxy)pyrrolidine, (9Z)-19-{[4-(dimethylamino)butyryl]oxy}pentadecano-9-enoic acid methyl ester, (9Z)-19-(dimethylamino)pentadecano-9-enoic acid methyl ester, (Z)-16-(2-(dimethylamino)-3-(nonoxy)propoxy)hexadec-7-enoic acid methyl ester, (2S)-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]decyl -2-amine, (12Z,15Z)-N,N-dimethyltetradec-12,15-diene-4-amine, methyl 7-(2-(dimethylamino)-3-((8-(2-(6-methoxy-6-epoxyhexyl)cyclopropyl)octyl)oxy)propoxy)heptanoate, methyl (9Z)-19-[(dimethylamino)methyl]octadec-9-enoate, methyl 2-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(4-(2-(dinonylamino)ethyl)piperidin-l-yl)aceto-1-one, 8-((2-(4-(dinonylglycyl)piperidin-l-yl)-2-epoxyethyl)(2-((8-methoxy-8-epoxyoctyl)(nonyl) Methyl octanoate, methyl 6-(2-(8-(2-(dimethylamino)-3-((5-methoxy-5-ephthyl)oxy)propoxy)octyl)cyclopropyl)hexanoate, ethyl 8-{2-[11-(dimethylamino)heptadecyl]cyclopropyl}octanoate, methyl 8-((2-(dinonylamino)ethyl)(2-(4-(dinonylglycyl)piperyl-1-yl)-2-ephthyl)amino)octanoate, methyl 6-(2-(8-(2-(dimethylamino)-3-(4-methoxy-4-ephthylbutoxy)propoxy)octyl)cyclopropyl)hexanoate, ethyl 8-{2-[11-(dimethylamino)octadecyl]cyclopropyl}octanoate 8-((2-((2-(4-(dinonylglycyl)piperazine-l-yl)-2-ephthylethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoate methyl ester, 8-{2-[11-(dimethylamino)nonadecanyl]cyclopropyl}octanoate ethyl ester, (Z)-16-(2-(dimethylamino)-3-((8-methoxy-8-ephthyl)oxy)propoxy)hexadec-7-enoate methyl ester, 4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-2-ephthylethyl)(nonyl)amino)pentyl butyrate, 8-{2-[11-(dimethylamino)eicosyl]cyclopropyl}octanoate ethyl ester,(Z)-16-(2-(dimethylamino)-3-((7-methoxy-7-heptaoxy)oxy)propoxy)hexadec-7-enoic acid methyl ester, 8-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-1-yl)-2-heptaoxyethyl)(nonyl)amino)octanoic acid methyl ester, 8-{2-[9-(dimethylamino)pentadecanyl]cyclopropyl}octanoic acid ethyl ester, (Z)-16-(2-(dimethylamino)-3-((5-methoxy-5-heptaoxy)oxy)propoxy)hexadec-7-enoic acid methyl ester, (11E,20Z,23Z)-N,N-dimethylnonadecanocarbo-11,20,23-trien-10-amine, N, N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, 8-{2-[9-(dimethylamino)hexadecyl]cyclopropyl} ethyl octanoate, 2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)-l-(5-(dinonylglycyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)ethyl-1-one, (Z)-16-(2-(dimethylamino)-3-(4-methoxy-4-butoxy)propoxy)hexadec-7-enoic acid methyl ester, 6-(2-(8-(2-(dimethylamino)-3-((6-methoxy-6-butoxy)propoxy)octyl)cyclopropyl)hexanoate methyl ester, 8-{ 2-[9-(dimethylamino)heptadecyl]cyclopropyl}ethyl octanoate, 2-(dinonylamino)-l-(5-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)ethyl-1-one, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N1,N1,N2-tris((9Z,12Z)-octadec-9,12-dien-l-yl)-N2-(2-(piperyl-l-yl)ethyl)ethyl-1,2-diamine, 8-{2-[9-(dimethylamino)octadecyl]cyclopropyl}ethyl octanoate, 1-[(1R,2S)-2-heptylcyclopropyl]-N, N-Dimethyloctadecano-9-amine, (Z)-16-(2-(dimethylamino)-3-((6-methoxy-6-hexyloxy)oxy)propoxy)hexadec-7-enoic acid methyl ester, N1,N1,N2-tris((Z)-octadecano-9-en-l-yl)-N2-(2-(piperyl-l-yl)ethyl)ethyl-1,2-diamine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecano-1-amine, 8-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)octanoic acid methyl ester, 4-{2-[11-(dimethylamino)eicosyl]cyclopropyl}ethyl butyrate,trans-1-methyl-3-[((9Z,12Z)-octadec-9,12-dienyl)oxy]-4-octoxy-pyrrolidone, (9Z)-19-(dimethylamino)hexadec-9-enoic acid methyl ester, (9Z)-19-{[4-(dimethylamino)butyryl]oxy}hexadec-9-enoic acid methyl ester, (Z)-16-(2-(dimethylamino)-3-(heptoxy)propoxy)hexadec-7-enoic acid methyl ester, (2R)-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]dodecane-2-amine, (13Z,16Z)-N,N-dimethyldocosahexadec-13,16-dien-5-amine, N,N-dimethyl-1 -[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, 7-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)heptanoate, 8-{2-[7-(dimethylamino)hexadecyl]cyclopropyl}octanoate, 2-(di-dodecylamino)-N-dodecyl-N-(2-(piperazinyl-l-yl)ethyl)acetamide, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N1-(2-(piperazinyl-l-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, 6-(2-(dimethylamino) Methyl hexanoate (-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)hexanoate), ethyl hexanoate (6-{2-[9-(dimethylamino)pentadecanyl]cyclopropyl}hexanoate), N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecanyl-10-amine), NN1,N2-tri-dodecyl-N2-(2-(piperyl-1-yl)ethyl)ethyl-1,2-diamine), methyl pentanoate (5-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)valerate), ethyl hexanoate (6-{2-[9-(dimethylamino)hexadecyl] Ethyl cyclopropyl hexanoate, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]eicosano-10-amine, NNN2-trinonyl-N2-(2-(piperyl-l-yl)ethyl)ethyl-1,2-diamine, methyl 4-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)butyrate, ethyl 6-{2-[9-(dimethylamino)heptadecyl]cyclopropyl}hexanoate, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecano-10-amine, N1,N1,N2-trihexyl-N2-(2-(piperyl-l-yl)ethyl)ethyl-1,2-diamine,8-(2-(dimethylamino)-3-((9Z,12Z)-octadec-9,12-dien-1-yloxy)propoxy)methyl octanoate, 6-{2-[9-(dimethylamino)octadecyl]cyclopropyl}hexanoate, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl)N1,N2,N2-tri-((9Z,12Z)-octadec-9,12-dien-1-yl)ethyl-1,2-diamine, 7-(2-(dimethylamino)-3-((9Z,12Z)-octadec-9,12-dien-1-yloxy)propoxy)methyl heptanoate, (9Z)-21-(dimethylamino)heptadec-9-enoate, 1-[(1 [S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecano-10-amine, 9-{[4-(dimethylamino)butyryl]oxy}octadecanoic acid 1-methyl ester 18-[(2Z)-non-2-en-1-] ester, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-((Z)-octadec-9-en-l-yl)ethyl-1,2-diamine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecano-8-amine, 6-(2-(dimethylamino)-3-((9Z,12Z)-octadec-9,12-dien-1-yloxy)propoxy)methyl hexanoate, (9Z)-21- (Dimethylamino)octadec-9-enoic acid ethyl ester, (9Z)-19-{[4-(dimethylamino)butyryl]oxy}heptadec-9-enoic acid dimethyl ester, N1-(2-(4-(2-(di-tetradecylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, 5-(2-(dimethylamino)-3-((9Z,12Z)-octadec-9,l2-dien-l-yloxy)propoxy)valerate methyl valerate, 8-{[4-(dimethylamino)butyryl]oxy}-15-(2-octylcyclopropyl)pentadecanoate ethyl ester, (9Z)-21-(dimethylamino)octadec-9-enoic acid ethyl ester, (13Z,16Z)- N,N-Dimethyl-3-nonyldocosa-13,16-dien-1-amine, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, 9-{[4-(dimethylamino)butyryl]oxy}-16-(2-octylcyclopropyl)hexadecanoate, methyl 4-(2-(dimethylamino)-3-((9Z,12Z)-octadec-9,12-dien-1-yloxy)propoxy)butyrate, (9Z)-21-(dimethylamino)trianedec-9-enoate, (12Z,15Z)-N,N-dimethyl-2-nonyldocosa-12,15-dien-1-amine,8-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)methyl octanoate, (9Z)-19-(dimethylamino)pentadecano-9-enoic acid ethyl ester, (18Z,21Z)-8-{[4-(dimethylamino)butyryl]oxy}heptadec-18,21-dienoic acid ethyl ester, (16Z)-N,N-dimethylpentadecano-16-en-8-amine, (9Z)-19-{[4-(dimethylamino)butyryl]oxy}heptadeco-18,21-dienoic acid ethyl ester, (9Z)-19-(dimethylamino)heptadec-9-enoic acid methyl ester, 2-(di-dodecylamino)-1-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperazine-1-yl)aceto-1-one, (Z)-16-(2-(dimethylamino)-3-(hexyloxy)propoxy)hexadec-7-enoic acid methyl ester, (2S)-1-[ [(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]dodecane-2-amine, (16Z,19Z)-N,N-dimethylpentadeca-16,19-dien-8-amine, N1-(2-(4-(2-(dinonylamino)ethyl)piperyl-1-yl)ethyl)-N1,N2^V2-tri-tetradecylethyl-1,2-diamine, 7-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy] Methyl propoxy)heptanoate, (19Z,22Z)-9-{[4-(dimethylamino)butyryl]oxy}octadec-19,22-dienoic acid methyl ester, (9Z)-19-(dimethylamino)hexadec-9-enoic acid ethyl ester, (22Z)-N,N-dimethyltridec-22-en-10-amine, N1-(2-(4-(2-(di((Z)-octadec-9-en-l-yl)amino)ethyl)piperidin-l-yl)ethyl)- !^^-Tri-dodecyl ethyl-1,2-diamine, methyl 5-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)valerate, (9Z)-19-(dimethylamino)heptadec-9-enoic acid ethyl ester, methyl 12-{[4-(dimethylamino)butyryl]oxy}eicosanoic acid (2-butylcyclopropyl) ester, (20Z)-N,N-dimethylhexadecano-20-en-10-amine, N1,N1,N2-tri-dodecyl- N2-(2-(4-(2-(dodecyl((9Z,12Z)-octadec-9,12-dienyl)amino)ethyl)piperazine-1-yl)ethyl)ethyl-1,2-diamine, methyl 4-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)butyrate, ethyl (9Z)-19-(dimethylamino)octadec-9-enoate, methyl 8-{[4-(dimethylamino)butyryl]oxy}heptadecanoate(2-octylcyclopropyl)methyl,(24Z)-N,N-Dimethyltriscarb-24-en-10-amine, N1-(2-(4-(2-(di-tetradecylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, (5Z)-17-(dimethylamino)hexadec-5-enoic acid ethyl ester, (Z)-8-(2-(dimethylamino)-3-(octadec-9-en-l-yloxy)propoxy)octanoic acid methyl ester, (2Z)-12-{[4-(dimethylamino)butyryl]oxy}tetaroyl Heptyl-2-en-l-ester of alkyl alkylate, (17Z)-N,N-dimethyl-2,9-neocodecano-17-en-10-amine, N1-(2-(4-(2-(di((Z)-dodec-6-en-l-yl)amino)ethyl)piperazine-l-yl)ethyl)N1,N2,N2-tri-dodecylethyl-1,2-diamine, (9Z)-17-(dimethylamino)hexadec-9-enoic acid ethyl ester, (Z)-7-(2-(dimethylamino)-3-(octadec-9-en-1-yloxy)propoxy)methyl heptanoate, (2Z)- 8-{[4-(dimethylamino)butyryl]oxy}heptadecanoic acid undec-2-en-l-ester, (14Z)-N,N-dimethyl-2-nonadecano-14-en-10-amine, (7Z)-17-(dimethylamino)tetrate-7-enoic acid ethyl ester, (Z)-N1-(2-(4-(2-dodec-6-en-l-yl(dodecyl)amino)ethyl)piperazine-N!^^-tri-dodecylethyl-1,2-diamine, (Z)-5-(2-(dimethylamino) methyl valerate (-3-(octadec-9-en-1-yloxy)propoxy)valerate, methyl 10-{[4-(dimethylamino)butyryl]oxy}nonadecanoate (2-hexylcyclopropyl) methyl ester, (15Z)-N,N-dimethylheptadec-15-en-10-amine, (7Z)-17-(dimethylamino)tetradec-7-enoate ethyl ester, (Z)-4-(2-(dimethylamino)-3-(octadec-9-en-1-yloxy)propoxy)butyrate, (2Z)- 10-{[4-(dimethylamino)butyryl]oxy}nonadecanoate non-2-en-l-ester, (20Z)-N,N-dimethylheptadec-20-en-10-amine, N1-(2-(4-(2-(dioctylamino)ethyl)piperazine-l-yl)ethyl)-N1,N2^V2-tri-dodecylethyl-1,2-diamine, 6-(2-(dimethylamino)-3 Methyl hexanoate (8-(2-octylcyclopropyl)octyl)oxy)propoxy)hexanoate, ethyl hexanoate (6-[2-(9-{[4-(dimethylamino)butyryl]oxy}octadecyl)cyclopropyl]hexanoate, ethyl (7Z)-17-(dimethylamino)pentadecano-7-enoate, ethyl (1-[(11Z,14Z)-1-nonyleicosico-11,14-dien-1-yl]pyrrolidine,(7Z)-17-(dimethylamino)hexadecyl-7-enoic acid ethyl ester, (20Z,23Z)-N-ethyl-N-methylhexadecyl-20,23-dien-10-amine, N,N-dimethylhexadecane-10-amine, methyl 6-{2-[ll-(dimethylamino)eicosyl]cyclopropyl}hexanoate, methyl 6-[2-(ll-{[4-(dimethylamino)butyryl]oxy}eicosyl)cyclopropyl]hexanoate, methyl 6-(3-(decoxy)-2-(dimethylamino)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, methyl 8-{2-[9-(dimethylamino)octadecyl]cyclopropyl}octanoate, methyl 8-[2-(9-{[4-(dimethylamino) Methyl octanoate, methyl 7-(2-(8-(2-(dimethylamino)-3-(octoxy)propoxy)octyl)cyclopropyl)heptanoate, representative of 8-((2-hydroxyethyl)(tetradecyl)amino)octanoate heptadecan-9-ester, 2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)-l-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)acetoone, (2S)-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]undecane-2-amine, (17Z,20Z)-N,N-dimethylhexadecane-17,20-dien-9-amine, ( 18Z)-4-(dimethylamino)butyrate heptadec-18-en-10-ester, (2S)-1-({6-[3B))-cholesterol-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-en-1-yloxy]prop-2-amine, methyl decanoate of 10-{2-[7-(dimethylamino)hexadecyl]cyclopropyl}decanoate, methyl decanoate of 10-[2-(7-{[4-(dimethylamino)butyryl]oxy}hexadecyl)cyclopropyl]decanoate, (2S)-N,N-dimethyl-1-({8-[(lR,2R)-2-{[(lS,2S)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)tetyl Alkyl-2-amine, methyl 6-(2-(dimethylamino)-3-(nonoxy)propoxy)hexanoate (2-octylcyclopropyl) ester, (19Z,22Z)-N,N-dimethyloctadec-19,22-diene-7-amine, dinonylglycine 4-((N-(2-(dinonylamino)ethyl)-N-nonylglycyl)oxy)pentan-2-ester, 3-hydroxybut-2-yl N-(2-(dinonylamino)ethyl)-N-nonyl, 8,8'-(26,28-dimethyl-11,24,30,43-tetraoxa-10,25,29,44-tetraoxa-19,35-diazapentatriane-19,35-diyl)dioctanoate di(heptadecan-9-yl) ester,8,8'-(26,27-dimethyl-11,24,29,42-tetraoxa-10,25,28,43-tetraoxa-19,34-diazapentadodecane-19,34-diyl)dioctanoate di(heptadecane-9-yl) ester, 8,8'-(11,24,29,42-tetraoxa-10,25,28,43-tetraoxa-19,34-diazapentadodecane-19,34-diyl)dioctanoate di(heptadecane-9-yl) ester, 8,8'-((piperazine-1,4-diylbis(5-oxapran-5,1-diyl))bis((8-(nonoxy)-8-sideoxyoctyl)ureadiyl))dioctanoate di(heptadecane-9-yl) ester, 15,18-dimethyl-9,2 4-bis(8-(nonoxy)-8-sideoxyoctyl)-14,19-disideoxy-9,15,18,24-tetraazatridodecanoic acid di(heptadecanoic-9-yl) ester, 15,19-dimethyl-9,25-bis(8-(nonoxy)-8-sideoxyoctyl)-14,20-disideoxy-9,15,19,25-tetraazatridodecanoic acid di(heptadecanoic-9-yl) ester, 15,18-diethyl-9,24-bis(8-(nonoxy)-8-sideoxyoctyl)-14,19-disideoxy-9,15,18,24-tetraazatridodecanoic acid di(heptadecanoic-9-yl) ester, N,N-dimethyl-3-{[(9Z,12Z)-octadec-9,12} -dien-1-yloxy]methyl}dodecane-1-amine, 8-[2-(ll-{[4-(dimethylamino)butyryl]oxy}octadecyl)cyclopropyl]octanoate methyl ester, 8-{2-[ll-(dimethylamino)heptadecyl]cyclopropyl}octanoate methyl ester (compound 18), 8-((2-hydroxyethyl)(8-(nonoxy)-8-sideoxyoctyl)amino)octanoate heptadecane-9-ester, 6-(2-(dimethylamino)-3-(heptoxy)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, (17Z)-N,N-dimethylhexadecane-17-en-9-amine, N1-(2-(4-(2-(di-dodecylamino)ethyl)piperazine-1-yl)ethyl)-N1^V2, N2-Trihexylethyl-1,2-diamine, N,N-Dimethyl-2-{[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]methyl}undecane-1-amine, 8-{2-[11-(dimethylamino)octadecyl]cyclopropyl}methyl octanoate, 6-(2-(dimethylamino)-3-(hexoxy)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, (18Z)-N,N-Dimethylheptadecane-18-en-10-amine, 2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl ester of tetradecanoate, 2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl ester of nonanoate, N-(2-(dinonylamino)ethyl)-N-nonylglycine tetradecane esterN-(2-(dinonylamino)ethyl)-N-nonylglycate, 4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)acetamino)butylvalerate, 1,1'-(piperazine-1,4-diyl)bis(5-(didecylamino)pentan-1-one), 2-((2-(dinonylamino)ethyl)(nonyl)amino)-N-tetradecylacetylamine, N-decyl-2-((2-(dinonylamino)ethyl)(nonyl)amino), N1-(3-(3-(dinonylamino)propoxy)propyl)-N1,N2,N2-tri-nonylethyl-1,2-diamine, N1-(2-(dinonylamino)ethyl)-N\N8,N8-trinonyloct-1,8-diamine, 8- [2-(ll-{[4-(dimethylamino)butyryl]oxy}nonadecanyl)cyclopropyl]octanoate methyl ester, 8-{2-[ll-(dimethylamino)nonadecanyl]cyclopropyl}octanoate methyl ester, (Z)-6-(3-(decoxy)-2-(dimethylamino)propoxy)hexanoate undec-2-en-l-ester, (2R,12Z,15Z)-N,N-dimethyl-1-(undecyloxy)tetradec-12,15-dien-2-amine, (21Z,24Z)-N,N-dimethyltridec-21,24-dien-9-amine, 2-(dinonylamino)-N-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N-methylacetylamino)butyl) -N-Methylacetamide, 7,10-dimethyl-13,16-dinonyl-6,11-dioxy-4-tetradecyl-4,7,10,13,16-pentazonopentadecyl ester, 2-(dinonylamino)-N-(2-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N-ethylacetamide)ethyl)-N-ethylacetamide, 2-(dinonylamino)-N-(3-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N-methylacetamide)propyl)-N-methylacetamide, 2-((2-(di((Z)-non-3-en-l-yl)amino)ethyl)((Z)-non-3-en-l-yl)amino)-N-(2-( 2-(dinonylamino)-N-methylacetamino)ethyl)-N-methylacetamide, 2-(dinonylamino)-N-(2-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl)acetamide, 8,1l-dimethyl-5,14,17-trinonyl-7,12-dioxy-5,8,11,14,17-pentazonohexadecanoate, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-N-methyl-N-(2-(methylamino)ethyl)acetamide, 2-(dinonylamino)-N-(2-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N-methylacetamino)ethyl)-N-methylacetamide,2-(dinonylamino)-N-methyl-N-(2-(methylamino)ethyl)acetamide, glycine 2-((N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)oxy)ethyl dinonyl ester, glycine 2-hydroxyethyl dinonyl ester, 8-[2-(11-{[4-(dimethylamino)butyryl]oxy}eicosyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[11-(dimethylamino)eicosyl]cyclopropyl]octanoic acid methyl ester [Cyclopropyl]octanoic acid methyl ester, (Z)-6-(2-(dimethylamino)-3-(nonoxy)propoxy)hexanoic acid undecano-2-en-1-ester, (2R,12Z,15Z)-1-(hexadecyloxy)-N,N-dimethyltetradecano-12,15-dien-2-amine, (22Z,25Z)-N,N-dimethyltetradecano-22,25-dien-10-amine, 4-(decylaminobutyric acid) L,L-(piperazine-l,4-diyl)bis(4-(didecylamino)but-l-one)tert-butyl ester, 5-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-5-heptylvalerate heptyl ester 5-(heptyloxy)-5-heptylvalerate, 5-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-5-heptylvalerate heptyl ester 5-(heptyloxy)-5-heptylvalerate Nonanoic acid (Z)-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-l-yl)-2-sideoxyethyl)(tetradecyl)amino)but-2-en-1-ester, nonanoic acid (Z)-4-hydroxybut-2-en-1-ester, decanoic acid (Z)-3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-l-yl)-2-sideoxyethyl)(tetradecyl-9-en-1- Methyl 8-[2-(9-{[4-(dimethylamino)butyryl]oxy}pentadecanyl)cyclopropyl]octanoate, methyl 8-{2-[9-(dimethylamino)pentadecanyl]cyclopropyl}octanoate, methyl 6-(2-(dimethylamino)-3-(heptoxy)propoxy)hexanoate (Z)-undec-2-en-l-ester, (2R,12Z,15Z)-1-(hexoxy)-N,N-dimethylamino)propoxy)hexanoate (Z)-undec-2-en-l-ester, (2R,12Z,15Z)-1-(hexyloxy)-N,N-dimethylamino)propoxy)propoxy)hexanoate (Z)-undecyl-2-en-l-ester, methyl 8-[2-(9-{[4-(dimethylamino)butyryl]oxy}pentadecanyl)cyclopropyl]propoxy)propoxy)hexanoate (Z)-undecyl-2-en-l-ester, methyl 8-[2-[9-(dimethylamino)pentadecanyl]cyclopropyl]propoxy)propoxy)hexanoate (Z)-undecyl-2-en-l-ester, methyl 8-[2R,12Z,15Z)-1-(hexyloxy)-N,N-dimethylamino)propoxy ... 2,15-dien-2-amine, (16Z,19Z)-N,N-dimethylpentacarbon-16,19-dien-6-amine, methyl 8-((2-(4-(N-(2-(di((Z)-non-3-en-l-yl)amino)ethyl)-N-((Z)-non-3-en-l-yl)glycinyl)piperazine-l-yl)-2-sideoxyethyl)(nonyl)amino)octanoate, tert-butyl 4-(nonylglycinyl)piperazine-1-carboxylate,(Z)-Dec-3-enoic acid 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-2-sideoxyethyl)(tetradecyl)amino)propyl ester, (Z)-dec-3-en-l-ol, 2-((2-(di((Z)-non-3-en-l-yl)amino)ethyl)((Z)-non-3-en-l-yl)amino)-l-(4-(dinonylglycyl)piperazine-1-yl)aceto-1-one(Z)-1- Nonyl-4-ene bromo, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-side-oxyethyl)(dodecyl)amino)propyl ester, tributyl dodecyl glycine, S-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-1-yl)-2-side-oxyethyl)(nonyl)amino)butyropentyl thiopentyl ester, 3-((2-(1-(N-(2-(dinonyl))ethyl)-N-nonylglycyl)piperazine-1-yl)-2-side-oxyethyl)(nonyl)amino)butyropentyl thiopentyl ester, 3-methylhexanoic acid 3-((2-(1-(N-(2-(dinonyl))ethyl)-(2-(dinonyl))-(2 ... (amino)ethyl)-N-nonylglycyl)piperidin-^yl)ethyl)(nonyl)amino)propyl ester, tributyl 4-(2-((3-((3-methylhexyl)oxy)propyl)(nonyl)amino)ethyl)piperidin-l-, hexanoic acid 3-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)-2-methylpropyl ester, 3-methylhexanoic acid 3-((2-(4-(N-(2-(dinonylamino)ethyl) (2-(2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-sideroethyl(nonyl)amino)propyl ester, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperyl-sideroethyl(nonyl)amino)-2-methylpropyl ester, methyl 8-[2-(9-{[4-(dimethylamino)butyryl]oxy}hexadecyl)cyclopropyl]octanoate, methyl 8-{2-[9-(dimethylamino)hexadecyl]cyclopropyl}octanoate, (Z)- 6-(2-(dimethylamino)-3-(hexoxy)propoxy)hexanoic acid undec-2-en-l-ester, (2R,12Z,15Z)-1-(decoxy)-N,N-dimethyltetradec-12,15-dien-2-amine, (17Z,20Z)-N,N-dimethylhexadec-17,20-dien-7-amine, 1-(dinonylglycyl)piperidine-4-carboxylic acid 2-((2-(dinonylamino) 4-(2-(2-(dinonylamino)ethyl)cyclohexane-1,4-dicarboxylic acid 1-(2-(dinonylamino)ethyl) ester, 2-(dinonylamino)ethanol-1-ol, 12-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)methyl dodecanoate,3-(2-((12-methoxy-12-sideoxydodecyl)(tetradecyl)amino)ethyl)pyrrolidin-l-carboxylic acid tributyl ester, 3-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)propyl ester, 3-(2-((3-(decyloxy)propyl)(tetradecyl)amino)ethyl)pyrrolidin-l-carboxylic acid tributyl ester, 6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)heptarate, 3-(2-((6-(heptoxy)-6-sideoxyheptarate) (tetradecyl)amino)ethyl)pyrrolidone-1-carboxylic acid tributyl ester, 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidone-3-yl)ethyl)(tetradecyl)amino)octanoic acid pentyl ester, 3-(2-(tetradecylamino)ethyl)pyrrolidone-1-carboxylic acid tributyl ester, 12-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)dodecanoic acid methyl ester, 3-(2-((12-methoxy-12-sideoxydodecyl)(tetradecyl)amino)ethyl)piperidin-1-carboxylic acid butyl ester, decanoic acid 3-((2-( l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)propyl ester, 6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)heptyl hexanoate, 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)pentyl octanoate, 6-((2-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperidin-l-yl)ethyl)(dodecyl)amino)pentyl hexanoate) 1: 6-Bromohexanoate methyl ester, 8-[2-(9-{[4-(dimethylamino)butyryl]oxy}heptadecyl)cyclopropyl]octanoate methyl ester, 8-{2-[9-(dimethylamino)heptadecyl]cyclopropyl}octanoate methyl ester, (2S,12Z,15Z)-N,N-dimethyl-1-(octoxy)tetradec-12,15-dien-2-amine, 6-(2-(dimethylamino)-3-(octoxy)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, (18Z,21Z)-N,N-dimethylheptadec-18,21-dien-8-amine, trans-1-methyl-3,4-bis(((Z)-hexadec-9-enoxy)methyl)pyrrolidine,(Z)-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl)-2-ethoxyethyl)(tetradecyl)amino)butyrate non-2-en-l-ester, trans-1-methyl-3,4-bis(((9Z,12Z)-octadec-9,12-dienyloxy)methyl)pyrrolidine, 12-((2-(4-(N-(2-( Dinonylamino(ethyl)-N-nonylglycyl)piperazine-1-yl)-2-epoxyethyl(tetradecyl)amino(methyl)dodecanoate, (7Z)-17-[2-(dimethylamino)ethyl]hexadec-7-enoic acid ethyl ester, trans-1-methyl-3,4-bis(((Z)-octadec-9-enoxy)methyl)pyrrolidine, 6-(2-{]11-^2-(dimethylamino) Methyl hexanoate (12-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)dodecanoate, methyl hexadecyl)cyclopropyl)decano ... Methyl heptaalkylcyclopropyl)octanoate, 2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl dinonyl ester, 4-(2-((dinonylglycinyl)oxy)ethyl)piperidin-1-carboxylic acid tributyl ester, 8-(2-{lLl-;2-(dimethylamino)ethyl]octadecyl}cyclopropyl)octanoate methyl ester, 8-(2-{l11- Methyl 2-(dimethylamino)ethyl]nonadecanyl}cyclopropyl)octanoate, l,-(piperazine-l,4-diyl)bis(2-(dinonylamino)ethyl-1-one), methyl 8-[2-{]11-^2-(dimethylamino)ethyl]eicosyl}cyclopropyl)octanoate, methyl 8-(2-{9-[2-(dimethylamino)ethyl]pentadecanyl}cyclopropyl)octanoate, (7Z)-19-{[4-(dimethylamino)butyryl]oxy}octadec-7-enoate, (7Z)-19-(dimethylamino)octadec-7-enoate, methyl cis-1-methyl Compound 11 contains: 3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-4-(octyloxy)pyrrolidone, 2-(di-dodecylamino)-l-(4-(N-(2-(di-dodecylamino)ethyl)-N-dodecylglycyl)piperyl-l-yl)acetoone, (Z)-6-(2-(dimethylamino)-3-(octyloxy)propoxy)hexanoic acid undec-2-en-l-ester, (2SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]dec-2-amine (compound 11).(19Z,22Z)-N,N-dimethyloctadec-19,22-dien-9-amine, methyl 8-(2-{9-[2-(dimethylamino)ethyl]hexadecyl}cyclopropyl)octanoate, methyl 5-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-sideoxyethyl)(nonyl)amino)pentyl methyl 8-(2-{9-[2-(dimethylamino)ethyl]heptadecyl}cyclopropyl)octanoate, methyl (7Z)-19-[2-(dimethylamino)ethyl]octadec-7-enoate, (Z)-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-l-yl) (2-Side-oxyethyl)(nonyl)amino)butyrate penta-2-en-l-ester, (1lZ)-19-[2-(dimethylamino)ethyl] octadec-11-enoic acid methyl ester, (9Z)-21-[2-(dimethylamino)ethyl] heptadec-9-enoic acid methyl ester, (9Z)-21-[2-(dimethylamino)ethyl] octadec-9-enoic acid methyl ester, (9Z)-21-[2-(dimethylamino)ethyl] nonadec-9-enoic acid methyl ester, glycine 2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl dinonyl ester, (9Z)-21-[2-(dimethylamino)ethyl] triadec-9-enoic acid methyl ester Esters, glycine (l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)methyl dinonyl ester, (9Z)-19-[2-(dimethylamino)ethyl]pentadecano-9-enoic acid methyl ester, (9Z)-19-[2-(dimethylamino)ethyl]hexadecadecano-9-enoic acid methyl ester, 6-(2-(8-(3-(decoxy)-2-(dimethylamino)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, (1lZ)-19-{[4-(dimethylamino)butyryl]oxy}octadecadecano-11-enoic acid methyl ester, (1lZ)-19-(dimethylamino)octadecadecano-11-enoic acid methyl ester, (2S)-N,N-dimethyl-1-[ (9Z,12Z)-octadec-9,12-dien-1-yloxy]dodecane-2-amine, (14Z,17Z)-N,N-dimethyltridecane-14,17-dien-4-amine, methyl di((9Z,12Z)-octadec-9,12-dienyl)amine, (9Z)-19-{[4-(dimethylamino)butyryl]oxy}octadecane-9-enoic acid methyl ester, (9Z)-19-(dimethylamino)octadecane-9-enoic acid methyl ester, (Z)-17-(2-(dimethylamino)-3-(octyloxy)propoxy)heptadecane-8-enoic acid methyl ester, (3R,4R)-3,4-bis((Z)-hexadecane-9-enyloxy)-1-methylpyrrolidone,(2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]undecane-2-amine, (20Z,23Z)-4-(dimethylamino)butyric acid 29octodec-20,23-dien-10-ester, (20Z,23Z)-N,N-dimethyl29octodec-20,23-dien-10-amine, 3-((6Z,9Z,28Z,31Z)-heptadec-6,9, 28,31-Tetraen-19-yloxy)-N,N-dimethylpropyl-1-amine, 3-((6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropyl-1-amine, (6Z,9Z,28Z,31Z)-4-(dimethylamino)butyric acid heptadec-6,9,28,31-tetraen-19-ester, 5-(dimethylamino)valerate (6Z,16 Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ester, (6Z,16Z)-5-(dimethylamino)valerate 12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ester, (6Z,16Z)-5-(dimethylamino)valerate 12-((Z)-dec-4-enyl)docosa-6,16-dien-11-ester, L-arginine-α-(2,3- (2,3-Dilauryloxy)propylamide, L-lysine-α-(2,3-dilauryloxy)propylamide, 2,3-dioleyloxypropylamide, 2,3-distearateyloxypropylamide, 2,3-dilauryloxypropylamide, flaxseed oil-methyl 4-(dimethylamino)propyl ether, flaxseed oil-methyl 4-(dimethylamino)butyl ether, and 2,2-flaxseed oil-4-(2-dimethylaminoethyl)-[1,3]-dioxane.

[0300] In some embodiments, at least one noncationic lipid comprises at least one phospholipid, at least one fusogenic lipid, at least one anionic lipid, at least one helper lipid, at least one neutral lipid, or any combination thereof. In some embodiments, the LNP may substantially lack at least one noncationic lipid. In some embodiments, the LNP may contain no amount of at least one noncationic lipid.

[0301] In some embodiments, at least one non-cationic lipid may be selected from, but is not limited to, at least one of the following: 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), DSPC, but each tail having 3 unsaturated double bonds (18:3 PC), acetylcarnitine (AC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), N-oleyl-SPM (C18:1), N-tetracosyl-SPM (C24:0), N-nervacyl-C (C24:l), aminomethyl]cholesterol (Cet-P), cholesterol hemisuccinate (CHEMS), cholesterol (Chol), cholesterol hemidodecanoic acid (Chol-C12), 12-cholesteroloxycarbonylaminododecanoic acid (Chol-C13N), cholesterol hemioxalate (Chol-C2), cholesterol hemimalonic acid (Chol-C3), N-(cholesterolyl-oxycarbonyl)glycine (Chol-C3N), cholesterol hemiglutarate (Chol-C5), cholesterol hemihexadiate (Chol-C6), cholesterol hemiheptanediate (Chol-C7), cholesterol hemicaprylate (Chol-C8), cardiolipin (Chol-C12), cholesterol hemicaprylate (Chol-C3), cholesterol hemicaprylate (Chol-C8), cardiolipin (Chol-C13N), cholesterol hemioxalate (Chol-C2), cholesterol hemimalonic acid (Chol-C3), cholesterol hemicaprylate (Chol-C8), cardiolipin (Chol-C13N), cholesterol hemicaprylate (Chol-C13N), cholesterol hemiglutarate (Chol-C5), cholesterol hemicaprylate (Chol-C6), cholesterol hemicaprylate (Chol-C7), cholesterol hemicaprylate (Chol-C8), cardiolipin (Chol-C13N), cholesterol hemicaprylate ... iolipid)(CL), 1,2-bis(tetradecano-10,12-diacyl)-sn-glycerol-3-phosphocholine (DC8-9PC), dihexadecanyl phosphate (DCP), dihexadecanyl phosphate (DCP1), 1,2-dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis(n-heptadecanoylphosphatidylcholine) (DHPC), dihexadecanylphosphatidylethanolamine (DHPE), 1,2-dilinolenic acid-sn-glycerol-3-phosphocholine (DLPC), 1,2-dilauryl-sn-glycerol-3-PE (DLPE), Dimyroxyglyceryl hemisuccinate (DMGS), Dimyroxyphosphatidylcholine (DMPC), Dimyroxyphosphoethanolamine (DMPE), Dimyroxyphosphatidylglycerol (DMPG), Dioleoxybenzyl alcohol (DOBA), 1,2-Dioleoxyglyceryl-3-hemisuccinate (DOGHEMS), N-[2~(2-{2-[2-(2,3-bis-octadec-9-enoxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-Trihydroxy-6-hydroxymethyl-tetrahydropyran-2-ylthioalkyl)-propionic acid (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine 4-(N-cis-butenediaminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2 Dioleoyl-sn-glycerol-3-(phospho-L-serine) (DOPS), non-cell-fusion phospholipid (DPhPE), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearate-dioleoylphosphatidylcholine (DSPC), distearate-dioleoylphospholipid Acetoethanolamine (DSPE), Distearate acetophosphate ethanolamine imidazole (DSPEI), 1,2-di-undecanoyl-sn-glycerol-phosphocholine (DUPC), Methiophanate-acetylcholine (EPC), N-histamine acetocholine carbamate (HCChol), Histamine distearate acetoglycerol (HDSG), N-histamine acetocholine hemisuccinate (HistChol), 1,2-dipalmitoylglycerol-hemisuccinate-Nα-histamine aceto-hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-tetradecanoic acid-diylacetamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-desinyl pyropheophorbide-a (HPPH), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dipalmitoylglycerol-Oα-histamine-Nα-hemisuccinate (IsohistsuccDG), mannosyl-dipalmitoylphosphatidylethanolamine (ManDOG), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-butenediaminomethyl)cyclohexane-methylamine] (MCC-PE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1-myristyl-2-hydroxy-sn-glycerol-phosphocholine (MHPC), athiol-reactive cis-butenidium-imino head-terminal lipids, such as 1,2-dioleyl-sn-glycerol-3-phosphate ethanolamine-N-[4-(p-cis-butenidium-phenyl)butyramine (MPB-PE), icosenoic acid (NA), sodium cholate (NaChol), l,2-Dioleoyl-sn-glycerol-3-[phosphoethanolamine-N-dodecanoyl (NC12-DOPE), as defined in the synthesis example in WO2008042973A2 (ND98), "N-pentadiylphosphatidylethanolamine of Formula 1" (NG-PE), N-hydroxysulfosuccinimide (NHS-'x'), "N-(co)-dicarboxylic acid-derived phosphatidylethanolamine covered by Formula 1" (NωPE-'x'), oleic acid (OA), 1-oleoyl-2-cholesterolylhemisuccinyl-sn-glycerol-3-phosphocholine (OChemsPC), phosphatidic acid (PA), phosphatidylethanolamine lipid (PE), and PE lipid conjugated with polyethylene glycol (PEG). Examples of PEG-PE include polyethylene glycol-distearate phosphatidylethanolamine lipid (PEG-PE), phosphatidylglycerol (PG), partially hydrogenated soybean phosphatidylcholine (PHSPC), phosphatidylinositol lipid (PI), phosphatidylinositol-4-phosphate (PIP), palmitoleyl oleate phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoleyl oleate phosphatidylglycerol (POPG), phosphatidylserine (PS), and lissamine rhodamine. B-phosphatidylethanolamine lipid (Rh-PE), purified soybean-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-transPE, 1-stearyl-2-oleyl-phosphatidylethanolamine (SOPE), soybean phosphatidylcholine (SPC), myelin (SPM), α,α'-caryophyllose 6,6'-disorbate (TDB), 1,2-ditransoleyl-sn-glycerol-3-phosphate ethanolamine (trans-DOPE), methyl phosphate ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-di-dihydropyrimidin-1(2 / - / )-yl)tetrahydrofuran-2-yl)methyl methyl ester, 1,2-dianarachidone Acryl-sn-glycerol-3-phosphocholine, 1,2-diarachidonicyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-docosahexaenoenyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-docosahexaenoenyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-linolenic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-di-linolenic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-di-linolenic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-diolenic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-distearate acetyl-sn-glycerol-3-phosphate ethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and diolenic acid phosphatidylethanolamine.

[0302] In some embodiments, the LNP includes at least one of the cationic lipids described in International Patent Publication No. WO2018118102 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, ATX-2, ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, and ATX-88. Methods for manufacturing such cationic lipids are also provided in International Patent Publication No. WO2018118102, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the cationic lipid in the LNP is ATX-88. As a non-limiting example, LNP comprises about 20 to 60% of ATX-2, ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87 or ATX-88; about 5 to 25% of non-cationic lipids; about 25 to 55% of sterols; and about 0.5 to 15% of PEG-modified lipids.

[0303] In some embodiments, the LNP includes at least one of the cationic lipids described in International Patent Publication No. WO2018119163 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, ATX-0002, ATX-0043, ATX-0057, ATX-0061, ATX-0063, ATX-0058, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0086, ATX-0087, ATX-0088, ATX-0091, ATX-0092, ATX-0093, ATX- 0094, ATX-0095, ATX-0096, ATX-0097, ATX-0098, ATX-0100, ATX-0101, ATX-0102, ATX-0106, ATX-0107, ATX-0108, ATX-0109, ATX-0110, ATX-01 11. ATX-0114, ATX-0115, ATX-0117, ATX-0118, ATX-0121, ATX-0122, ATX-0123, ATX-0124, ATX-0125, ATX-0126, ATX-0129, ATX-0132 and ATX-0134. Methods for manufacturing such cationic lipids are also provided in International Patent Publication No. WO2018119163, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the cationic lipid in LNP is ATX-88.As a non-limiting example, LNP includes approximately 20% to 60% of ATX-0002, ATX-0043, ATX-0057, ATX-0061, ATX-0063, ATX-0058, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0086, ATX-0087, ATX-0088, ATX-0091, ATX-0092, ATX-0093, ATX-0094, ATX-0095, ATX-0096, ATX-0097, ATX-0098, ATX-0100, and ATX-0098. X-0101, ATX-0102, ATX-0106, ATX-0107, ATX-0108, ATX-0109, ATX-0110, ATX-0111, ATX-0114, ATX-0115, ATX-0117, ATX-0118, ATX-0121, ATX-0122, ATX-0123, ATX-0124, ATX-0125, ATX-0126, ATX-0129, ATX-0132 or ATX-0134; approximately 5 to 25% non-cationic lipids; approximately 25 to 55% sterols; and approximately 0.5 to 15% PEG-modified lipids.

[0304] In some embodiments, LNP is included in International Patent Publications Nos. WO2012019168, WO2012045082, WO2012135805, WO2012158736, WO2013039857, WO2013039861, WO2013052523, WO2013090186, WO2013090648, WO2013096709, WO2013101690, WO2013106496, WO2013130161, WO2013151663, WO2013151664, WO2013151665, and WO20131516. 66. WO2013151667, WO2013151668, WO2013151669, WO2013151670, WO2013151671, WO2013151672, WO2013151736, WO2014028429, WO2014081507, WO20 14093574, WO2014093924, WO2014113089, WO2014144039, WO2014144711, WO2014144767, WO2014152027, WO2014152030, WO2014152031, WO201415221 1. WO2014152540, WO2014158795, WO2014159813, WO2014164253, WO2015006747, WO2015034925, WO2015034928, WO2015038892, WO2015048744, WO201 5051169, WO2015051173, WO2015051214, WO2015058069, WO2015085318, WO2015089511, WO2015105926, WO2015164674, WO2015196118, WO2015196128 , WO2015196130, WO2015199952, WO2016011222, WO2016011226, WO2016011306, WO2016014846, WO2016022914, WO2016036902, WO2016077123, WO2016 077125, WO2016100812, WO2016118724, WO2016118725, WO2016164762, WO2016176330, WO2016201377, WO2017004143, WO2017015457, WO2017015463,WO2017015630、WO2017019935、WO2017020026、WO2017031232、WO2017049074、WO2017049245、WO2017049275、WO2017049286、WO2017062513、WO2017070601、WO2017070613、WO2017070616、WO2017070618、WO2017070620、WO2017070622、WO2017070623、WO2017070624、WO2017070626、WO2017075531、WO2017099823、WO2017106799、WO2017112865、WO2017112943、WO2017117528、WO2017120612、WO2017127750、WO2017180917、WO2017201317、WO2017201325、WO2017201328、WO2017201332、WO2017201333、WO2017201340、WO2017201342、WO2017201346、WO2017201347、WO2017201348、WO2017201349、WO2017201350、WO2017201352、WO2017214175、WO2017218704、WO2017223135、WO2017223176、WO2018053209、WO2018075980、WO2018078053、WO2018081459、WO2018081480、WO2018081638、WO2018089540、WO2018089851、WO2018107026、WO2018107088、WO2018144082、WO2018144775、WO2018144778、WO2018151816、WO2018157009、WO2018170245、WO2018170256、WO2018170260、WO2018170270、WO2018170306、WO2018170336、WO2018170347、WO2018175783、WO2018187590、WO2018191657、WO2018191719、WO2018200737、WO2018200943、WO2018213731、WO2018213789、WO2018231990、WO2018232006、WO2018232120、WO2018232355、WO2018232357、WO2019018765、WO2019023179、WO2019036000、WO2019036008、WO2019036028、WO2019036030、WO2019036670、WO2019036682、WO2019046809、WO2019055807、WO2019089818、WO2019089828、WO2019103993、WO2019104152、WO2019104160、WO2019104195、WO2019136241、WO2019143910、WO2019148101、WO2019152557、WO2019200171、WO2019226650、WO2020023390、WO2020047201、WO2020056147、WO2020056155、WO2020056239、WO2020056304、WO2020056370、WO2020061284、WO2020061295、WO2020061317、WO2020061332、WO2020061367、WO2020061426、WO2020061457、WO202006242、WO2020069169、WO2020081938、WO2020097291、WO2020097409、WO2020146805、WO2020160397、WO2020160430、WO2020185811、WO2020190750、WO2020227510、WO2020227537、WO2020227615、WO2020227642、WO2020227690、WO2020243561、WO2020263883、WO2020263985、WO2021022173、WO2021026358、WO2021030701、WO2021050864、WO2021050986、WO2021055833、WO2021055835、WO2021055849、WO2021076805、WO2021076811、WO2021142280、WO2021154763、WO2021155243、WO2021155267、WO2021155274、At least one of the cationic lipids described in WO2021159040 and WO2021159130. As a non-limiting example, LNP comprises about 20 to 60% cationic lipids; about 5 to 25% non-cationic lipids; about 25 to 55% sterols; and about 0.5 to 15% PEG-modified lipids.

[0305] In some embodiments, the LNP comprises an ionizable lipid or lipid-like material. As non-limiting examples, the ionizable lipid may be C12-200, CKK-E12, 5A2-SC8, BAMEA-016B, or 7C1. Other ionizable lipids are known in this art and may be used herein.

[0306] In some embodiments, the LNP contains phospholipids. As a non-limiting example, the phospholipid (assistant) may be DOPE, DSPC, DOTAP, or DOTMA.

[0307] In some embodiments, the LNP comprises a PEG derivative. As a non-limiting example, the PEG derivative may be lipid-anchored, such as PEG being C14-PEG2000, C14-PEG1000, C14-PEG3000, C14-PEG5000, C12-PEG1000, C12-PEG2000, C12-PEG3000, C12-PEG5000, C16-PEG1000, C16-PEG2000, C16-PEG3000, C16-PEG5000, C18-PEG1000, C18-PEG2000, C18-PEG3000, or C18-PEG5000.

[0308] In some embodiments, at least one sterol comprises at least one cholesterol or cholesterol derivative. In some embodiments, LNP may be substantially free of at least one sterol. In some embodiments, LNP may contain no amount of at least one sterol.

[0309] In some embodiments, at least one additional LNP functional component comprises at least one component that reduces particle aggregation, at least one component that reduces LNP clearance from the individual's internal circulation, at least one component that increases the ability of LNP to cross the mucus layer, at least one component that reduces the individual's immune response to administered LNP, at least one component that modifies the membrane fluidity of LNP, at least one component that promotes LNP stability, or any combination thereof. In some embodiments, the LNP may substantially lack at least one additional LNP functional component. In some embodiments, the LNP may not contain any amount of at least one additional LNP functional component.

[0310] In some embodiments, the additional LNP functional component may be composed of a polymer. In some embodiments, the polymer containing the additional LNP functional component may be composed of at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-isozoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

[0311] In some embodiments, the average molecular weight of the polymer portion (e.g., PEG) may be between 500 and 20,000 Daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 20,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000. 0, 9,000 to 20,000, 9,500 to 20,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000 to 20,000 17,500 to 20,000, 18,000 to 20,000, 18,500 to 20,000, 19,000 to 20,000, 19,500 to 20,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to 19,500, 3,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500, 5,500 to 19,500, 6,000 to 19,500, 6,500 to 19,500 0, 7,000 to 19,500, 7,500 to 19,500, 8,000 to 19,500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to 19,500, 10,500 to 19,500, 11,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500, 13,000 to 19,500, 13,500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 to 19,500, 16,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to 19,500, 18,500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2, 000 to 19,000, 2,500 to 19,000, 3,000 to 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to 19,000, 5,500 to 19,000, 6,000 to 19,000, 6,500 to 19,000 000, 7,000 to 19,000, 7,500 to 19,000, 8,000 to 19,000, 8,500 to 19,000, 9,000 to 19,000, 9,500 to 19,000, 10,000 to 19,000, 10,500 to 19,000, 11,000 to 19,000 11,500 to 19,000, 12,000 to 19,000, 12,500 to 19,000, 13,000 to 19,000, 13,500 to 19,000, 14,000 to 19,000, 14,500 to 19,000, 15,000 to 19,000, 15,500 to 19,000 16,000 to 19,000, 16,500 to 19,000, 17,000 to 19,000, 17,500 to 19,000, 18,000 to 19,000, 18,500 to 19,000, 1,500 to 18,500, 2,000 to 18,500, 2,500 to 18,500 3,000 to 18,500, 3,500 to 18,500, 4,000 to 18,500, 4,500 to 18,500, 5,000 to 18,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500, 7,000 to 18,500, 7,500 to 18 500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to 18,500, 10,000 to 18,500, 10,500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500 00, 12,500 to 18,500, 13,000 to 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to 18,500, 15,000 to 18,500, 15,500 to 18,500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to 18,500, 17,500 to 18,500, 18,000 to 18,500, 1,500 to 18,000, 2,000 to 18,000, 2,500 to 18,000, 3,000 to 18,000, 3,500 to 18,000, 4,000 to 18,000 4,500 to 18,000, 5,000 to 18,000, 5,500 to 18,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 to 18,000, 7,500 to 18,000, 8,000 to 18,000, 8,500 to 18,000, 9,000 to 18 ,000, 9,500 to 18,000, 10,000 to 18,000, 10,500 to 18,000, 11,000 to 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 to 18,000, 13,000 to 18,000, 13,500 to 1 8,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to 18,000, 15,500 to 18,000, 16,000 to 18,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000, 1,500 to 1 7,500, 2,000 to 17,500, 2,500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, 4,000 to 17,500, 4,500 to 17,500, 5,000 to 17,500, 5,500 to 17,500, 6,000 to 17,500, 6 500 to 17,500, 7,000 to 17,500, 7,500 to 17,500, 8,000 to 17,500, 8,500 to 17,500, 9,000 to 17,500, 9,500 to 17,500, 10,000 to 17,500, 10,500 to 17,500, 11,000 to 17,500, 11,500 to 17,500, 12,000 to 17,500, 12,500 to 17,500, 13,000 to 17,500, 13,500 to 17,500, 14,000 to 17,500, 14,500 to 17,500, 15,000 to 17,500, 15,500 0 to 17,500, 16,000 to 17,500, 16,500 to 17,500, 17,000 to 17,500, 1,500 to 17,000, 2,000 to 17,000, 2,500 to 17,000, 3,000 to 17,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to 17,000, 5,000 to 17,000, 5,500 to 17,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 to 17,000, 8,000 to 17,000, 8,500 to 17,000, 9,0 00 to 17,000, 9,500 to 17,000, 10,000 to 17,000, 10,500 to 17,000, 11,000 to 17,000, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 to 17,000, 13,5 00 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to 17,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 To 16,500, 3,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to 16,500, 5,500 to 16,500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500 7,500 to 16,500, 8,000 to 16,500, 8,500 to 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to 16,500, 10,500 to 16,500, 11,000 to 16,500, 11,500 to 16,500, 12, 000 to 16,500, 12,500 to 16,500, 13,000 to 16,500, 13,500 to 16,500, 14,000 to 16,500, 14,500 to 16,500, 15,000 to 16,500, 15,500 to 16,500, 16,000 to 16,500, 1, 500 to 16,000, 2,000 to 16,000, 2,500 to 16,000, 3,000 to 16,000, 3,500 to 16,000, 4,000 to 16,000, 4,500 to 16,000, 5,000 to 16,000, 5,500 to 16,000, 6,000 to 16,000 00, 6,500 to 16,000, 7,000 to 16,000, 7,500 to 16,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000, 9,500 to 16,000, 10,000 to 16,000, 10,500 to 16,000, 11,000 to 16,000, 11,500 to 16,000, 12,000 to 16,000, 12,500 to 16,000, 13,000 to 16,000, 13,500 to 16,000, 14,000 to 16,000, 14,500 to 16,000, 15,000 to 16,000, 1 5,500 to 16,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 to 15,500, 3,000 to 15,500, 3,500 to 15,500, 4,000 to 15,500, 4,500 to 15,500, 5,000 to 15,500, 5,500 to 15 500, 6,000 to 15,500, 6,500 to 15,500, 7,000 to 15,500, 7,500 to 15,500, 8,000 to 15,500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to 15,500, 10,000 to 15,500, 10 ,500 to 15,500, 11,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500, 12,500 to 15,500, 13,000 to 15,500, 13,500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 1 5,000 to 15,500, 1,500 to 15,000, 2,000 to 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000, 4,000 to 15,000, 4,500 to 15,000, 5,000 to 15,000, 5,500 to 15,000 ,000, 6,000 to 15,000, 6,500 to 15,000, 7,000 to 15,000, 7,500 to 15,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to 15,000, 9,500 to 15,000, 10,000 to 15,000, 10 500 to 15,000, 11,000 to 15,000, 11,500 to 15,000, 12,000 to 15,000, 12,500 to 15,000, 13,000 to 15,000, 13,500 to 15,000, 14,000 to 15,000, 14,500 to 15,000, 1 500 to 14,500, 2,000 to 14,500, 2,500 to 14,500, 3,000 to 14,500, 3,500 to 14,500, 4,000 to 14,500, 4,500 to 14,500, 5,000 to 14,500, 5,500 to 14,500, 6,000 to 14,500, 6,500 to 14,500, 7,000 to 14,500, 7,500 to 14,500, 8,000 to 14,500, 8,500 to 14,500, 9,000 to 14,500, 9,500 to 14,500, 10,000 to 14,500, 10,500 to 14,500, 1 1,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500, 13,000 to 14,500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000, 2,000 to 14,000, 2, 500 to 14,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to 14,000, 5,000 to 14,000, 5,500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000 00, 7,500 to 14,000, 8,000 to 14,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to 14,000, 10,500 to 14,000, 11,000 to 14,000, 11,500 to 14,000, 1 2,000 to 14,000, 12,500 to 14,000, 13,000 to 14,000, 13,500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to 13,500, 3,000 to 13,500, 3,500 to 13,500, 4,000 To 13,500, 4,500 to 13,500, 5,000 to 13,500, 5,500 to 13,500, 6,000 to 13,500, 6,500 to 13,500, 7,000 to 13,500, 7,500 to 13,500, 8,000 to 13,500, 8,500 to 13,500 9,000 to 13,500, 9,500 to 13,500, 10,000 to 13,500, 10,500 to 13,500, 11,000 to 13,500, 11,500 to 13,500, 12,000 to 13,500, 12,500 to 13,500, 13,000 to 13,500, 1 500 to 13,000, 2,000 to 13,000, 2,500 to 13,000, 3,000 to 13,000, 3,500 to 13,000, 4,000 to 13,000, 4,500 to 13,000, 5,000 to 13,000, 5,500 to 13,000, 6,000 to 13,000000, 6,500 to 13,000, 7,000 to 13,000, 7,500 to 13,000, 8,000 to 13,000, 8,500 to 13,000, 9,000 to 13,000, 9,500 to 13,000, 10,000 to 13,000, 10,500 to 13,000, 1 1,000 to 13,000, 11,500 to 13,000, 12,000 to 13,000, 12,500 to 13,000, 1,500 to 12,500, 2,000 to 12,500, 2,500 to 12,500, 3,000 to 12,500, 3,500 to 12,500, 4,000 To 12,500, 4,500 to 12,500, 5,000 to 12,500, 5,500 to 12,500, 6,000 to 12,500, 6,500 to 12,500, 7,000 to 12,500, 7,500 to 12,500, 8,000 to 12,500, 8,500 to 12,500 9,000 to 12,500, 9,500 to 12,500, 10,000 to 12,500, 10,500 to 12,500, 11,000 to 12,500, 11,500 to 12,500, 12,000 to 12,500, 1,500 to 12,000, 2,000 to 12,000, 2,5 00 to 12,000, 3,000 to 12,000, 3,500 to 12,000, 4,000 to 12,000, 4,500 to 12,000, 5,000 to 12,000, 5,500 to 12,000, 6,000 to 12,000, 6,500 to 12,000, 7,000 to 12,000 0, 7,500 to 12,000, 8,000 to 12,000, 8,500 to 12,000, 9,000 to 12,000, 9,500 to 12,000, 10,000 to 12,000, 10,500 to 12,000, 11,000 to 12,000, 11,500 to 12,000, 1, 500 to 11,500, 2,000 to 11,500, 2,500 to 11,500, 3,000 to 11,500, 3,500 to 11,500, 4,000 to 11,500, 4,500 to 11,500, 5,000 to 11,500, 5,500 to 11,500, 6,000 to 11,500 00, 6,500 to 11,500, 7,000 to 11,500, 7,500 to 11,500, 8,000 to 11,500, 8,500 to 11,500, 9,000 to 11,500, 9,500 to 11,500, 10,000 to 11,500, 10,500 to 11,500, 11,000 to 11,500, 1,500 to 11,000, 2,000 to 11,000, 2,500 to 11,000, 3,000 to 11,000, 3,500 to 11,000, 4,000 to 11,000, 4,500 to 11,000, 5,000 to 11,000, 5,500 to 11,000 00, 6,000 to 11,000, 6,500 to 11,000, 7,000 to 11,000, 7,500 to 11,000, 8,000 to 11,000, 8,500 to 11,000, 9,000 to 11,000, 9,500 to 11,000, 10,000 to 11,000, 10,5 00 to 11,000, 1,500 to 10,500, 2,000 to 10,500, 2,500 to 10,500, 3,000 to 10,500, 3,500 to 10,500, 4,000 to 10,500, 4,500 to 10,500, 5,000 to 10,500, 5,500 to 10,500 0, 6,000 to 10,500, 6,500 to 10,500, 7,000 to 10,500, 7,500 to 10,500, 8,000 to 10,500, 8,500 to 10,500, 9,000 to 10,500, 9,500 to 10,500, 10,000 to 10,500, 1,500 To 10,000, 2,000 to 10,000, 2,500 to 10,000, 3,000 to 10,000, 3,500 to 10,000, 4,000 to 10,000, 4,500 to 10,000, 5,000 to 10,000, 5,500 to 10,000, 6,000 to 10,000 6,500 to 10,000, 7,000 to 10,000, 7,500 to 10,000, 8,000 to 10,000, 8,500 to 10,000, 9,000 to 10,000, 9,500 to 10,000, 1,500 to 9,500, 2,000 to 9,500, 2,500 to 9,500 0, 3,000 to 9,500, 3,500 to 9,500, 4,000 to 9,500, 4,500 to 9,500, 5,000 to 9,500, 5,500 to 9,500, 6,000 to 9,500, 6,500 to 9,500, 7,000 to 9,500, 7,500 to 9,500, 8,0 00 to 9,500, 8,500 to 9,500, 9,000 to 9,500, 1,500 to 9,000, 2,000 to 9,000, 2,500 to 9,000, 3,000 to 9,000, 3,500 to 9,000, 4,000 to 9,000, 4,500 to 9,000, 5,000 to 9,000, 5,500 to 9,000, 6,000 to 9,000, 6,500 to 9,000, 7,000 to 9,000, 7,500 to 9,000, 8,000 to 9,000, 8,500 to 9,000, 1,500 to 8,500, 2,000 to 8,500, 2,500 to 8,500 3,000 to 8,500, 3,500 to 8,500, 4,000 to 8,500, 4,500 to 8,500, 5,000 to 8,500, 5,500 to 8,500, 6,000 to 8,500, 6,500 to 8,500, 7,000 to 8,500, 7,500 to 8,500, 8,000 To 8,500, 1,50...

Claims

1. A polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is a spike (S) protein, and wherein the polynucleotide comprises a first sequence region comprising a nucleic acid sequence having at least 80% identity with members of the group consisting of SEQ ID NO: 7, 20, 26, 27 and 32.

2. The polynucleotide of claim 1, wherein the first sequence region is at least 95% identical to SEQ ID NO:

7.

3. The polynucleotide of claim 2, wherein the first sequence region is at least 99% identical to SEQ ID NO:

7.

4. The polynucleotide of claim 3, wherein the first sequence region is composed of SEQ ID NO:

7.

5. The polynucleotide of claim 4, wherein the encoded spike protein has the protein sequence of SEQ ID NO:

2.

6. The polynucleotide of any one of claims 1 to 5, wherein at least 50% of the polynucleotide sequence is codon-optimized.

7. The polynucleotide of claim 5, wherein the polynucleotide is DNA.

8. The polynucleotide of claim 5, wherein the polynucleotide is RNA.

9. The polynucleotide of claim 8, wherein the polynucleotide is mRNA.

10. The polynucleotide of claim 9, comprising a 5'UTR and a 3'UTR, wherein the 5'UTR comprises SEQ ID NO: 47 and the 3'UTR comprises SEQ ID NO:

48.

11. The polynucleotide of claim 10, wherein at least one uracil nucleotide is modified to N1-methylpseuuridine.

12. The polynucleotide of claim 11, wherein all uridine nucleotides are modified to N1-methylpseuuridine.

13. A nucleic acid vaccine comprising the polynucleotides as claimed in claim 12.

14. The nucleic acid vaccine, as requested in item 13, is formulated in lipid nanoparticles (LNP).

15. The nucleic acid vaccine as claimed in claim 14, wherein the LNP comprises (a) 50 to 85 mol% of cationic lipids; (b) 13 to 49.5 mol% of non-cationic lipids; and (c) 0.5 to 2 mol% of conjugated lipids.

16. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) 50 to 65 mol% cationic lipids; (b) non-cationic lipids comprising 4 to 10 mol% phospholipids and 30 to 40 mol% cholesterol; and (c) 0.5 to 2 mol% conjugated lipids.

17. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 30 to 60 mol% cationic lipids; (b) about 0 to 30 mol% non-cationic lipids; (c) about 18.5 to 48.5 mol% sterols; and (d) about 0 to 10 mol% polyethylene glycol-modified lipids.

18. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 35 to 55 mol% cationic lipids; (b) about 5 to 25 mol% non-cationic lipids; (c) about 30 to 40 mol% sterols; and (d) about 0 to 10 mol% polyethylene glycol-modified lipids.

19. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 35 to 45 mol% cationic lipids; (b) about 25 to 35 mol% non-cationic lipids; (c) about 20 to 30 mol% sterols; and (d) about 0 to 10 mol% polyethylene glycol-modified lipids.

20. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 45 to 65 mol% cationic lipids; (b) about 5 to 10 mol% non-cationic lipids; (c) about 25 to 40 mol% sterols; and (d) about 0.5 to 10 mol% polyethylene glycol-modified lipids.

21. The nucleic acid vaccine as claimed in claim 14, wherein the LNP comprises (a) about 40 to 60 mol% cationic lipids; (b) about 5 to 15 mol% non-cationic lipids; (c) about 35 to 45 mol% sterols; and (d) about 0.5 to 3 mol% polyethylene glycol-modified lipids.

22. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 30 to 60 mol% cationic lipids; (b) about 0 to 30 mol% non-cationic lipids; (c) about 15 to 50 mol% sterols; and (d) about 0.01 to 10 mol% polyethylene glycol-modified lipids.

23. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 10 to 75 mol% cationic lipids; (b) about 0.5 to 50 mol% non-cationic lipids; (c) about 5 to 60 mol% sterols; and (d) about 0.1 to 20 mol% polyethylene glycol-modified lipids.

24. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 50 to 65 mol% cationic lipids; (b) about 3 to 15 mol% non-cationic lipids; (c) about 30 to 40 mol% at least one sterol; and (d) about 0.5 to 2 mol% polyethylene glycol-modified lipids.

25. The nucleic acid vaccine as claimed in claim 14, wherein the LNP comprises (a) about 50 to 85 mol% cationic lipids; (b) about 3 to 15 mol% non-cationic lipids; (c) about 30 to 40 mol% sterols; and (d) about 0.5 to 2 mol% polyethylene glycol-modified lipids.

26. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 25 to 75 mol% cationic lipids; (b) about 0.1 to 15 mol% non-cationic lipids; (c) about 5 to 50 mol% sterols; and (d) about 0.5 to 20 mol% polyethylene glycol-modified lipids.

27. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 50 to 65 mol% cationic lipids; (b) about 5 to 10 mol% non-cationic lipids; (c) about 25 to 35 mol% sterols; and (d) about 5 to 10 mol% polyethylene glycol-modified lipids.

28. The nucleic acid vaccine of claim 14, wherein the LNP comprises (a) about 20 to 60 mol% cationic lipids; (b) about 5 to 25 mol% non-cationic lipids; (c) about 25 to 55 mol% sterols; and (d) about 0.5 to 15 mol% polyethylene glycol-modified lipids.

29. The nucleic acid vaccine as claimed in claim 14, wherein the LNP comprises (a) 45 to 55 mmol of 3D-P-DMA; (b) 8 to 10 mmol of DSPC; (c) 36 to 42 mmol of cholesterol; and (d) 1.4 to 1.8 mmol of PEG-DMA.

30. The nucleic acid vaccine as claimed in claim 29, wherein the LNP comprises (a) 49.96 mmol of 3D-P-DMA; (b) 9.97 mmol of DSPC; (c) 38.44 mmol of cholesterol; and (d) 1.61% of PEG-DMA.

31. A pharmaceutical composition comprising a nucleic acid vaccine as claimed in claim 30 and a pharmaceutically acceptable excipient.

32. The pharmaceutical composition of claim 31, comprising about 0.2 mg / mL of the mRNA as described in claim 9.

33. The pharmaceutical composition of claim 32 is suitable for intramuscular (IM) injection.

34. The pharmaceutical composition of claim 32 is packaged in a 3 mL glass vial with 2 mL filler.

35. A method for inducing an immune response in an individual, comprising administering a nucleic acid vaccine as claimed in any one of claims 13 to 30 or a pharmaceutical composition as claimed in any one of claims 31 to 34.

36. The method of claim 35, wherein the immune response comprises a T-cell response.

37. The method of claim 35, wherein the immune response comprises a B cell response.

38. The method of claim 35, further comprising administering a reinforcing agent after the first administration.

39. The method of claim 35, wherein the individual’s immune response is dose-dependent.

40. The method of claim 39, wherein the dose-dependent immune response comprises one or more of the SARS-CoV-2 spike protein-specific IgG, IgG1, IgG2a, IgG2b, IgM and IgA antibodies that induce the individual.

41. The method of claim 39, wherein the dose-dependent immune response comprises inducing one or more of IL-2+ T cells, IL-4+ T cells and IFN-γ+ T cells.

42. A method of treating an individual with COVID-19, comprising administering to the individual a nucleic acid vaccine as claimed in any one of claims 13 to 30 or a pharmaceutical composition as claimed in any one of claims 31 to 34.

43. A method for preventing an individual from contracting COVID-19, comprising administering to the individual a nucleic acid vaccine as claimed in any one of claims 13 to 30 or a pharmaceutical composition as claimed in any one of claims 31 to 34.

44. A method for alleviating or improving the physiological effects or symptoms of COVID-19 in an individual, comprising administering to the individual a nucleic acid vaccine as claimed in any of claims 13 to 30 or a pharmaceutical composition as claimed in any of claims 31 to 34.

45. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 5 µg to about 100 µg.

46. ​​The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 16 µg.

47. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 40 µg.

48. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 100 µg.

49. The method of any one of claims 42 to 44, wherein the volume of the pharmaceutical composition administered to the individual is about 0.025 mL to about 0.5 mL.

50. The method of any one of claims 42 to 44, wherein the volume of the pharmaceutical composition administered to the individual is about 0.025 mL, 0.05 mL, 0.08 mL, 0.2 mL, or 0.5 mL.

51. The method of any of claims 42 to 50, comprising administering a second dose of the nucleic acid vaccine or pharmaceutical composition between about one week and about five weeks after the first dose.

52. The method of claim 51, wherein the second dose is administered approximately four weeks after the first dose.

53. The method of any of claims 42 to 44, wherein anti-spike protein IgG antibodies are detected in the individual up to day 28 after receiving the first dose of the nucleic acid vaccine or pharmaceutical composition.

54. The method of claim 53, wherein, up to day 42 following the administration of the second dose of the nucleic acid vaccine or pharmaceutical composition, the amount of the anti-spike protein IgG antibody present in the individual increases.

55. The method of claim 53, wherein the anti-spike protein IgG antibody value in the individual is more than 10 times the anti-spike protein IgG antibody value of a serum sample from a SARS-CoV-2 convalescent patient.

56. A method for inducing the production of SARS-CoV-2 neutralizing antibodies in an individual, comprising administering to the individual a nucleic acid vaccine as claimed in any one of claims 13 to 30 or a pharmaceutical composition as claimed in any one of claims 31 to 34.

57. The method of claim 56, wherein the SARS-CoV-2 neutralizing antibody can be detected in the individual up to day 28 after administration.

58. The method of claim 57, wherein on day 42 after receiving a second dose of the nucleic acid vaccine or pharmaceutical composition, the production of the SARS-CoV-2 neutralizing antibody in the individual increases.

59. The method of claim 57, wherein the level of the SARS-CoV-2 neutralizing antibody increases tenfold up to day 42.

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