TW202330923A - Compositions and methods for the prevention and/or treatment of covid-19 - Google Patents

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

Compositions and methods for preventing and/or treating COVID-19

The present disclosure relates generally to nucleic acid vaccines, and specifically to compositions, formulations and methods of nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2. /or uses for the prevention, mitigation and/or treatment and/or prevention of COVID-19, including alleviating the physiological effects of infection and/or symptoms. Cross-references to related applications

This application claims a Canadian application filed on September 28, 2021 with the title Composition and Methods for the Prevention and/or Treatment of COVID-19 No. 3,132,191 and Application No. 3,146,392 titled Compositions and Methods for Preventing and/or Treating COVID-19 filed on January 21, 2022, the contents of each of these applications shall be cited in their entirety. Incorporated by reference. sequence list

This application is filed together with the sequence listing in electronic format. The sequence listing file titled 20921007PCTSEQLST.xml was created on September 8, 2022, and is 130,296 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

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 (referred to as “COVID-19”), which affects mammals in different ways, ranging from asymptomatic individuals to those with symptoms ranging from mild symptoms to severe illness or death. Individuals with widespread symptoms.

Vaccines are an effective way to provide preventive protection against infectious diseases. Currently, there are limited vaccines available to prevent, mitigate, and/or treat COVID-19. Treatment for COVID-19 is limited to managing 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.

This disclosure provides nucleic acid vaccines, compositions and formulations containing nucleic acid vaccines, and methods of using them to prevent coronavirus infection to prevent, mitigate and treat COVID-19. Nucleic acid vaccines may include polynucleotides encoding at least one antigenic protein of SARS-CoV-2, fragments or variants thereof. SARS-CoV-2 antigen protein is the structural protein of SARS-CoV-2. Structural proteins can be spike proteins, membrane proteins, nucleocapsid phosphoproteins, or envelope proteins. Non-limiting examples of the amino acid sequences of these structural proteins are shown in Table 1 (SEQ ID Nos: 1 to 6 and 15 to 19).

Provided herein are COVID-19 nucleic acid vaccines for use in methods of vaccinating individuals against COVID-19, wherein the nucleic acid vaccines may include at least one polynucleotide encoding at least one structural protein of SARS-CoV-2 or a fragment thereof.

Provided herein are methods of inducing an immune response in an individual by administering a nucleic acid vaccine described herein in an amount effective to produce an immune response. 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 can be generated by a single administration of a nucleic acid vaccine described herein. As another non-limiting example, an immune response can be generated by booster administration of a nucleic acid vaccine described herein. Administration of pharmaceutical compositions can produce a dose-responsive immune response in an individual. As a non-limiting example, a dose-responsive immune response may comprise inducing one or more SARS-CoV-2 spike protein-specific IgG, IgGl, IgG2a, IgG2b, IgM and IgA antibodies in an individual. As another non-limiting example, a dose-responsive immune response may include induction of 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 effects in the subject.

Provided herein are methods of treating and/or preventing COVID-19 in an individual by administering a nucleic acid vaccine described herein.

This article provides pharmaceutical compositions and formulations of nucleic acid vaccines for the treatment and prevention of COVID-19.

The nucleic acid vaccines described herein may be formulated in one or more lipid nanoparticles (LNPs).

In some embodiments, the LNPs comprise (a) 50 to 85 mol % cationic lipids; (b) 13 to 49.5 mol % noncationic lipids; and (c) 0.5 to 2 mol % Conjugated lipids.

In some embodiments, the LNP includes (a) 50 to 65 mol% cationic lipids; (b) noncationic lipids including 4 to 10 mol% phospholipids and 30 to 40 mol% cholesterol; and (c) 0.5 to 2 mol% conjugated lipids.

In some embodiments, the LNP includes (a) about 30 to 60 mol% cationic lipids; (b) about 0 to 30 mol% noncationic lipids (c) about 18.5 to 48.5 mol% sterols ; and (d) about 0 to 10 mol% of pegylated lipid.

In some embodiments, the LNPs comprise (a) about 35 to 55 mol% cationic lipids; (b) about 5 to 25 mol% noncationic lipids; (c) about 30 to 40 mol% solids. alcohol; and (d) about 0 to 10 mole % of pegylated lipid.

In some embodiments, the LNPs comprise (a) about 35 to 45 mol% cationic lipids; (b) about 25 to 35 mol% noncationic lipids; (c) about 20 to 30 mol% solids. alcohol; and (d) about 0 to 10 mole % of pegylated lipid.

In some embodiments, the LNPs comprise (a) about 45 to 65 mol% cationic lipids; (b) about 5 to 10 mol% noncationic lipids; (c) about 25 to 40 mol% solids. alcohol; and (d) about 0.5 to 10 mol% of PEGylated lipid.

In some embodiments, the LNPs comprise (a) about 40 to 60 mol% cationic lipids; (b) about 5 to 15 mol% noncationic lipids; (c) about 35 to 45 mol% solids. alcohol; and (d) about 0.5 to 3 mol% of PEGylated lipid.

In some embodiments, the LNPs comprise (a) about 30 to 60 mol% cationic lipids; (b) about 0 to 30 mol% noncationic lipids; (c) about 15 to 50 mol% solids. alcohol; and (d) about 0.01 to 10 mol% of PEGylated lipid.

In some embodiments, the LNPs comprise (a) about 10 to 75 mol% cationic lipids; (b) about 0.5 to 50 mol% noncationic lipids; (c) about 5 to 60 mol% solids. alcohol; and (d) about 0.1 to 20 mol% of PEGylated lipid.

In some embodiments, the LNPs comprise (a) about 50 to 65 mol% of cationic lipids; (b) about 3 to 15 mol% of noncationic lipids; (c) about 30 to 40 mol% of at least a sterol; and (d) about 0.5 to 2 mol% of a pegylated lipid.

In some embodiments, the LNPs comprise (a) about 50 to 85 mol% cationic lipids; (b) about 3 to 15 mol% noncationic lipids; (c) about 30 to 40 mol% solids. alcohol; and (d) about 0.5 to 2 mol% of PEGylated lipid.

In some embodiments, the LNPs comprise (a) about 25 to 75 mol% cationic lipids; (b) about 0.1 to 15 mol% noncationic lipids; (c) about 5 to 50 mol% solids. alcohol; and (d) about 0.5 to 20 mol% of PEGylated lipid.

In some embodiments, the LNPs comprise (a) about 50 to 65 mol% cationic lipids; (b) about 5 to 10 mol% noncationic lipids; (c) about 25 to 35 mol% solids. alcohol; and (d) about 5 to 10 mole % of pegylated lipid.

In some embodiments, the LNPs comprise (a) about 20 to 60 mol% cationic lipids; (b) about 5 to 25 mol% noncationic lipids; (c) about 25 to 55 mol% solids. alcohol; and (d) about 0.5 to 15 mol% of PEGylated lipid.

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

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

Also provided herein is a nucleic acid vaccine for COVID-19, which includes about 0.2 mg/mL mRNA, wherein the mRNA includes a coding region having a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the mRNA of the nucleic acid vaccine disclosed herein includes a coding region having the nucleic acid sequence set forth in SEQ ID NO: 7. Nucleic acid vaccines can be formulated as 2 mL filler in 3 mL glass vials.

In some embodiments, administering the nucleic acid vaccine to the individual includes administering about 5 µg to about 100 µg of mRNA to the individual. For example, a method may comprise administering approximately 16 µg of mRNA to an individual. Alternatively, the method may comprise administering about 40 µg of mRNA to the individual. Alternatively, the method may comprise administering about 100 µg of mRNA to the individual.

In some embodiments, administering the nucleic acid vaccine to the individual includes administering about 0.025 mL to about 0.5 mL of the nucleic acid vaccine to the individual. For example, a method may comprise administering about 0.025 mL of a nucleic acid vaccine to an individual, administering about 0.05 mL of a nucleic acid vaccine to an individual, administering about 0.08 mL of a nucleic acid vaccine to an individual, administering about 0.2 mL of a nucleic acid vaccine to an individual, or administering about 0.5 mL of a nucleic acid vaccine to an individual.

In some embodiments of the provided methods, administering includes intramuscular (IM) injection of the nucleic acid vaccine into the individual.

The nucleic acid vaccine may be administered to an individual in the form of a first dose of the nucleic acid vaccine, followed by a second dose of the nucleic acid vaccine about 1 to about 5 weeks later. In some embodiments, the second dose of the nucleic acid vaccine is administered approximately 4 weeks after the first dose.

In some embodiments, anti-spike protein IgG antibodies are detected in the individual as of day 28 after receiving the first dose of the nucleic acid vaccine.

In some embodiments, anti-spike protein IgG antibodies are detected in the individual as of day 28 after receiving the first dose of the nucleic acid vaccine, and are enhanced in the individual as of day 42 after receiving the second dose of the nucleic acid vaccine .

In some embodiments, anti-spike protein IgG antibodies in an individual are increased up to 10-fold compared to the mean anti-spike protein IgG antibodies in serum samples from SARS-CoV-2 convalescent patients.

In some implementations, SARS-CoV-2 neutralizing antibodies are detected in the individual as of day 28 after the first dose of the nucleic acid vaccine.

In some embodiments, SARS-CoV-2 neutralizing antibodies increase in the individual by day 42 after the second dose of the nucleic acid vaccine.

Accordingly, the present disclosure provides a COVID-19 nucleic acid vaccine for use in a method of vaccinating an individual against COVID-19, wherein the nucleic acid vaccine contains approximately 0.2 mg/mL mRNA, wherein the mRNA contains at least 95% identity to SEQ ID NO: 7 The nucleic acid sequence, and wherein the nucleic acid vaccine is formulated for intramuscular (IM) injection and is formulated in lipid nanoparticles (LNP).

Details of various implementation aspects are set forth in the following description. Other features, objects and advantages will be apparent from the description and scope of the claims.

Detailed description I.Introduction

The following description sets forth exemplary compositions, methods, parameters, and the like. It should be appreciated, however, that this description is not intended to limit the scope of the present disclosure, but is instead provided as a description of illustrative implementations.

Described herein are nucleic acid vaccines, specifically polynucleotides (e.g., mRNA), compositions, formulations, and methods of nucleic acid vaccines comprising polynucleotides encoding one or more SARS-CoV-2 antigenic proteins, fragments, or variants thereof. and/or uses for the prevention, mitigation and/or treatment of COVID-19. The antigenic protein may be the structural protein of SARS-CoV-2. Structural proteins may be spike (S) proteins, membrane (M) proteins, nucleocapsid (N) phosphoproteins, or envelope (E) proteins.

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 the structural protein of SARS-CoV-2. The polynucleotide can be an RNA polynucleotide, such as an mRNA polynucleotide.

In some embodiments, the nucleic acid vaccine includes at least one mRNA polynucleotide encoding at least one of a structural protein of SARS-CoV-2 or a fragment or variant of a structural protein.

In some embodiments, polynucleotides can be designed to encode one or more polypeptides of interest from SARS-CoV-2, or fragments or variants thereof. Such polypeptides of interest for SARS-CoV-2 may include, but are not limited to, intact polypeptides, multiple polypeptides or polypeptide fragments, or polypeptide variants, which independently may be composed of one or more regions of a polynucleotide from SARS-CoV-2 or partially or fully encoded. As used herein, the term "polypeptide of interest" refers to any polypeptide selected to be encoded within a polynucleotide described herein or whose function is affected by a polynucleotide described herein. Any of the peptides or polypeptides described herein may be antigenic (also referred to as immunogenic).

As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) linked together most commonly 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.

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, it will be at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants and analogs of each of the foregoing. Polypeptides may be single molecules or may be multimolecular complexes, such as dimers, trimers, or tetramers. It may also comprise single or multi-chain polypeptides, such as antibodies or insulin, and may 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 of the amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

The term "polypeptide variant" refers to molecules whose amino acid sequence differs from the native or reference sequence. Amino acid sequence variants may have substitutions, deletions and/or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, a variant will have at least about 50% identity (homology) to 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 85% identity to the native or reference sequence. 90%, or even better at least about 95% identical (same origin).

In some implementations, "variant mimics" are provided. As used herein, the term "variant mimetic" is a mimetic containing one or more amino acids that will mimic the activation sequence. For example, glutamate esters can serve as mimetics of phospho-threonine and/or phospho-serine. Alternatively, a variant mimetic may cause deactivation or deactivate a product containing the mimetic, for example, phenylalanine may act as a deactivating substitution for tyrosine; or alanine may act as a deactivating substitution for serine.

As applied to amino acid sequences, "homology" is defined as the difference between a residue in a candidate amino acid sequence and an amine group in a second sequence after aligning the sequences and introducing gaps if necessary to achieve maximum percent homology. Percent identity of residues in an acid sequence. Methods and computer programs for comparison are well known in the art. It is understood that homology is determined by calculation of percent identity, but its value may vary due to gaps and penalties introduced in the calculation.

When applied to a polypeptide sequence, "homologue" refers to a corresponding sequence in another species that is substantially identical to a second sequence in a second species.

As used herein, "analog" is intended to include polypeptide variants that differ by one or more amino acid changes, such as amino acid residue substitutions that maintain one or more properties of the parent or starting polypeptide, added or missing.

In some embodiments, the present disclosure encompasses several types of polypeptide-based compositions, including variants and derivatives. These include substitution, insertion, 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 or starting molecule.

For example, a sequence tag or amino acid, such as one or more lysines, can be added to a peptide sequence described herein (eg, at the N- or C-terminus). Sequence tags can be used for peptide purification or localization. Lysine can be used to increase peptide solubility or allow biotin labeling. Alternatively, amino acid residues located in the carboxyl and amino terminal regions of the amino acid sequence of the peptide or protein can optionally be deleted, thereby providing a truncated sequence. Alternatively, depending on the intended use of the sequence, for example, the sequence appears as part of a larger sequence that is soluble or linked to a solid support, with certain amino acids (eg, C-terminal or N-terminal residues) deleted.

When referring to a polypeptide, a "substitutional variant" is one in which at least one amino acid residue in the native or starting sequence is removed and a different amino acid is inserted in its place at the same position. The substitution may be monosubstituted, where only one amino acid in the molecule is substituted, or the substitution may be polysubstituted, where two or more amino acids in the same molecule are substituted.

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 of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, and leucine for another non-polar residue. Similarly, examples of conservative substitutions include replacing one polar (hydrophilic) residue with another polar residue, such as between arginine and lysine, between glutamic acid and asparagine , and between glycine and serine. In addition, a basic residue such as lysine, arginine or histidine is substituted for another basic residue, or an acidic residue such as aspartic acid or glutamic acid is substituted for another acidic residue. Additional examples of conservative substitutions. Examples of non-conservative substitutions include substitution of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, methionine with such as cysteine, gluten Polar (hydrophilic) residues of amino acid, glutamic acid or lysine and/or substitution of polar residues with non-polar residues.

When referring to a polypeptide, an "insertional variant" is a variant in which one or more amino acids are inserted immediately adjacent to an amino acid at a specific position in the native or starting sequence. "Immediately adjacent" to an amino acid means linked to the alpha-carboxy or alpha-amino functional group of the amino acid.

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

When referring to polypeptides, "covalent derivatives" include modifications and/or post-translational modifications of the native or starting protein with organic proteinaceous or non-proteinaceous derivatizing agents. Covalent modification has traditionally been accomplished by reacting the targeted amino acid residues of the protein with an organic derivatizing agent capable of reacting with selected side chains or terminal residues, or by utilizing recombinant host cells of choice. introduced by the post-translational modification mechanism. The resulting covalent derivatives are suitable for use in programs directed toward the identification of residues important for biological activity, immunoassays, or the preparation of anti-protein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the capabilities of those skilled in the art and can be performed without undue experimentation.

When referring to polypeptides, "characteristics" are defined as the unique amino acid sequence-based components of the molecule. Characteristics of the polypeptides encoded by the polynucleotides described herein include surface appearance, local conformational shape, folds, loops, half-rings, domains, half-domains, sites, termini, or any combination thereof.

As used herein, when referring to a polypeptide, the term "surface expression" refers to the polypeptide-based components of a protein that appear on the outermost surface.

As used herein, when referring to a polypeptide, the term "local conformational shape" means a representation of the polypeptide-based protein structure that is located within a definable protein space.

As used herein, when referring to a polypeptide, the term "folding" refers to the resulting configuration of an amino acid sequence when energy is minimized. Folding can occur at the second or third level of the folding process. Examples of secondary sheets include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed due to the aggregation or separation of higher energy forces. Regions formed in this manner include hydrophobic and hydrophilic pockets, and the like.

As used herein, the term "turn" when relating to polypeptide conformation means changing the direction of the backbone of a peptide or polypeptide and may involve the bending of one, two, three, or more amino acid residues.

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 direction of the backbone of the peptide or polypeptide. When a loop is found in a polypeptide and only changes the direction of the backbone, it may contain four or more amino acid residues. Oliva et al. have identified at least five types of protein rings ( J. Mol Bio., 1 266 (4): 814-830; 1997). Rings can be open or closed. Closed or "cyclic" rings can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids between bridging moieties. Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical of polypeptides with disulfide bonds, or the bridging moiety may be non-protein based, such as dibromide as used herein. agent.

As used herein, when referring to a polypeptide, the term "hemicycle" refers to a portion of an identified ring that has at least half of the amino acid residues of the ring from which the portion is derived. It is understood that a ring may not always contain an even number of amino acid residues. Therefore, in those cases where a ring contains or is identified as containing an odd number of amino acids, the half-ring of the odd-numbered ring will contain either the integer portion of the ring or the next integer portion (number of amino acids in the ring/2 + /-0.5 amino acids).

As used herein, when referring to a polypeptide, the term "domain" refers to a polypeptide moiety that has one or more identifiable structural or functional features or properties (e.g., binding capacity, serving as a protein-protein interaction site).

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

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 amino acid-based embodiments. Site refers to a position within a peptide or polypeptide that can be modified, manipulated, altered, derivatized or altered within a polypeptide-based molecule described herein.

As used herein, when referring to a polypeptide, the term "termini" or "terminus" refers to the end of the peptide or polypeptide. Such terminal ends are not limited to the first or last position of a peptide or polypeptide, but may include other amino acids in the terminal region. Polypeptide-based molecules described herein may be characterized by having an N-terminus (terminated by an amino acid having a free amine group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). In some cases, the proteins described herein are composed of multiple polypeptide chains held together by disulfide bonds or by non-covalent forces (multimers, oligomers). Proteins of this class will have multiple N-termini and C-termini. Alternatively, the ends of the polypeptide may be modified so that, as the case may be, they begin or end with a non-polypeptide-based moiety, such as an organic conjugate.

Once any feature is identified or defined as a required component of a polypeptide to be encoded by a polynucleotide described herein, several manipulations of such features can be performed by moving, swapping, inverting, deleting, randomizing or duplicating and /or any of the modifications. Furthermore, it is understood that feature manipulation can produce the same results as molecular modifications described herein. For example, manipulations involving domain deletions will result in changes in the length of the molecule, as will modification of a nucleic acid to encode a less than full-length molecule.

In a polypeptide, the term "modification" refers to a modification compared to the standard set of 20 amino acids. The modification can be a variety of different modifications. In some embodiments, these regions may contain one, two, or more (optionally different) modifications.

Modification and manipulation can be accomplished by methods known in the art, such as, but not limited to, site-directed mutagenesis or a priori incorporation during chemical synthesis. The resulting modified molecules can then be tested for activity using in vitro or in vivo assays, such as those described herein or any other suitable screening assay known in the art.

In some embodiments, polypeptides may comprise common sequences discovered through several rounds of experiments. As used herein, a "common" sequence is a single sequence that represents a collection of sequences that allow for variation at one or more sites.

As those skilled in the art will recognize, protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of related polypeptides. For example, 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) is provided herein. 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, about 20, about 30, about 40, about 50, or about 100 amino acids or more amino acids are included. Any protein with an elongated segment that is about 40%, about 50%, about 60%, about 70%, about 80% identical to any of the sequences described herein can be used in accordance with the nucleic acid vaccines described herein. , about 85%, about 90%, about 95%, or about 100% consistent. In certain embodiments, the polypeptides used according to the nucleic acid vaccines described herein include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any sequence provided or referenced herein.

Accordingly, the polynucleotides of the present disclosure encode peptides or polypeptides that contain substitutions, insertions and/or additions, deletions and covalent modifications relative to reference sequences, in particular the peptide or polypeptide sequences disclosed herein. Polynucleotides may also contain substitutions, insertions and/or additions, deletions and covalent modifications relative to the polynucleotide reference sequence.

The reference molecule (polypeptide or polynucleotide) may share some identity with the designed molecule (polypeptide or polynucleotide). The term "identity" as known in the art refers to the relationship between the sequences of two or more peptides, polypeptides or polynucleotides, as determined by comparing such sequences. In this technology, identity also means the degree of sequence relatedness between them, as determined by the number of matches between two or more strings of amino acid residues or nucleosides. Identity measures the percentage of identical matches between the smaller of two or more sequences using gap alignments (if present) that are determined by specific mathematical models or computer programs (such as "algorithms"). ") processing. The identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, N.Y., 1988 ; Biocomputing: Informatics and Genome Projects, edited by Smith, D. W., Academic Press, N.Y., 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, A. M. and Griffin, H. G., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, N.Y, 1991; and Carillo et al., SIAM J. Applied Math. 48: 1073;1988).

In some embodiments, encoded polypeptide variants may have the same or similar activity as a reference polypeptide. Alternatively, a variant may have altered activity (eg, increased or decreased) relative to a reference polypeptide. In general, variants of a particular polynucleotide or polypeptide described herein will be identical to a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, but less than 100% sequence identity. Such comparison tools include those of the BLAST suite of programs (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 herein, specifically in the definition of "consistency." II. Components of the Disclosure SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus that causes coronavirus disease 2019 known as "COVID-19". COVID-19 affects mammals in different ways, ranging from asymptomatic individuals to individuals with a wide range of symptoms ranging from mild symptoms to severe illness or death. To date, approximately 80% of COVID-19 patients have mild to moderate symptoms, while approximately 20% may 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 so far include fever or chills, cough, shortness of breath or breathing Difficulty, fatigue, body aches, muscle aches, headache, sore throat, congestion or runny nose, nausea and/or vomiting, diarrhea, and new loss of taste or smell.

The genome of SARS-CoV-2 encodes four structural proteins: spike (S), envelope (E), membrane (M) and nucleocapsid (N), as well as non-structural proteins (named nsp1 to nsp16) and Auxiliary protein. The spike protein on the surface of the virus is usually the main mediator for the virus to enter cells. The SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) via its receptor binding domain (RBD) and is proteolytically activated by human proteases. Another feature of the SARS-CoV-2 spike protein is that the protein has a functional furin cleavage site at the S1-S2 boundary (S1 is the receptor binding unit and S2 is the membrane fusion unit), which can predict Activates entry of many viruses, including SARS-CoV-2. The SARS-CoV-2 spike has been used as a protective antigen to elicit neutralizing antibodies in various vaccine development strategies. Membrane and envelope proteins are used for virus assembly. Envelope protein (E) can form a homopentameric 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; the contents of which are incorporated herein by reference in their entirety). In addition, the nucleocapsid protein in SARS-CoV-2 can regulate host cell mechanisms and participate in the regulation of the virus life cycle.

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 critical for binding to the ACE2 receptor, and the SARS-CoV-2 genome appears to have an RBD with high receptor homology to humans, ferrets, cats, and others species with high affinity binding to ACE2 (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) .

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode full-length polypeptides of structural proteins, or fragments or variants of structural proteins of SARS-CoV-2, such as spike protein, nucleocapsid protein, capsid protein, Membrane protein or membrane protein.

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode more than one fragment or variant of a structural protein of SARS-CoV-2, such as spike protein, nucleocapsid protein, envelope protein, and/or Membrane Protein.

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a mutant variant of one of the structural proteins, or a fragment of a mutant variant of the structural protein of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change from aspartate to glycine, one of the structural proteins of SARS-CoV-2.

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a 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 from aspartate to glycine in the spike protein of SARS-CoV-2. As a non-limiting example, a variant may be a single amino acid change from aspartate to glycine (D614G) at position 614 in the spike protein of SARS-CoV-2 (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).

In some embodiments, the nucleic acid vaccines described herein may encode one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2. Non-limiting examples of proteins, peptides, fragments or variants of structural proteins of SARS-CoV-2 are provided in Table 1. In the table, if known, the NCBI reference number is also provided. Table 1. Structural protein sequence of SARS-CoV-2 Sequence 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 protein (NCBI reference number: YP_009724392.1) protein 4 Membrane protein (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 B.1.17 (UK) variant spike protein with L452R mutation protein 18 B.1.351 (South Africa) variant spike protein with L452R mutation protein 19 P.1 (Brazilian) variant spike protein protein

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.

In some embodiments, the nucleic acid vaccine can be an mRNA vaccine that, when translated, produces one or more proteins, peptides, fragments, or variants of the structural proteins of SARS-CoV-2. Therefore, the polynucleotide of the mRNA vaccine is an mRNA polynucleotide encoding one or more proteins, peptides, fragments or variants thereof of the structural protein of SARS-CoV-2.

In one embodiment, the coding sequence of the mRNA vaccine described herein may be based on the spike ( S) protein coding sequence. In some embodiments, a code change of a single amino acid change from D614 to G614 is introduced to match the amino acids of the currently prevalent virus strains.

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

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

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

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

Any of the sequences mentioned in Tables 1 to 3, or variants thereof, may also be used in the memory booster vaccines described herein.

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

Sequences of SARS-CoV-2 proteins or fragments or variants thereof can be obtained from any source. In some embodiments, the sequence of the SARS-CoV-2 protein or fragment or variant thereof is derived from a strain capable of infecting or at risk of infecting human individuals.

In some embodiments, the sequence of a SARS-CoV-2 protein or fragment or variant thereof may be modified or optimized (such as codon optimization) for expression in a particular cell or host organism.

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

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 (eg, 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 (eg, between the S1 and S2 boundaries). In some viral envelope proteins, disruption of the furin cleavage site was found to enhance performance and stability. In some embodiments, the nucleic acid vaccine encodes the S protein sequence (eg, full length, fragment, or variant) of SARS-CoV-2, wherein the S protein includes the D614G mutation. Nucleic acid vaccines encoding the S protein of SARS-CoV-2, fragments or variants thereof may also include a signal peptide and/or at least one linker (such as GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be encoded by codons optimization.

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 a topological domain of the M protein of SARS-CoV-2 (eg, a region on the virion surface or within the virion). 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 the M protein of SARS-CoV-2 (eg, full-length protein or fragment). Nucleic acid vaccines encoding the M protein of SARS-CoV-2, fragments or variants thereof may also include a signal peptide and/or at least one linker (such as GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be codon-coded optimization.

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 an N protein fragment 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 the dimerization 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 (eg, full-length protein or fragment). Nucleic acid vaccines encoding the N protein of SARS-CoV-2, fragments or variants thereof may also include a signal peptide and/or at least one linker (such as GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be codon-coded optimization.

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 an E protein fragment of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a topological domain of the E protein of SARS-CoV-2 (eg, a region on the virion surface or within the virion). In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the E protein of SARS-CoV-2 (eg, full-length protein or fragment). Nucleic acid vaccines encoding the E protein of SARS-CoV-2, fragments or variants thereof may also include a signal peptide and/or at least one linker (such as a GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may be encoded by codons optimization.

In some embodiments, the nucleic acid vaccine encodes two different structural proteins of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the S protein of SARS-CoV-2, fragments or variants thereof and the M protein of SARS-CoV-2, fragments or variants thereof. In some embodiments, the nucleic acid vaccine encodes the S protein of SARS-CoV-2, fragments or variants thereof and the N protein of SARS-CoV-2, fragments or variants thereof. In some embodiments, the nucleic acid vaccine encodes the S protein of SARS-CoV-2, fragments or variants thereof and the E protein of SARS-CoV-2, fragments or variants thereof. In some embodiments, the nucleic acid vaccine encodes the M protein of SARS-CoV-2, fragments or variants thereof and the N protein of SARS-CoV-2, fragments or variants thereof. In some embodiments, the nucleic acid vaccine encodes the M protein of SARS-CoV-2, fragments or variants thereof and the E protein of SARS-CoV-2, fragments or variants thereof. In some embodiments, the nucleic acid vaccine encodes the N protein of SARS-CoV-2, fragments or variants thereof and the E protein of SARS-CoV-2, fragments or variants thereof. Nucleic acid vaccines encoding two different structural proteins of SARS-CoV-2, fragments or variants thereof may also include signal peptides and/or at least one linker (such as GSG linker) sequence, and one or more sequences of the nucleic acid vaccine may Codon optimized. In some embodiments, the sequences encoding two different structural proteins of SARS-CoV-2 or fragments or variants thereof of the nucleic acid vaccine are constructed as a single polynucleotide.

In some embodiments, the nucleic acid vaccine encodes at least three different sequences of structural protein fragments of SARS-CoV-2 or variants thereof. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment or variant sequences of SARS-CoV-2 and 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 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 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 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 S protein, fragment or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E protein, fragment or variant sequences of SARS-CoV-2 and S 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 N 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 E 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 M 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 E protein, fragment or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E protein, fragment or variant sequences of SARS-CoV-2 and N 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 M protein, fragment or variant sequence of SARS-CoV-2, and the N protein of SARS-CoV-2, Fragment or variant sequence. In some embodiments, 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 of SARS-CoV-2, Fragment or variant sequence. 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 of SARS-CoV-2, Fragment or variant sequence. In some 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 of SARS-CoV-2, Fragment or variant sequence. Nucleic acid vaccines encoding at least three different sequences of structural protein fragments of SARS-CoV-2 or variants thereof may also include a signal peptide and/or at least one linker (such as GSG linker) sequence, and one or more of the nucleic acid vaccines Sequences can be codon optimized. SARS-CoV-2 variants

SARS-CoV-2 is a member of the Macroviridae family. Multiple variants (sometimes called “strains” or “lineages”) of SARS-CoV-2 have been identified globally. The nomenclature of SARS-CoV-2 variants used in this specification is consistent with the PANGO nomenclature of new virus 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 data related to PANGO SARS-CoV-2 lineages or variants can be accessed online using Nextstrain’s SARS-CoV-2 Analysis User Interface (nextstrain.org/ncov/global).

As of this disclosure, multiple PANGO lineage variants of SARS-CoV-2 have been identified, including the following (numbers in parentheses represent the number of cases for each submitted PANGO lineage:

From an epidemiological perspective, variants are usually classified into 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, see, for example, cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html.

VOIs may have certain genetic markers associated with changes in receptor binding, reduced neutralization of antibodies generated in response to previous infection or vaccination, reduced therapeutic efficacy, potential diagnostic impact, or predicted increased transmissibility or disease severity. In some cases, VOIs have specific genetic markers predicted to affect transmission, diagnosis, treatment, or immune evasion, or lead to increased proportions of cases or unique outbreak clusters. SARS-CoV-2 VOI includes, for example, PANGO lineage 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.

VOCs can include variants with evidence of increased transmissibility, more severe disease (increased hospitalizations or deaths), significantly reduced neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or failed diagnostic tests . In some cases, VOCs have evidence of impact on diagnostics, treatments, and vaccines; widespread interference with diagnostic test targets; evidence of significant increased resistance to one or more classes of therapy; evidence of increased resistance to one or more classes of therapy; presence of antibodies generated during previous infection or vaccination and evidence of a significant reduction in efficacy; evidence of reduced vaccine-induced protection against severe disease; 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).

VOHC may have clear evidence that preventive measures or medical countermeasures (MCMs) have been significantly reduced in effectiveness relative to previously circulating variants. In some cases, VOHC has impacted medical countermeasures (MCM), demonstrating diagnostic failure, with evidence of significantly reduced vaccine effectiveness, a disproportionately high number of vaccine breakthrough cases, minimal protection against vaccine-induced severe disease, and risk of multiple emergencies. Significantly reduced sensitivity, more severe clinical disease, and increased hospitalization rates for EUA or approved therapeutics.

The nucleic acid vaccines disclosed herein may encode one or more polypeptides of any SARS-CoV-2 variant described herein, such as one or more proteins, peptides, fragments or variants thereof. In some embodiments, the nucleic acid vaccines 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 or variants thereof. In some embodiments, the nucleic acid vaccine encodes a polypeptide comprising a specific mutation known as D614G.

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

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

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

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

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise one or more mutations or substitutions present in the P.1 SARS-CoV-2 variants (i.e., gamma variants), such as the following One or more: Spike protein substitutions K417N/T, E484K, N501Y and/or D614G.

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise one or more mutations or substitutions present in the B.1.351 SARS-CoV-2 variant (i.e., the beta variant), such as the following One or more: Spike protein substitution K417N, E484K, N501Y and/or D614G. The B.1.351 variant is also known as the South African variant because it first originated in South Africa.

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise one or more mutations or substitutions present in B.1.427 SARS-CoV-2 variants, such as one or more of the following: Protein replaces L452R and/or D614G.

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise one or more mutations or substitutions present in B.1.429 SARS-CoV-2 variants, such as one or more of the following: The spike protein replaces S13I, W152C, L452R and/or D614G.

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise 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 replaces G142D, E154K, L452R, E484Q, D614G, P681R and/or Q1071H.

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise 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 that comprise 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 replaces V70F, A222V, W258L and/or K417N.

In some embodiments, the nucleic acid vaccine encodes one or more polypeptides that comprise 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 replaces T19R, G142D, L452R, E484Q, D614G, P681R and/or D950N.

In some embodiments, the nucleic acid vaccine encodes a SARS-CoV-2 spike protein, such as a protein, peptide, fragment or variant selected from one or more substitutions and/or deletions selected from the following: 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.

In some embodiments, the nucleic acid vaccine encodes SARS-CoV-2 ORF1a selected from one or more substitutions and/or deletions: L3201P, T265I, T2007I, L3468V, Δ3675-3677, and L3930F.

In some embodiments, the nucleic acid vaccine encodes a SARS-CoV-2 ORF1b selected from one or more substitutions: P314F, P314L, and Q1011H.

In some embodiments, the nucleic acid vaccine encodes SARS-CoV-2 ORF3a selected from one or more substitutions: P42L and Q57H.

In some embodiments, the nucleic acid vaccine encodes a T11I substituted SARS-CoV-2 ORF8.

In some embodiments, the nucleic acid vaccine encodes an R81C substituted SARS-CoV-2 5'UTR.

In some embodiments, the nucleic acid vaccine encodes a SARS-CoV-2 M protein including an I82T substitution, such as a protein, peptide, fragment or variant.

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

In some embodiments, a polynucleotide described herein encodes at least one polypeptide of interest, such as one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2. The SARS-CoV-2 proteins, peptides, fragments or variants thereof of the present disclosure may be wild-type, wherein they are derived from infectious agents or modified (for example, the structural proteins or fragments and variants thereof are engineered, designed or artificial ). It may have any combination of features described herein.

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, and are used to prevent, mitigate and/or treat COVID-19. As a non-limiting example, such peptides or polypeptides may serve as antigens or antigenic molecules (also preferably as immunogenic molecules). The term "nucleic acid" in its broadest sense includes any compound and/or substance containing a polymer of nucleotides. Such polymers are often called polynucleotides.

Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), Including LNA with β-D-ribose configuration, α-LNA with a-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-a-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

In some embodiments, in vitro transcription (IVT) enzyme synthesis methods can be used to prepare linear polynucleotides (referred to as " IVT polynucleotide").

In some embodiments, nucleic acid vaccines may include "chimeric polynucleotides" that have portions or regions that differ in size and/or encode proteins (eg, structural proteins of SARS-CoV-2). A "chimera" is an entity having two or more incongruous or heterogeneous parts or regions. As used herein, a "portion" or "region" of a polynucleotide is defined as any portion of a polynucleotide that is less than the entire length of the polynucleotide.

In some embodiments, nucleic acid vaccines include polynucleotides of about 30 to about 100,000 nucleotides in length (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, 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, 5 00 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).

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

In some embodiments, the length of at least one peptide or polypeptide of interest of a polynucleotide encoding a 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, 1,300, 1,400, 0 00, 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 "region codes."

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)" refers to any polynucleoside that encodes at least one peptide or polypeptide of interest and is capable of being translated to produce the encoded peptide or polypeptide of interest in vitro, in vivo, in situ, or ex vivo acid.

The minimum length of the polynucleotide region of the nucleic acid vaccine may be a length sufficient to encode a nucleic acid sequence encoding a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, the length may be sufficient to encode 2 to 30 amino acids, such as 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids of peptides. The length may be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or no longer than 40 amino acids, such as no longer than 35, 30, 25, 20 , 17, 15, 14, 13, 12, 11, or 10 amino acid peptides. Examples of dipeptides that may be encoded by the polynucleotide sequence include, but are not limited to, carnosine and methamphetamine.

The polynucleotide region of a nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for preventing, mitigating and/or treating COVID-19 may be greater than about 30 nucleotides in length acid. 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, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,0 00, 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, the region includes about 30 to about 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, 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

The nucleic acid vaccine described herein may be an mRNA vaccine. An mRNA vaccine includes at least one mRNA molecule that, when translated, produces at least one peptide or polypeptide of interest for use in preventing, mitigating, and/or treating COVID-19. Generally speaking, an mRNA molecule generally includes at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' end cap, and a poly-A tail. mRNA components: start codon and stop codon

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

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

In some embodiments, the length of the region that initiates or terminates translation can independently range from 3 to 40, such as 5 to 30, 10 to 20, 15, or at least 4, or 30, or less nucleotides. within the range. Additionally, in addition to start and/or stop codons, these regions may also contain one or more signal and/or restriction sequences.

In some embodiments, a masking agent can be used to mask the first initiation codon or alternative initiation codon to increase translation initiation of the initiation codon or alternative initiation codon downstream of the masked initiation codon or alternative initiation codon. The probability of initiation at a codon.

In some embodiments, the initiation codon can be removed from the polynucleotide sequence such that translation of the polynucleotide begins at a codon other than the initiation codon. Translation of the polynucleotide can begin at a codon subsequent to the removed initiation codon or at a downstream initiation codon or an alternative initiation codon. The polynucleotide sequence from which the initiation codon is removed may further comprise at least one masking agent for the downstream initiation codon and/or for the replacement initiation codon in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide. mRNA components: coding region

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 or variants thereof of SARS-CoV-2 for use in preventing, mitigating and/or treating COVID-19. mRNA components: untranslated region

The polynucleotides of the nucleic acid vaccines described herein may include one or more regions or portions that serve as or function as untranslated regions (UTRs). The wild-type UTR of the gene is transcribed but not translated. In mRNA, the 5' UTR begins at the start of transcription and continues to but not including the start codon; whereas the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. While not wishing to be bound by theory, UTRs may play a role in the stability and translation of nucleic acid molecules and translation. Variants of the UTR may be utilized in which one or more nucleotides are added or removed to the terminus, including A, T, C, or G.

In some embodiments, the length of the UTR of the polynucleotide of the nucleic acid vaccine can 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).

Wild-type 5' UTRs include features that play a role in translation initiation because these 5' UTRs include sequences such as the Kozak sequence, which are known to be involved in how ribosomes initiate translation of many genes. The 5' UTR is also known to form secondary structures involved in binding of elongation factors. Other non-UTR sequences, such as introns or portions of intronic sequences, may also be used as regions or subregions that may increase protein yield and polynucleotide content.

The native or wild-type 3' UTR is known to have adenosine and uridine stretches embedded within it. Such AU-rich tags are particularly prevalent in genes with high turnover rates. The introduction, removal or modification of 3'UTR AU-rich elements (AREs) can be used to modulate the stability of polynucleotides in nucleic acid vaccines.

A UTR from any gene can be incorporated into a region of the polynucleotide of a nucleic acid vaccine. Alternatively, artificial UTRs that are not variants of the wild-type region can also be used in the polynucleotides of nucleic acid vaccines. These UTRs, or portions thereof, may be placed in the same orientation as in the transcript from which they are selected, or their orientation or position may vary. As used herein, the term "change" when relating to a UTR sequence means that the UTR has changed in some way relative to the reference sequence. As non-limiting examples, the 5' or 3' UTR may be inverted, shortened or elongated by one or more other 5' UTRs or 3' UTRs from different parent sequences.

In some embodiments, the flanking regions are selected from a family of transcripts whose proteins share common functional, structural, and property characteristics. For example, the polypeptide of interest may belong to a family of proteins expressed in a particular cell, tissue, or at some time during development. The UTR from any of these genes can be exchanged with any other UTR of the same or a different protein family to generate new polynucleotides. 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, localization, origin, or mode of expression.

The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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 5' to the start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. In some embodiments, the 5' UTR is 1, 2, 3, 4, 5, 6, or more nucleotides away from the 5' start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine acid; for example, 1, 2, 3, 4, 5, 6, or more inter-nucleotide spacer sequences separate the 5'UTR from the start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise a 5'UTR that has at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5'UTR having at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5'UTR that has at least 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). sequence.

The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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 3' to the start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. In some embodiments, the 3' UTR is 1, 2, 3, 4, 5, 6, or more nucleotides away from the 3' start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. acid; for example, 1, 2, 3, 4, 5, 6, or more inter-nucleotide spacer sequences separate the 3'UTR from the start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR having at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). sequence.

The polynucleotide of the nucleic acid vaccine disclosed herein may comprise 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 3' to the start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. In some embodiments, the 3' UTR is 1, 2, 3, 4, 5, 6, or more nucleotides away from the 3' start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. acid; for example, 1, 2, 3, 4, 5, 6, or more inter-nucleotide spacer sequences separate the 3'UTR from the start codon of the sequence encoding the SARS-CoV-2 polypeptide of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR having at least 94% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise 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). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR having at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3'UTR that has at least 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). sequence. mRNA components: Cap and IRES sequences

In some embodiments, the polynucleotides of the nucleic acid vaccines disclosed herein may include a 5' end cap structure. The 5' end cap structure of natural mRNA participates in nuclear export, increases the stability of mRNA, and binds to the mRNA end cap binding protein (CBP). This protein associates with poly(A) binding protein through CBP to form a mature circular mRNA species and is responsible for the expression of mRNA in the nucleus. Stability and translational capacity in cells. End caps further aid in the removal of 5' proximal introns during mRNA splicing.

In some embodiments, the 5' end capping region of the polynucleotide of the nucleic acid vaccine may comprise a single end cap or a series of nucleotides forming the end cap. The length of the capping region may be 1 to 10, such as 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2, or 10 or less nucleotides. In some examples, the capping region can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some implementations, the end cap is absent.

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 can be chemical (eg, non-enzymatic) or enzymatically synthesized, with a chemical structure that differs from the native (eg, endogenous, wild-type, or physiological) 5'-end cap, but which retains the end cap function.

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 comprise a guanine analog. Suitable guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine (m1G), 2'fluoro-guanosine, 7-deaza-guanosine, 8-side oxy-guanosine, 2- Amino-guanosine, LNA-guanosine and 2-azido-guanosine.

Those skilled in the art will appreciate that the 5' end cap can be generated enzymatically or by other synthetic methods. The endogenous mRNA molecule is end-capped at the 5'-end, and 5'-ppp-5'-triple is generated between the terminal guanosine cap residue and the sense nucleotide transcribed at the 5'-end of the mRNA molecule. Phosphate bond. This 5'-guanylate end cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugar of the transcribed nucleotide at the 5' end of the mRNA and/or the front end may also be 2'-O-methylated. 5'-decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

The polynucleotides, eg, mRNA, of the nucleic acid vaccines described herein can be modified to include a non-hydrolyzable end cap structure, thereby preventing decapping and thus increasing the mRNA half-life. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester bond, modified nucleotides can be used during the capping reaction. For example, poxvirus capping enzyme available from, for example, New England Biolabs (Ipswich, MA) can be used with alpha-thio-guanosine nucleotides according to the manufacturer's instructions to cap the 5'-ppp-5' end. Phosphorothioate bonds are produced. Additional modified guanosine nucleotides may be used, such as alpha-methyl-phosphonate and seleno-phosphate nucleotides.

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

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, differ from natural (i.e., endogenous) end-cap analogs in their chemical structure. , wild-type or physiological) 5'-end cap while retaining end cap function. End cap analogs can be chemically (eg, non-enzymatically) or enzymatically synthesized and attached to nucleic acid molecules, such as mRNA molecules.

For example, the anti-reverse end-cap analogue (ARCA) end-cap contains two guanines linked by a 5'-5'-triphosphate group, one of which contains an N7 methyl group and a 3'-O-methyl group ( That is, 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). Another 3'-O atom of unmodified guanine is connected to the 5'-terminal nucleotide of a capped nucleic acid molecule (such as mRNA). N7- and 3' -O-Methylated guanine provides capping of the terminal portions of nucleic acid molecules (eg, mRNA).

Another exemplary end cap is mCAP, which is similar to ARCA but has a 2'-O-methyl on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5 '-Guanosine, m7Gm-ppp-G).

Although cap analogs allow concomitant capping of nucleic acid molecules in in vitro transcription reactions, up to 20% of transcripts can remain uncapped. This, along with structural differences between end-cap analogues and the endogenous 5'-end cap structure, can lead to reduced translational capacity and reduced cell stability.

In illustrative aspects of the present disclosure, polynucleotides (eg, mRNA) can be post-transcriptionally capped using enzymes. For example, recombinant pox virus plus capping enzyme and recombinant 2'-O-methyltransferase can generate a typical 5'-5'-triple between the 5'-terminal nucleotide of the mRNA and the guanine capping nucleotide. Phosphate bond, in which the end-cap guanine contains N7 methylation and the 5'-terminal nucleotide of the mRNA contains 2'-O-methyl. This type of structure is called Cap 1 structure. In some embodiments, the Cap 1 structure provides greater translational capacity and cellular stability and reduced cellular pro-inflammatory cytokines compared to, for example, other 5' end cap analog structures known in the art. activation. Cap structure includes 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')N1mpNp (Cap 1) and 7mG(5')-ppp(5')N1mpN2mp (Cap 2) .

In one embodiment, the polynucleotides of the nucleic acid vaccines described herein comprise the Cap 1 structure.

Because polynucleotides, such as mRNA, can be capped post-transcriptionally, and because this process is more efficient, up to 100% of the polynucleotides, such as mRNA, can be capped. This contrasts with approximately 80% for end-cap analogues when attached to mRNA during in vitro transcription reactions.

In some embodiments, the polynucleotide of the nucleic acid vaccine may contain an internal ribosome entry site (IRES) sequence. While not wishing to be bound by theory, IRES play an important role in initiating protein synthesis in the absence of a 5' cap structure. An IRES may serve as the sole ribosome binding site or may serve as one of multiple ribosome binding sites on the mRNA. mRNA components: tail region

In some embodiments, the polynucleotide of the nucleic acid vaccine, such as mRNA, includes a tailing region. Non-limiting examples of tailing regions include poly-A sequences, poly-C sequences, and/or polyA-G quadruplexes.

In some embodiments, the mRNA includes chain-terminating nucleosides. Non-limiting examples of chain-terminating nucleosides include 2'-O methyl, F, and locked nucleic acids (LNA).

In some embodiments, the length of the sequence of the tailing region of the polynucleotide of the nucleic acid vaccine may range from none 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 may be described in units of poly-A binding protein binding or as a function of poly-A binding protein binding.

In some implementations, the poly-A tail can also be added after the construct has been exported from the core.

In some embodiments, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides, such as mRNA molecules, to increase stability during RNA processing. Immediately following transcription, the 3' end of the transcript can be cleaved to release the 3' hydroxyl group. Next, poly-A polymerase adds the adenine nucleotide strand to the RNA. A process called polyadenylation adds a poly-A tail that 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.

In some embodiments, the poly-A tail (when present) is greater than 30 nucleotides in length (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 includes about 30 to about 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).

In some embodiments, the poly-A tail is about 99 nucleotides in length (SEQ ID NO: 44).

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 particular feature or region, or the length of the final product expressed from the polynucleotide.

In this case, the length of the poly-A tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the polynucleotide or feature thereof. The poly-A tail can also be designed as part of the polynucleotide to which it belongs. In this case, the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60% of the total length of the structure, the area of the structure, or the total length of the structure minus the poly-A tail , 70%, 80%, or 90%, or greater. In addition, engineered binding sites and polynucleotide binding of poly-A binding proteins can enhance performance. signal sequence

In some embodiments, the polynucleotides of the nucleic acid vaccine may also encode additional features that facilitate transport of the polypeptide to therapeutically relevant sites. One such feature that aids in protein transport is the signal sequence. As used herein, a "signal sequence" or "signal peptide" is a polynucleotide or polypeptide, respectively, of about 9 to 200 nucleotides (3 to 60 amino acids) in length, which is incorporated 5' of the coding region, respectively. terminal or encoded N-terminal polypeptide. In some embodiments, the addition of such sequences allows for transport of the encoded polypeptide to the endoplasmic reticulum via one or more secretory pathways. After transporting the protein, some signal peptides are cleaved from the protein by signal peptidases.

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

The polynucleotide of the nucleic acid vaccine, its region or part or sub-region may be codon optimized. Codon optimization methods are known in the art and may be adapted in an effort to achieve one or more of several goals. These goals include, but are not limited to, matching codon frequencies in the target and host organisms to ensure correct folding; altering GC content to increase mRNA stability or reduce secondary structure; minimizing errors that may impair gene architecture or expression. Tandemly repeat codons or base runs; customize transcription and translation control regions; insert or remove protein transport sequences; remove/add post-translational modification sites (such as glycosylation sites) in encoded proteins; add, move Eliminate or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; modulate translation rates to allow proper folding of individual domains of the protein; or reduce or eliminate problematic secondary levels within the polynucleotide. structure. Codon optimization tools, algorithms and services are known in the art, 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 usage for each amino acid is 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 glutamate GAA, GAG F Phenylalanine 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 asparagine AAT, AAC P proline CCT, CCC, CCA, CCG Q Glutamine CAA, CAG R Arginine CGT, CGC, CGA, CGG, AGA, AGG s Serine TCT, TCC, TCA, TCG, AGT, AGC Sec Selenium Cysteine UGA in mRNA in the presence of selenocysteine insertion element (SETS) terminate 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

In some embodiments, the nucleic acid vaccine is vectorized after codon optimization. Non-limiting examples of vectors include, but are not limited to, plastids, viruses, myxoplasts, and artificial chromosomes. Modify

Nucleic acid vaccines (including mRNA vaccines) of the present disclosure may include one or more modifications. The term "modified" or "modified" as appropriate refers to modification of A, G, U or C ribonucleotides. In general, as used herein, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5'-terminal cap portion of the mRNA. In a polypeptide, the term "modification" refers to a modification compared to the standard set of 20 amino acids.

As used herein, "nucleoside" is defined as a compound containing a combination of a sugar molecule (eg, pentose or ribose) or a derivative thereof and an organic base (eg, purine or pyrimidine) or a derivative thereof ("nucleobase"). As used herein, "nucleotide" is defined as a nucleoside that includes a phosphate group or other backbone linkage (internucleoside linkage).

The modification can be a variety of different modifications. In some embodiments, the coding region, untranslated region, flanking region, and/or terminal or tailing region may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the nucleic acid vaccines of the present disclosure include one or more modifications that make the nucleic acid molecule more resistant to degradation in cells when introduced into cells and/or compared to unmodified polynucleotides. Or more stable in cells.

The polynucleotides of the nucleic acid vaccines described herein may include any useful modifications, such as modifications to sugars, nucleobases, or internucleoside linkages (eg, to connecting phosphate/phosphodiester linkages/phosphodiester backbones). One or more atoms of the pyrimidine nucleobase may be, for example, optionally substituted amine, optionally substituted thiol, optionally substituted alkyl (such as methyl or ethyl), optionally substituted Halogen (eg chlorine or fluorine) atoms or groups are substituted or substituted. In certain embodiments, modifications (eg, one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present disclosure may be ribonucleic acid (RNA) modified into deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or mixtures thereof things. This article describes additional modifications.

In some embodiments, modifications include 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides (2'-OMe and 2'-MOE modifications, respectively).

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can comprise at least one modification described herein.

The polynucleotides of the nucleic acid vaccines described herein may include a combination of modifications to sugars, nucleobases, and/or internucleoside linkages.

Modifications of polynucleotides (eg, RNA polynucleotides, such as mRNA polynucleotides) that may be used in the vaccines of the present disclosure include, but are not limited to, any modifications as described in PCT Publication WO2017070626, the contents of which are incorporated by reference in their entirety. Included herein are modifications or deletions of, for example, nucleotides (or codons) encoding one or more N-linked glycosylation sites of the translated polypeptide. Modifications useful in the vaccines of the present disclosure 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 of the present disclosure may further include PCT patent application publications WO2020255063, WO2020182869, WO2016011222, WO2016011226, WO2016005004, WO2016000792, WO2015176737, WO2015085318, WO2015048744 and WO2 015034925, as well as those described in U.S. Patent Application Publications US20200254086, US20200206362, US20180311336, and US20180303929 features or modifications; each such content is incorporated herein by reference in its entirety.

For example, polynucleotides comprising the mRNA molecules of the nucleic acid vaccines described herein may include the following modifications. The internucleoside linkages of a polynucleotide may be partially or completely modified. A polynucleotide may contain modifications to one or more nucleobases. The polynucleotide may contain 5-methylcytosine in place of all cytosine nucleobases/cytidine nucleotides. Additionally, a polynucleotide may have one or more modifications to one or more sugar subunits of the nucleoside. The sugar modification can be one or more locked nucleic acid (LNA) or 2'-O-methoxyethyl modifications ("2'-MOE") modifications. Polynucleotides can be designed into patterned arrays with sugar, nucleobase, or bond modifications. In some embodiments, polynucleotides may contain modifications that maximize stability. In some embodiments, the polynucleotide can be completely modified with 2'-MOE-sugar. modified nucleobase

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.

In some embodiments, the modified nucleobase is modified uracil. Exemplary nucleobases and nucleosides with modified uracil include pseudouridine (ψ), pyridin-4-one nucleosides, 5-aza-uridine, 6-aza-uridine, 2-sulfide Base-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio- Pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (such as 5-iodo-uridine or 5-bromo-uridine) , 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), uridine 5-oxyacetate (cmo ⁵ U), uridine 5-oxyacetate methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxymethylhydroxymethyl-uridine (chm ⁵ U), 5-carboxymethyl Hydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 5-methylaminomethyl -2-Thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-aminoformylmethyl-uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5 -Proynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyl-uridine (τm ⁵ U), 1-taurinemethyl-pseudouridine, 5-taurine Acid methyl-2-thio-uridine (τm ⁵ s ² U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m ⁵ U, that is With nucleobases deoxythymine), 1-methylpseudouridine (m ¹ ψ), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-sulfide Base-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl -pseudouridine, 1-methyl-1-desa-pseudouridine, 2-thio-1-methyl-1-desa-pseudouridine, dihydrouridine (D), dihydropseudouridine Glycoside, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methyl Oxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl- Pseudouridine (also known as 1-methylpseudouridine (m ¹ ψ)), 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3- (3-Amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(prenylaminomethyl)uridine (inm ⁵ U), 5-(prenylaminomethyl) Methyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl methyl-uridine (m ⁵ Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s ² Um), 5-methoxy Carbonylmethyl-2'-O-methyl-uridine (mcm ⁵ Um), 5-carboxymethyl-2'-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethyl Aminomethyl-2'-O-methyl-uridine (cmnm ⁵ Um), 3,2'-O-dimethyl-uridine (m ³ Um), 5-(prenylamino Methyl)-2'-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2'-F-arabinose-uridine, 2'-F-uridine Glycosides, 2'-OH-arabinose-uridine, 5-(2-methoxycarbonylvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is modified cytosine. Exemplary nucleobases and nucleosides with modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m ³ C), N4-acetyl-cytidine (ac ⁴ C), 5-formyl-cytidine (f ⁵ C), N4-methyl-cytidine (m ⁴ C), 5-methyl-cytidine (m ⁵ C), 5-halo-cytidine (e.g. 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm ⁵ C), 1-methyl-pseudo-cytidine, pyrro-cytidine , pyrrolo-pseudoisocytidine, 2-thio-cytidine (s ² C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-Methyl-pseudoisocytidine, 4-thio-1-methyl-1-desa-pseudoisocytidine, 1-methyl-1-desaza-pseudoisocytidine, zebrarine ( zebularine), 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2- Methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, raisin Ridine (k ₂ C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m ⁵ Cm), N4- Acetyl-2'-O-methyl-cytidine (ac ⁴ Cm), N4,2'-O-dimethyl-cytidine (m ⁴ Cm), 5-formyl-2'-O- Methyl-cytidine (f ⁵ Cm), N4,N4,2'-O-trimethyl-cytidine (m ⁴ ₂ Cm), 1-thio-cytidine, 2'-F-arabinose-cytidine glycosides, 2'-F-cytidine, and 2'-OH-arabinose-cytidine.

In some embodiments, the modified nucleobase is modified adenine. Exemplary nucleobases and nucleosides with modified adenine include 2-amino-purine, 2,6-diaminopurine, 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-deaza-adenine , 7-desaza-8-aza-adenine, 7-desaza-2-amino-purine, 7-desaza-8-aza-2-amino-purine, 7-desaza-2, 6-Diaminopurine, 7-desaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m ¹ A), 2-methyl-adenine (m ² A ), N6-methyl-adenosine (m ⁶ A), 2-methylthio-N6-methyl-adenosine (ms ² m ⁶ A), N6-prenyl-adenosine (i ⁶ A) , 2-methylthio-N6-isopentenyl-adenosine (ms ² i ⁶ A), N6-(cis-hydroxyisopentenyl)adenosine (io ⁶ A), 2-methylthio- N6-(cis-hydroxyisopentenyl)adenosine (ms ² io ⁶ A), N6-glycinylamide methanoyl-adenosine (g ⁶ A), N6-threonylamine acylamine Formyl-adenosine (t ⁶ A), N6-methyl-N6-threonamide carboxyl-adenosine (m ⁶ t ⁶ A), 2-methylthio-N6-threonamide Aminoformyl-adenosine (ms ² g ⁶ A), N6, N6-dimethyl-adenosine (m ⁶ ₂ A), N6-hydroxyvalamine-methylaminoformyl-adenosine (hn ⁶ A), 2-Methylthio-N6-hydroxyvalinyl-aminoformyl-adenosine (ms ² hn ⁶ A), N6-acetyl-adenosine (ac ⁶ A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6, 2'-O-dimethyl-adenosine (m ⁶ Am), N6,N6,2'-O-trimethyl-adenosine (m ⁶ ₂ Am), 1,2'-O-dimethyl- Adenosine (m ¹ Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azide Nitrogen-adenosine, 2'-F-arabinose-adenosine, 2'-F-adenosine, 2'-OH-arabinose-adenosine, and N6-(19-amino-pentoxa-19 Alkyl ester)-adenosine.

In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides with modified guanine include inosine (I), 1-methyl-inosine (m ¹ I), Wyosine (imG), methyl Wyosine (mimG) , 4-desmethyl-wyosin (imG-14), iso-wyosin (imG2), wyosin (yW), peroxy-wyosin (o ₂ yW), hydroxywyosin (OHyW), unmodified hydroxywyosine (OHyW*), 7-deaza-guanosine, Q nucleoside (Q), epoxy Q nucleoside (oQ), galactosyl-Q nucleoside (galQ ), mannosyl-Q nucleoside (manQ), 7-cyano-7-deaza-guanosine (preQ ₀ ), 7-aminomethyl-7-deaza-guanosine (preQ ₁ ), ancient Purine (G ⁺ ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-guanosine -8-aza-guanosine, 7-methyl-guanosine (m ⁷ G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine Glycoside, 1-methyl-guanosine (m ¹ G), N2-methyl-guanosine (m ² G), N2,N2-dimethyl-guanosine (m ² ₂ G), N2,7-di Methyl-guanosine (m ^2,7 G), N2,N2,7-dimethyl-guanosine (m ^2,2,7 G), 8-side oxy-guanosine, 7-methyl-8 -Pendant 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 ² Gm), N2,N2-dimethyl -2'-O-methyl-guanosine (m ² ₂ Gm), 1-methyl-2'-O-methyl-guanosine (m ¹ Gm), N2,7-dimethyl-2'- O-methyl-guanosine (m ^2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m ¹ Im), and 2 '-O-Riboguanosine (phosphate) (Gr(p)).

The nucleobase of the nucleotide can be independently selected from purine, pyrimidine, purine analogs or pyrimidine analogs. For example, the nucleobases may each independently be selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, nucleobases may also include, for example, naturally occurring and synthetic derivatives of bases, 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- of adenine and guanine Propyl and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymus Pyrimidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen (e.g. 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and Other 8-substituted adenine and guanine, 5-halogen (specifically 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7 -Methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaguanine, 7-deazaguanine , 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 trioxone, 9-deazapurine, imidazo[4,5 -d]pyridine, thiazolo[4,5-d]pyrimidine, pyridine-2-one, 1,2,4-triphenidate, thiazolo[4,5-d]pyrimidine; and 1,3,5triphenidate.

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (eg, backbone structures) can be introduced at various positions in the polynucleotides described herein. Those skilled in the art will appreciate that nucleotide analogs or other modifications can be located at any position on a polynucleotide such that the function of the polynucleotide is not substantially reduced. The polynucleotides of the present disclosure 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, namely A, G, T/ any one or more of U or C) or any intermediate percentage (such as 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% 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%).

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be modified into circular nucleic acids. The ends of the polynucleotide can be connected by chemical reagents or enzymes to produce a cyclic polynucleotide without free ends. Circular polynucleotides are expected to be more stable than their linear counterparts and resistant to exonuclease digestion. Cyclic polynucleotides may further comprise other structural and/or chemical modifications regarding A, G, T/U or C ribonucleotides/deoxyribonucleotides.

In some embodiments, the polynucleotide is at least 50% modified, for example, at least 50% of the nucleotides are modified. In some embodiments, the polynucleotide is at least 75% modified, for example, at least 75% of the nucleotides are modified. It will be understood that since the nucleotides (sugar, base and phosphate moieties, such as bonds) may each be modified, any modification to any portion of the nucleotide or nucleoside will constitute a modification.

In some embodiments, the polynucleotide is modified in at least 10% of only one component of the nucleotides, where such component is a linkage between nucleobases, sugars, or nucleosides. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleobases, sugars, or linkages of the polynucleotides described herein may be modified. % modified.

As a non-limiting example, all uracil nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications may be the same or different. In some embodiments, all guanine nucleotides of the polynucleotide of the nucleic acid vaccine are modified. The modifications may be the same or different. In some embodiments, all guanine nucleotides of the polynucleotide of the nucleic acid vaccine are modified. The modifications may be the same or different. In some embodiments, all cytosine nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications may be the same or different. In some embodiments, all adenine nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications may be the same or different.

In one embodiment of the present disclosure, the polynucleotide of the nucleic acid vaccine is modified to include N1-methyl-pseudouridine nucleotide. sugar modification

Modified nucleosides and nucleotides that can be incorporated into polynucleotides (eg, RNA or mRNA, as described herein) can be modified on the sugars of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryl Oxy group; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl -C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; sugar (such as ribose, pentose or any sugar described herein); polyethylene glycol (PEG), -O (CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is 0 to 20 (e.g., 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) Integer; "locked" nucleic acid (LNA) in which the 2'-hydroxyl group is linked to the 4'-carbon of the same ribose by a C1-6 alkylene or C1-6 heteroalkylene bridge, where exemplary bridges include methylene base, propyl, ether or amine bridge; aminoalkyl; aminoalkoxy; amine; and amino acid.

In some embodiments, polynucleotides, such as the mRNA of the nucleic acid vaccines described herein, comprise at least one sugar modification. In general, RNA includes the sugar ribose as a 5-membered ring with oxygen. Exemplary non-limiting modified nucleotides include replacing the oxygen in ribose (e.g., with S, Se, or an alkylene group, such as methylene or ethylene); adding a double bond (e.g., with a cyclopentenyl group). or cyclohexenyl replacing ribose); Ring condensation of ribose (for example, to form a 4-membered ring of cyclobutane or oxygen); Ring expansion of ribose (for example, to form a 6- or 7-membered ring with additional carbons or heteroatoms) Rings such as anhydrous hexitol, altritol, mannitol, cyclohexyl, cyclohexenyl and N-𠰌linyl (also with an aminophosphate backbone); polycyclic forms (e.g. tricyclic and "unlocked" forms such as glycol nucleic acid (GNA) (e.g. R-GNA or S-GNA, in which ribose is replaced by a glycol unit linked to a phosphodiester bond), threose nucleic acid ( TNA, in which ribose is replaced by α-L-threose furanosyl-(3'→2')) and peptide nucleic acid (PNA, in which 2-amino-ethyl-glycine bond replaces ribose and phosphodiester) chain). A sugar moiety may also contain one or more carbons that have the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, polynucleotide molecules (including mRNA) as described herein may include molecules containing, for example, arabinose Nucleotides that are sugars.

Non-limiting examples of sugar modifications may include those provided in Table 5. Polynucleotides of the present disclosure may have one or more nucleotides carrying modifications as provided in Table 5. In some embodiments, each of the polynucleotides described herein carries any of the modifications as provided in Table 5, or does not carry any of the modifications as provided in Table 5 . Table 5. Nucleotide sugar modifications

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

The polynucleotides of the present disclosure may include any modification to the internucleoside linkages (eg, to the connecting phosphate/phosphodiester linkage/phosphodiester backbone). In the context of a polynucleotide backbone, the phrases "phosphate ester" and "phosphodiester" are used interchangeably. The backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. Additionally, modified nucleosides and nucleotides may include a complete replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphate, selenophosphate, boranophosphate, borano phosphate ester, hydrophosphonate, Amino phosphates, diamino phosphates, alkyl or aryl phosphonates and phosphate triesters. Both non-attached oxygens of phosphorodithioates are replaced by sulfur. Phosphate linkers can also be modified by replacing the connecting oxygen with nitrogen (bridging aminophosphate), sulfur (bridging phosphorothioate), and carbon (bridging methylene-phosphonate).

Alpha-thio-substituted phosphate moieties are provided to confer stability to RNA and DNA polynucleotides via non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently have a longer half-life in the cellular environment. Phosphorothioate-linked polynucleotide molecules are also expected to reduce innate immune responses through weaker binding/activation of cellular innate immune molecules.

In certain embodiments, modified nucleosides include alpha-thio-nucleosides (e.g., 5'-O-(1-phosphorothioate)-adenosine, 5'-O-(1-phosphorothioate) ester)-cytidine (α-thio-cytidine), 5'-O-(1-phosphorothioate)-guanosine, 5'-O-(1-phosphorothioate)-uridine or 5 '-O-(1-phosphorothioate)-pseudouridine).

In some embodiments, a polynucleotide contains at least one phosphorothioate or methylphosphonate linkage between nucleotides.

In some embodiments, the polynucleotide includes at least one 5'-( E )-vinyl phosphonate (5'- E -VP), a phosphate mimetic, as a modification.

In one embodiment of the present disclosure, the polynucleotide (eg, mRNA) of the COVID-19 nucleic acid vaccine can be modified. Price

The valence of the nucleic acid vaccines of the present disclosure may vary. "Valence" refers to the number of antigenic components in a nucleic acid vaccine or a polynucleotide of a nucleic acid vaccine. The antigenic components of a nucleic acid vaccine can be on the same polynucleotide or they can be on different polynucleotides. In some implementations, the nucleic acid vaccine can be monovalent. In some embodiments, the nucleic acid vaccine can be bivalent. In some implementations, the nucleic acid vaccine can be trivalent. In some embodiments, the nucleic acid vaccine can be multivalent, which can include 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. synthesis enzymatic method In vitro transcriptase synthesis

In vitro transcription (IVT) systems can be used to transcribe cDNA encoding the polynucleotides of the nucleic acid vaccines described herein. The system typically includes transcription buffer, nucleotide triphosphates (NTPs), ribonuclease inhibitors, and polymerases. NTP can be made in-house, selected from a supplier, or synthesized as described herein. The NTP may be selected from, but is 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.

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

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

Polynucleotide or nucleic acid synthesis reactions can be performed by enzymatic methods using polymerases. Polymerase catalyzes the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain. Currently known DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis. DNA polymerase I (pol I) or A polymerase family, including the Klenow fragment of Escherichia coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerase, and T7 RNA and DNA polymerase are among these families One of the most thoroughly studied families. Another large family is the DNA polymerase a (pol a) or B polymerase family, which includes all eukaryotic replicating DNA polymerases and polymerases from bacteriophages T4 and RB69. Although these polymerase families employ similar catalytic mechanisms, they differ in acceptor specificity, acceptor analog binding efficiency, extent and rate of primer extension, DNA synthesis mode, exonuclease activity, and sensitivity to inhibitors. There are differences in aspects. solid phase chemical synthesis

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be produced entirely or partially using solid phase techniques. 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 solution of reactants. Impurities and excess reagents are washed away and no purification is required after each step. Automation of this process can be performed on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows the rapid production of polynucleotides or nucleic acids on relatively large scales, making some commercially available.

In some embodiments, automated solid-phase synthesis is used, in which strands are synthesized in the 3' to 5' direction. The hydroxyl group in the 3' end of the nucleoside is tethered to the solid support via a chemically or photocleavable 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 support for binding of nucleosides. At the end of the synthesis, a cleavage agent such as ammonia or ammonium hydroxide is added to remove all protecting groups and release the polynucleotide strands from the solid support. Light can also be applied to cleave polynucleotide strands. The product can then be further purified using high pressure liquid chromatography (HPLC) or electrophoresis. liquid phase chemical synthesis

Synthesis of the polynucleotides of the nucleic acid vaccines described herein may be performed in liquid phase by the sequential addition of monomer building blocks. Covalent bonds are formed between monomers or between terminal functional groups of a growing chain and incoming monomers. Functional groups that do not participate in the reaction must be temporarily protected. After adding each monomer building block, the reaction mixture must be purified before adding the next monomer building block. The functional group at one end of the chain must be deprotected in order to react with the next monomer to build the block. Liquid-phase synthesis is time-consuming and laborious, and cannot be automated. Despite its limitations, solution-phase synthesis can be used to prepare short polynucleotides on a large scale. Since the system is homogeneous, it does not require large excesses of reagents and is cost-effective in this regard. Quantification and purification

In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be quantified in exosomes or when derived from one or more body fluids. As used herein, "body fluid" includes 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 pre-ejaculation fluid, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitium fluid, menstrual fluid, pus, sebum, vomitus, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, blastocoel fluid and umbilical cord blood. Alternatively, exosomes can be obtained from an organ selected from the group consisting of: lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testicle, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

In the exosome quantification method, samples of no more than 2 mL are obtained from individuals and separated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity purification, and microfluidic separation. or a combination thereof to isolate exosomes. In an assay, the level or concentration of the polynucleotide can be the expressed amount, presence, absence, truncation, or alteration of the administered construct. This level correlates favorably with analysis of one or more clinical phenotypes or with human disease biomarkers. The analysis can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, and for exosomes using immunohistochemistry, Separation methods such as enzyme-linked immunosorbent assay (ELISA). Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation or a combination thereof.

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 the endogenous form due to structural modifications.

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.). Quantified polynucleotides can be analyzed to determine whether the polynucleotide is of the appropriate size and to check that the polynucleotide has not been degraded. 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, reverse phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography-mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE).

Purification of polynucleotides of nucleic acid vaccines described herein may include, but is not limited to, polynucleotide clean-up, quality assurance, and quality control. Cleanup can be performed by methods known in the art, such as, but not limited to, AGEN-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, reverse phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC). The term "purified" when used in connection with a polynucleotide, such as "purified polynucleotide" refers to a polynucleotide that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that renders another unsuitable, impure or inferior. Accordingly, purified polynucleotides (such as DNA and RNA) exist in a form or configuration different from that found in nature or in a form or configuration different from that present before they are subjected to processing or purification methods. .

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

The nucleic acid vaccines described herein can be used as therapeutic or prophylactic agents. In some embodiments, the present disclosure provides pharmaceutical compositions that include at least one pharmaceutically acceptable carrier and a nucleic acid vaccine, that is, a nucleic acid vaccine for COVID-19. Therefore, pharmaceutical compositions including the nucleic acid vaccines described herein can be used to prevent, mitigate and/or treat COVID-19.

Provided herein are nucleic acid vaccines and pharmaceutical compositions thereof that can be used in combination with one or more pharmaceutically acceptable excipients. The pharmaceutical composition may optionally contain one or more additional active substances, for example therapeutic and/or prophylactic active substances. The pharmaceutical compositions of the nucleic acid vaccines described herein may be sterile and/or pyrogen-free.

In some embodiments, the compositions are 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, e.g., encoding one or more proteins, peptides, fragments thereof of SARS-CoV-2 or variant polynucleotides for preventing, mitigating and/or treating COVID-19.

Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as to non- Administration to human animals, such as non-human mammals. It is fully understood that pharmaceutical compositions suitable for administration to humans are modified in order to render the compositions suitable for administration to a variety of animals, and that an ordinarily skilled veterinary pharmacologist may use only ordinary experimental design and/or or make such modifications. 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. Preparations

Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, which as used herein include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid vehicles suitable for the particular dosage form desired, dispersion or Suspension aids, surfactants, isotonic agents, thickening or emulsifiers, preservatives and the like. Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st ed., A. R. Gennaro, Lippincott, Williams & Wilkins , Baltimore, MD, 2006; incorporated herein by reference in its entirety). Unless any conventional carrier medium is incompatible with the substance or its derivatives, such as produces any adverse biological effects or otherwise interacts in a deleterious manner with any other component of the pharmaceutical composition, the use of conventional carrier media is contemplated within the scope of this disclosure. Formulation medium.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological art or hereafter developed. Generally, such preparation methods include the steps of combining the active ingredient with an excipient and/or one or more other accessory ingredients, and then dividing, shaping and/or encapsulating the product as necessary and/or desired. Single dose or multiple dose units required.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and/or sold in bulk, in single unit dosage form, and/or in a plurality of single unit dosage forms. As used herein, a "unit dose" is a discrete quantity of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient will generally equal the dose of active ingredient to be administered to the individual and/or an appropriate fraction of this dose, such as one-half or one-third of such dose.

The relative amounts of active ingredients, pharmaceutically acceptable excipients, and/or any additional ingredients in pharmaceutical compositions according to the present disclosure will depend on the identity, size, and/or condition of the individual being treated and further upon the administration of the composition. changes along the way. 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%, at least 80% (w/w) of the active ingredient.

In some embodiments, the formulations described herein can contain at least one nucleic acid vaccine composition, such as a nucleic acid vaccine for COVID-19, such as an mRNA vaccine for COVID-19. As a non-limiting example, the formulation may contain 1, 2, 3, 4, or 5 nucleic acid vaccine compositions with different sequences, such as 1, 2, 3, 4, or 5 mRNA vaccine compositions with different sequences. . In some embodiments, the formulation contains at least two nucleic acid vaccine (eg, mRNA vaccine) compositions with different sequences. In some embodiments, the formulation contains at least three nucleic acid vaccine (eg, mRNA vaccine) compositions with different sequences. In some embodiments, the formulation contains at least four nucleic acid vaccine (eg, mRNA vaccine) compositions with different sequences. In some embodiments, the formulation contains at least five nucleic acid vaccine (eg, mRNA vaccine) compositions with different sequences.

The nucleic acid vaccine composition of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g., from storage of the nucleic acid vaccine composition library formulations); (4) alter biodistribution (e.g., targeting nucleic acid vaccine compositions to specific tissues or cell types); (5) increase translation of encoded proteins in vivo; and/or (6) alter expression of encoded proteins in vivo Release features.

In addition to traditional excipients (such as any and all solvents, dispersion media, diluents or other liquid vehicles), the present disclosure includes dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifiers, preservatives, etc. Agents and excipients may include but are not limited to lipids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nucleic acid vaccine compositions transfected (for example, for Transplanted into an individual) cells, hyaluronidase, nanoparticle mimics, and combinations thereof. Accordingly, the formulations of the present disclosure may include one or more excipients, each taken together to increase the stability of the nucleic acid vaccine composition and/or to increase the amount of cell transfection of the nucleic acid vaccine composition. In addition, the nucleic acid vaccine composition of the present disclosure can be formulated using self-assembled nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients and delivery agents for nucleic acids that can be used to formulate the nucleic acid vaccine compositions of the present disclosure are disclosed in PCT Patent Application Publication WO 2013/090648, the content of which is incorporated by reference in its entirety. method is incorporated into this article. lipidoid

The nucleic acid vaccine composition of the present disclosure can be formulated using one or more lipids.

The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suitable for delivery of 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; the contents of which are incorporated herein by reference in full).

Although these lipids have been used to effectively 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 which are incorporated herein by reference in their entirety), but this disclosure encompasses formulations thereof and delivery of at least one pharmaceutically acceptable carrier , including use in nucleic acid vaccines. Complexes, micelles, liposomes or particles can be prepared that contain such lipids and thus can effectively deliver nucleic acid vaccine compositions following injection of lipid formulations via local and/or systemic routes of administration. Lipid complexes containing nucleic acid vaccine compositions can be administered in various ways, including but not limited to intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), and intrathecal (IT) , or intracerebroventricular (ICV) administration.

In vivo delivery of nucleic acids can be affected by many parameters, including, but not limited to, formulation composition, nature of particle PEGylation, degree of loading, 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 changes in the length of the anchor chains of poly(ethylene glycol) (PEG) lipids can have significant effects on in vivo efficacy. Available for use with different lipid classes, including but not limited to penta[3-(1-lauraminopropyl)]-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 formulations of MD1 were tested for in vivo activity.

The lipid, referred to herein as "98N12-5", was disclosed by Akinc et al., Mol Ther. 2009, 17:872-879, the contents of which are incorporated herein by reference in their entirety.

Lipids, referred to herein as "C12-200", are 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 the documents are incorporated into this article by full-text citation. Lipoid formulations may include particles containing 3 or 4 or more components in addition to the nucleic acid vaccine composition. For example, formulations with certain lipidoids include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol, and 10% PEG (C14 alkyl chain length). As another example, formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% distearylphosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

In some embodiments, nucleic acid vaccine compositions formulated with lipidoids are used for systemic intravenous administration. For example, the final optimized intravenous formulation, which uses a nucleic acid vaccine composition and contains 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid, may result in greater than 90% distribution to the liver. composition, having a final weight ratio of total lipid:nucleic acid vaccine composition of approximately 7.5:1 and a C14 alkyl chain length on the PEG lipid, 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, an intravenous formulation using C12-200 lipidoids (see PCT Patent Application Publication WO2010129709, the contents of which are incorporated by reference in its entirety) may have a C12 of 50/10/38.5/1.5 -200/distearylphosphatidylcholine/cholesterol/PEG-DMG molar ratio, where a total lipid:nucleic acid weight ratio of 7:1 and an average particle size of 80 nm can effectively deliver nucleic acid vaccine compositions (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).

In some embodiments, formulations containing MD1 lipidoids can be used to effectively deliver nucleic acid vaccine compositions to hepatocytes in vivo. The properties of optimized lipidoid formulations for the intramuscular or subcutaneous route can vary significantly depending on the target cell type and the ability of the formulation to diffuse through the extracellular matrix into the bloodstream. Although due to the size of endothelial fenestrae, particle sizes less than 150 nm may be required for effective hepatocyte delivery (see Akinc et al., Mol Ther. 2009, 17:872-879, the contents of which are incorporated by reference in their entirety herein), but the use of lipid-formulated nucleic acid vaccine compositions to deliver formulations to other cell types (including but not limited to endothelial cells, bone marrow cells, muscle cells) may not be subject to similar size restrictions.

In vivo delivery of siRNA to other non-hepatocytes, such as myeloid cells and endothelial cells, has been reported using lipid-like formulations (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; The full text is incorporated into this article by reference). For efficient delivery to bone marrow cells, such as monocytes, lipidoid formulations may have similar molar ratios of components. Different ratios of lipids to other components, including but not limited to distearylphosphatidylcholine, cholesterol, and PEG-DMG, can be used to optimize the formulation of nucleic acid vaccine compositions for delivery to different cell types, including But it is not limited to liver cells, bone marrow cells, muscle cells, etc. For example, the molar ratio of components may include, but is not limited to, 50% C12-200, 10% distearylphosphatidylcholine, 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 lipidoid formulations may not require all formulation components required for systemic delivery, and thus may only include lipidoid and nucleic acid vaccine compositions. liposomes

The nucleic acid vaccine composition of the present disclosure can be formulated using one or more liposomes.

In some embodiments, the pharmaceutical composition of the nucleic acid vaccine composition includes liposomes. Liposomes are artificially prepared vesicles that may consist primarily of lipid bilayers and may be used as delivery vehicles for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes, such as, but not limited to, multilamellar vesicles (MLV), which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, small single cells, which can be less than 50 nm in diameter. vesicles (SUV) and large unilamellar vesicles (LUV) with diameters between 50 nm and 500 nm. Liposome design may include, but is not limited to, opsonins or ligands to improve liposome attachment to unhealthy tissue or activation events, such as, but not limited to, endocytosis. Liposomes can contain low or high pH to improve delivery of pharmaceutical formulations.

The formation of liposomes can depend on physicochemical characteristics such as, but not limited to, the coating pharmaceutical formulation and liposome ingredients; the nature of the medium in which the lipid vesicles are dispersed; the effective concentration of the coated substance and its potential toxicity; Any other processes involved during application and/or delivery of the vesicles; Optimization of the size, polydispersity and shelf life of the vesicles for the intended application; and Batch-to-batch reproducibility and large-scale production of safe and efficient lipids product possibilities.

In some embodiments, pharmaceutical compositions comprising nucleic acid vaccines described herein may include, but are not limited to, liposomes, such as those composed of 1,2-dioleyloxy-N,N-dimethylaminopropane ( Liposomes formed from DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), SMARTICLES®/NOV340 (Marina Biotech, Bothell), 1,2-dilinoleyloxy-3-dimethyl Aminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2- DMA) and MC3 (U.S. Patent Application Publication US20100324120; the contents of which are incorporated herein by reference in their entirety), neutral DOPC (1,2-dioleyl-sn-glyceryl-3-phosphocholine) based 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 lipids (Quiet Therapeutics, Israel) and liposomes that can deliver small molecule drugs, such as but not limited to DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

In some embodiments, pharmaceutical compositions comprising 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) , these particles 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; deFougerolles Hum Gene Ther. 2008, 19:125- 132; the contents of which are each incorporated herein in their entirety). The original fabrication method by Wheeler et al. was the detergent dialysis method, which was subsequently modified by Jeffs et al. and is known as the spontaneous vesicle formation method. In addition to nucleic acid vaccine compositions, liposome formulations can be composed of 3 to 4 lipid components. As a non-limiting example, liposomes may contain, but are not limited to, 55% cholesterol, 20% distearyl phospholipid choline (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy -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 lipid, wherein the cationic lipid may be 1,2-distearyl Oxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA or 1,2-dilinoxy-3-dimethylaminopropane (DLenDMA) as described by Heyes et al. . In another example, the nucleic acid-lipid particle may comprise from about 50 mol% to about 85 mol% of the cationic lipid of the total lipids present in the particle; from about 13 mol% to about 49.5 mol% of a noncationic lipid; and about 0.5 mol% to about 2 mol% of a conjugated lipid that inhibits particle aggregation, based on the total lipids present in the particle, as described in WO2009127060 to Maclachlan et al.; Its contents are incorporated herein by reference in their entirety. In another example, the nucleic acid-lipid particle can be any nucleic acid-lipid particle disclosed in US2006008910 to Maclachlan et al.; the contents of which are incorporated herein by reference in their entirety. As non-limiting examples, nucleic acid-lipid particles may include cationic lipids of Formula I, non-cationic lipids, and conjugated lipids that inhibit particle aggregation.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in lipid vesicles, which can have cross-links between functionalized lipid bilayers.

In some embodiments, liposomes may contain sugar-modified lipids as disclosed in US Pat. No. 5,595,756 to Bally et al., the contents of which are incorporated herein by reference in their entirety. The lipid may be gangliosides and cerebroglycerides in an amount of about 10 molar %.

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

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in lipid-polycation complexes. Formation of lipid-polycation complexes can be accomplished by methods known in the art and/or as described in US Publication No. 20120178702, the contents of which are incorporated herein by reference in their entirety. As non-limiting examples, polycations may include cationic peptides or polypeptides such as, but not limited to, polylysine, polyornithine, and/or polyarginine, and the cations described in PCT Patent Application Publication No. WO2012013326 Peptides; the contents of which are incorporated herein by reference in their entirety. In some embodiments, nucleic acid vaccine compositions can be formulated in lipid-polycation complexes, which can further include neutral lipids such as, but not limited to, cholesterol or dioleyl phospholipid acylylethanolamine (DOPE).

Liposome formulations can be affected by factors that are not limited to the selection of cationic lipid components, cationic lipid saturation, nature of PEGylation, ratios of all components, and biophysical parameters such as size. In one example from Semple et al. (Semple et al. Nature Biotech. 2010, 28:172-176; the contents of which are incorporated by reference in their entirety), the liposome formulation consisted of 57.1% cationic lipids, 7.1% dihydrogen It is composed of palmityl phospholipid choline, 34.3% cholesterol and 1.4% PEG-c-DMA.

In some embodiments, pharmaceutical compositions may be formulated with any of the amphoteric liposomes disclosed in PCT Patent Application Publication No. WO 2008043575 to Panzner and U.S. Patent No. 2 to Essler et al. (Marina Biotech) No. 8,580,297, the contents of which are incorporated herein by reference in their entirety. Amphipathic liposomes may contain a mixture of lipids including cationic amphiphilic molecules, anionic amphiphilic molecules, and optionally one or more neutral amphiphilic molecules. Amphiphilic liposomes may comprise amphipathic compounds based on amphiphilic molecules whose head groups are substituted with one or more amphiphilic groups. In some embodiments, pharmaceutical compositions may be formulated with amphipathic lipids containing one or more amphipathic groups having an isoelectric point of 4 to 9, as described in U.S. Patent Application Publication No. 1 to Essler et al. (Marina Biotech). No. US20140227345, the content of which is incorporated herein by reference in its entirety. In some embodiments, pharmaceutical compositions may be formulated with liposomes containing sterol derivatives as disclosed in U.S. Patent No. 7,312,206 to Panzner et al. (Novosom), the contents of which are incorporated by reference in their entirety. in this article. In some embodiments, the pharmaceutical composition may be an amphoteric liposome comprising at least one amphipathic cationic lipid, at least one amphiphilic anionic lipid, and at least one neutral lipid, or at least one having a positive charge and a negative charge. A liposome formulation of the two amphipathic lipids 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. 7780983 issued to Panzner et al. (Novosom), Its contents are incorporated herein by reference in their entirety. In some embodiments, pharmaceutical compositions can be formulated with liposomes capable of encapsulating the nucleic acid vaccine compositions of the present disclosure, such liposomes comprising the lipids taught in U.S. Patent Application Publication No. US 20110076322 to Panzner et al. of serum-stable mixtures, the contents of which are incorporated herein by reference in their entirety. The lipid mixture includes phosphatidyl choline and phosphatidyl ethanolamine in a ratio ranging from about 0.5 to about 8. The lipid mixture may also include pH-sensitive anionic and cationic amphiphilic molecules such that the mixture is amphipathic, negatively or neutrally charged 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, the lipid system loaded with the nucleic acid vaccine composition of the present disclosure as a load is prepared by the method disclosed in U.S. Patent Application Publication No. US 20120021042 issued to Panzner et al., the content of which is The full text is incorporated by reference into this article. The method includes the steps of mixing an aqueous solution of a polyanionic active agent and an alcoholic solution of one or more amphiphilic molecules and buffering the mixture to an acidic pH, wherein the one or more amphiphilic molecules are prone to form amphoteric liposomes at an acidic pH, thereby Formation of amphoteric liposomes in suspension encapsulating active agents lipoplex

The nucleic acid vaccine composition of the present disclosure can be formulated using one or more lipoplexes.

In some embodiments, the nucleic acid vaccine composition can be formulated as a lipoplex, such as but not limited to ATUPLEX ^™ system, DACC system, DBTC system and other siRNA-lipoplex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT ^TM 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 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 USA . 2007, 6;104:4095-4100; deFougerolles Hum Gene Ther. 2008, 19:125-132; Attribute each such content to its respective The full text is incorporated into this article by reference). Lipid Nanoparticles (LNP)

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in lipid nanoparticles (LNPs). In general, LNPs can be characterized as small solid or semi-solid particles that have an external lipid layer with a hydrophilic outer surface exposed to the non-LNP environment; they can be aqueous (vesicle-like) or non-aqueous ( micelle-like) internal space; and at least one hydrophobic intermembrane space. LNP films can be lamellar or non-lamellar, and can be composed of 1, 2, 3, 4, 5, or more layers. In some embodiments, an LNP can contain a load or payload into its interior space, into its intermembrane space, into its exterior surface, or any combination thereof.

LNPs suitable for use herein are known in the art and typically contain cholesterol (which aids in stability and promotes membrane fusion), phospholipids (which provide structure to the LNP bilayer and also aid in endosomal escape), polyethylene glycol Alcohol (PEG) derivatives (which reduce LNP aggregation and "protect" LNP from non-specific endocytosis by immune cells) and ionizable lipids (which complex with negatively charged RNA and enhance endosomal escape), which form LNP form composition.

The components of the LNP can be selected based on desired goals, tropism, loading, size, or other desired characteristics or properties.

The LNPs may be lipid nanoparticles as described in PCT Patent Application Publication No. WO2012170930, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in LNPs including at least one cationic lipid.

In some embodiments, cationic lipids useful in the formulations of the present disclosure may be selected from, but are not limited to, cationic lipids described in PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, Nos. WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, 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 which is incorporated herein by reference in its entirety. The cationic lipid may also be selected from, but is not limited to, Formula A as described in: PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259 No. WO2012054365 and WO2012044638; each of these contents is incorporated herein by reference in its entirety. Alternatively, the cationic lipid may be selected from, but is not limited to, Formula CLI-CLXXIX in PCT Patent Application No. WO2008103276, Formula CLI-CLXXIX in U.S. Patent No. 7,893,302, Formula CLI-CLXXXXII in U.S. Patent No. 7,404,969, and U.S. Patent Publication No. Formulas I-VI of US20100036115; each such content is incorporated herein by reference in its entirety. The cationic lipid may be a multivalent cationic lipid, such as those 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. Cationic lipids may have a positively charged head group including two quaternary amine groups and a hydrophobic moiety including four hydrocarbon chains, as described in US Patent No. 7,223,887 to Gaucheron et al. Cationic lipids can be biodegradable, such as those disclosed in US Patent Application Publication No. US20130195920 to Maier et al., the contents of which are incorporated herein by reference in their entirety. The cationic lipid 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 can also be US20130156845 to Manoharan et al., US 20130129785 to Manoharan et al., WO 2012047656 to Wasan et al., WO 2010144740 to Chen et al., WO 2010144740 to Ansell. et al., or WO 2012016184 to Manoharan et al., each of which is incorporated herein by reference in its entirety.

As a non-limiting example, the cationic lipid may be selected from the group consisting of (20Z,23Z)-N,N-dimethylecos-20,23-diene-10-amine, (17Z,20Z)-N,N- Dimethylpentadeca-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentadeca-16,19-diene-8-amine, (13Z,16Z )-N,N-dimethyldococ-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethyldococ-12,15-diene- 4-amine, (14Z,17Z)-N,N-dimethyltetracarbon-14,17-diene-6-amine, (15Z,18Z)-N,N-dimethyltetracarbon -15,18-diene-7-amine, (18Z,21Z)-N,N-dimethyl27-18,21-diene-10-amine, (15Z,18Z)-N,N -Dimethylicosac-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethylicosac-14,17-diene-4-amine, ( 19Z,22Z)-N,N-dimethyloctadecanoic-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethyloctadecanoic-18,21- Diene-8-amine, (17Z,20Z)-N,N-dimethylhexadecanoic-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethyldi Pentadecane-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethyltriadenocarbon-22,25-diene-10-amine, (21Z,24Z)- N,N-dimethyltriacona-21,24-diene-9-amine, (18Z)-N,N-dimethyltriacona-18-en-10-amine, (17Z)- N,N-dimethyloctadecanoic-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctadecanoic-19,22-diene-7-amine, N ,N-dimethylheptadecane-10-amine, (20Z,23Z)-N-ethyl-N-methyloctadecane-20,23-diene-l0-amine, 1-[( 11Z,14Z)-l-nonyleicosac-11,14-dien-l-yl]pyrrolidine, (20Z)-N,N-dimethyleicosac-20-en-l0-amine , (15Z)-N,N-dimethylnac-15-en-l0-amine, (14Z)-N,N-dimethylnac-14-en-l0-amine, ( 17Z)-N,N-dimethyltriacon-17-en-l0-amine, (24Z)-N,N-dimethyltriacon-24-ene-l0-amine, (20Z) -N,N-dimethylnonadenocarbon-20-en-l0-amine, (22Z)-N,N-dimethylnonadenocarbon-22-en-l0-amine, (16Z)-N ,N-dimethylpentac-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylpentac-12,15-diene-1 -Amine, (13Z,16Z)-N,N-dimethyl-3-nonyldococacan-l3,16-diene-l-amine, N,N-dimethyl-l-[(lS ,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10- Amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R )-2-octylcyclopropyl] unicosan-10-amine, N, N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentyl Cyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8- Amine, N,N-dimethyl-[(lR,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-dimethyloctadecyl Alk-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-l-[ (1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene- 1-yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1- baseoxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1- [(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yl Oxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-di En-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3 -[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl -3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3 -[(9Z,12Z)-octadecanoic-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadecanoic- 9-en-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadecane -6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-eicosan-11,14 -Dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z, 14Z)-Eicosane-11,14-diene-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-Eicosane-11,14 -Dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-bicos-13,16- Dien-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-bicos-13 ,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-bicos- 13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-dococ-13-ene-1- baseoxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadecan-9-en-1-yloxy]-N, N-Dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z, 12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy) Oxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan- 2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E , 20Z, 23Z)-N,N-dimethyl nonacarbo-l1,20,2-triene-10-amine, or its pharmaceutically acceptable salt or stereoisomer. Lipid Nanoparticle (LNP) Composition

In some embodiments, lipid nanoparticles can be composed 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, lipid nanoparticles can be composed 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, LNP can be composed of at least one cationic lipid, at least one non-cationic lipid, and at least one sterol. In some embodiments, LNP can be composed of at least one cationic lipid, at least one non-cationic lipid, and at least one additional LNP functional component. In some embodiments, LNP can be composed of at least one noncationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, LNP can be composed of at least one cationic lipid and at least one non-cationic lipid. In some embodiments, LNP can be composed of at least one cationic lipid and at least one sterol. In some embodiments, LNP can be composed of at least one cationic lipid and at least one additional LNP functional component. In some embodiments, LNP can be composed of at least one non-cationic lipid and at least one sterol. In some embodiments, LNP can be composed of at least one non-cationic lipid and at least one additional LNP functional component. In some embodiments, LNP can be composed of at least one sterol and at least one additional LNP functional component. In some embodiments, LNPs can be composed of at least one cationic lipid. In some embodiments, the LNP can be composed of at least one non-cationic lipid. In some embodiments, the LNP can be composed of sterols. In some implementations, LNP can be composed of additional LNP functional components.

In some embodiments, the at least one cationic lipid can include at least one ionizable cationic lipid, at least one amine lipid, at least one saturated cationic lipid, at least one unsaturated cationic lipid, at least one zwitterionic lipid, at least one polypeptide cationic lipids or any combination thereof. In some embodiments, the LNP may be substantially free of at least one cationic lipid. In some embodiments, LNPs may not contain any amount of at least one cationic lipid.

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-(l,2-bis-tetradecyloxy-propyl-3-dimethyl Ethoxylammonium bromide)-propan-2-ol ((R)-PLC-2)), 2-(dinonylamino)ethan-1-ol (17-10), 2-(di-deca) Dialkylamino)ethane-1-ol (17-11), 3-(di-dodecylamine)propan-1-ol (17-12), 4-(di-dodecylamine yl)butan-1-ol (17-13), 2-(hexyl((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (17- 2), 2-(nonyl((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (17-3), 2-(dodecane ((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (17-4), 2-(((9Z,12Z)-octadecanoic acid) -9,12-dien-1-yl)(tetradecyl)amino)ethan-1-ol (17-5), 2-(((9Z,12Z)-octadecane-9,12- Dien-1-yl)(octadecyl)amino)ethan-1-ol (17-6), 2-(ditetradecylamine)ethan-1-ol (17-7), 2-(Di((Z)-octadec-9-en-1-yl)amino)ethan-1-ol (17-8), (9Z,12Z)-N-(2-methoxyethane base)-N-((9Z,12Z)-octadeca-9,12-diene-1-yl)octadeca-9,12-diene-1-amine (17-9), N-nonane Base-N-(2-(piperidine-1-yl)ethyl)non-1-amine (19-1), N-dodecyl-N-(2-(piperidine-1-yl)ethyl) yl)dodecane-1-amine (19-2), (9Z,12Z)-N-((9Z,12Z)-octadecan-9,12-dien-1-yl)-N-(2 -(Piper-1-yl)ethyl)octadecy-9,12-diene-1-amine (19-3), N-dodecyl-N-(2-(4-methylpiper 𠯤-1-yl)ethyl)dodecyl-1-amine intermediate 1: 2-(di-dodecylamine)ethan-1-ol (19-4), N-dodecyl- N-(2-(4-(4-methoxybenzyl)piperidine-1-yl)ethyl)dodecane-1-amine (19-5), (9Z,12Z)-N-( 2-(4-Dodecylpiperidine-1-yl)ethyl)-N-((9Z,12Z)-octadecanoic-9,12-dien-1-yl)octadecanoic-9, 12-diene-1-amine (19-6), (3-((6Z,9Z,28Z,31Z)-trioctacarbon-6,9,28,31-tetraen-19-yloxy) -N,N-dimethylpropan-1-amine) (1-Bl 1), N-(2-(di-dodecylamine)ethyl)-N-dodecylglycine ( 20-1), 8,8'-((2-(Dodecyl(2-hydroxyethyl)amino)ethyl)ureadiyl)dioctanoic acid dinonyl ester (20-10), 3-( (2-(di-tetradecylamine)ethyl)(dodecyl)amino)propan-1-ol (20-11), 2-((2-(ditetradecylamine) Base)ethyl)(tetradecyl)amino)ethan-1-ol (20-12), 2-((2-(bis((9Z,12Z)-octadeca-9,12-diene) -1-yl)amino)ethyl)(dodecyl)amino)ethan-1-ol (20-13), 2-((2-(bis((9Z,12Z)-octadecane- 9,12-dien-1-yl)amino)ethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethan-1-ol (20- 14), 2-((2-(di-dodecylamine)ethyl)(hexyl)amino)ethan-1-ol (20-15), 2-((2-(dinonylamine) ethyl)(nonyl)amino)ethan-1-ol (20-16), 2-((2-(di-dodecylamine)ethyl)(nonyl)amino)ethyl -1-ol (20-17), 2-((2-(dinonylamino)ethyl)(dodecyl)amino)ethan-1-ol (20-18), 2-(( 2-(di-dodecylamine)ethyl)amino)ethan-1-ol (20-19), 6-(dodecyl(2-(dodecyl(2-hydroxyethyl) )Amino)ethyl)amino)pentylhexanoate (20-2), 2-((2-(di-dodecylamine)ethyl)(dodecyl)amino)ethyl- 1-ol (20-20), 3-((2-(di-dodecylamine)ethyl)(dodecyl)amino)propan-1-ol (20-21), 4- ((2-(di-dodecylamine)ethyl)(dodecyl)amino)butan-1-ol (20-22), (Z)-2-((2-(di- Dodecylamine)ethyl)dodec-6-en-1-yl)amino)ethan-1-ol (20-23), 2-((2-(di-dodecylamine Base)ethyl)(tetradecyl)amino)ethan-1-ol(20-24), 2-((2-(di-dodecylamine)ethyl)((9Z,12Z) -Octadec-9,12-dien-1-yl)amino)ethan-1-ol (20-25), 6-((2-(di-dodecylamine)ethyl)( 2-Hydroxyethyl)amino)pentylhexanoate (20-3), 6,6'-((2-(dodecyl(2-hydroxyethyl)amino)ethyl)ureadiyl) Dipentyl dicaproate (20-4), 6,6'-((2-((6-(heptyloxy)-6-side oxyhexyl)(2hydroxyethyl)amino)ethyl) Ureadiyl)diheptyl dihexanoate (20-5), 6-((2-(dinonylamino)ethyl)(2-hydroxyethyl)amino)pentylhexanoate (20-6 ), 6-(Dodecyl(2-(Dodecyl(2-hydroxyethyl)amino)ethyl)amino)heptylhexanoate (20-7), 8-((2-( Di-dodecylamine)ethyl)(2-hydroxyethyl)amino)octanoic acid nonyl ester (20-8), l8-((2-(di-dodecylamine)ethyl) (2-Hydroxyethyl)amino)octadecan-9-octanoate (20-9), 1-(2,2-di((9Z,12Z)-octadecan-9,12-diene- 1-yl)cyclopropyl)-N,N-dimethylmethylamine (21-1), 4-(dimethylamino)butanoic acid 3,3-bis((9Z,12Z)-octadecane -9,12-dien-1-yl)cyclobutyl ester (21-2), 3-(dimethylamino)propionic acid 3,3-bis((9Z,12Z)-octadecane-9, 12-Dien-1-yl)cyclopentyl ester (21-3), 4-(dimethylamino)butyric acid 3,3-bis((9Z,12Z)-octadeca-9,12-di En-1-yl)cyclopentyl ester (21-4), 1-(2,3-di((8Z,11Z)-heptadecan-8,11-dien-1-yl)cyclopropyl)- N,N-dimethylmethylamine (21-6), unknown (75-016B), poly{4-((2-(dimethylamino)ethyl)thio)tetrahydro-2H-pyridine Pyran-2-one}-r-poly{4-(octylthio)tetrahydro-2H-pyran-2-one}(A7), (3aR5s,6aS)-N,N-dimethyl-2, 2-Di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopent-1,3-dioxon-5-amine (ALN100), (3aR,5s, 6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3] Dioxyer-5-amine (ALN1001), ((3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadeca-9,12-diene (ALNY-100), tetrahydro-3aH-cyclopenta[d][1,3]dioxon-5-amine)) (ALNY-100), dimyristyltrimethylammonium propane (amine lipid 6), Benzamiπdiπ -Dialkyl-formic acid (BAADACA), N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), N,N-bis- (2-Hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonylamino-ethyl)ammonium bromide (BHEM-Chol1), 2-{4-[(3β)-cholesterol- 5-en-3-yloxy]butoxy}-iV?N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan -1-amine (butyl-CLinDMA), (2JR)-2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-Λr^dimethyl-3 -[(9Z,12Z)-octadeca-9,12-diene-l-yloxypropan-1-amine (butyl-CLinDMA (2R)), (25)-2-{4-[( 3β)-Cholesterol-5-en-3-yloxy]butoxy}-iVy/V-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-l -yloxy]propan-1-amine (butyl-CLinDMA (2S)), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl))amine Base)ethyl)(2-hydroxydodecyl)amino)ethyl)piperidine-l-yl)ethylureadiyl)di-dodecan-2-ol (C 12-200), 1 ,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl) Piperanyl-1-yl)ethyl)ureadiyl)bis(dodecan-2-ol)(C12-200), cholesteryl-succinylsilane (C2), bis(octadecane-9,12 -Dienoic acid)(9Z,9'Z,12Z,12'Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanyl )oxy)propane-1,3-diester (cationic lipid A2), octadeca-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-cholesteryl)-oxy Carbonyl-aminomethylimidazole (CHIM), [(2-morpholin-4-yl-ethylaminemethyl)methyl]-cholesterylcarbamic acid ester (Chol-C3N-Mo2), [(2-methyl Cholin-4-yl-ethylamineformyl)-ethyl]-carbamic acid cholesteryl ester Chol-DMC3N-Mo2[l-methyl-2-(2-morpholin-4-yl-ethylamineformyl) Cholesteryl)-propyl]-carbamic acid ester (Chol-C4N-Mo2), 4-(dimethylamino)butyric acid 1,17-bis(2-octylcyclopropyl)heptadecan-9- Ester (CL), 4-(dimethylamino)-butanoic acid 6,9,28,31-tetraene-19-ester (CL01), 3-(dimethylamino)propane Cholesteryl acid ester (CL06), 2-(dimethylamino)cholesteryl acetate (CL08), N,N-dimethyl-2,3-bis(((9Z,12Z)-octadecacarbon-9, 12-dien-1-yl)oxy)propan-1-amine (CL-1), N-methyl-2-(((9Z,12Z)-octadeca-9,12-diene-1) -yl)oxy)-N-(2-((((9Z,12Z)-octadeca-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-methylpyrrolidine (compound CL-12) (CL -12), 2-(dimethylamino)-N-((6Z,9Z,28Z,31Z)-37-6,9,28,31-tetraen-19-yl)acetamide (CL-13), (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoic acid)-3-(dimethylamino)propan-1,2- Diester (CL-14), (9Z,12Z)-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)amine (CL-15), di-dodecanoic acid 7-Hydroxy 7-(4-((1-methylpiperidin-4-carbonyl)oxy)butyl)tridecane-1,13-diester (CL15B6), 7-hydroxyditetradecanoate 7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diester (CL15C6), dipalmitic acid 7-hydroxy 7-(4-( (1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diester (CL15D6), dioleic acid 7-hydroxy 7-(4-((1-methylpiperdine) Bis(2-(((9Z,12Z)-octadeca-9,12-diene-1)bis(2-(((9Z,12Z)-octadeca-9,12-diene) -yl)oxy)ethyl)amine (CL-16), (9Z,12Z)-N-methyl-N-(2-(((9Z,12Z)-octadeca-9,12-diene) -1-yl)oxy)ethyl)octadeca-9,12-diene-1-amine (CL-17), (9Z,12Z)-N-(3-(((9Z,12Z)- Octadeca-9,12-diene-1-yl)oxy)propyl)octadec-9,12-diene-1-amine (CL-18), bis((11Z,14Z)-di Decacarbon-11,14-dien-1-yl)carbamic acid (1-methylpiperidin-3-yl)methyl ester (CL-19), N-methyl-N,N-bis(2-( (Z)-Hexadecacarbon-9-alkenyloxy)ethyl)amine (CL-2), (13Z,16Z)-N,N-dimethyl-4-((9Z,12Z)-octadeca Carbon-9,12-diene-1-yl)dococ-3,13,16-triene-1-amine (CL-20), (S)-2-amino-3-hydroxy-N ,N-bis(2-(((Z)-octadec-9-en-1-yl)oxy)ethyl)propanamide (CL-21), C2:N,N-di-hexadecan Alkyl-N'-(3-triethoxysilylpropyl)succinimide (CL3), trans-1-methyl-3,4-bis(((Z)-octadecacarbon- 9-en-1-yl)oxy)methyl)pyrrolidine (CL-3), bis(methylene)(9Z,9'Z,12Z,12'Z)-bis(octadecacarbon-9, 12-Dienoic acid) trans-1-methylpyrrolidine-3,4-diester (CL-4), ditetradecanoic acid 7-(4-(diisopropylamino)butyl) -7-Hydroxytridecane-1,13-diester (CL4C6), dipalmitic acid 7-(4-(diisopropylamino)butyl)-7-hydroxytridecane-1,13-di Ester (CL4D6), dioleic acid 11-(4-(diisopropylamino)butyl)-11-hydroxyhexadecane-1,21-diester (CL4H10), dioleic acid 7-(4 -(Diisopropylamino)butyl)-7-hydroxytridecane-1,13-diester (CL4H6), dioleic acid 9-(4-(diisopropylamino)butyl)- 7-Hydroxyheptadecane-1,17-diester (CL4H8), 4-(dimethylamino)butyric acid (6Z, 9Z, 28Z, 31Z)-trioctadecane-6,9,28,31 -Tetraene-19-ester (CL-5), 2-(dimethylamino)-N-(2-(((Z)-octadec-9-en-1-yl)oxy)ethyl base)-N-((9Z,12Z)-octadeca-9,12-dien-1-yl)acetamide (CL-53), 3-((2-(((Z)-octadeca) Carbo-9-en-1-yl)oxy)ethyl) ((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)propane-1-All (CL-54 ), 1-methyl-3,3-bis((((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)methyl)azetidine (CL- 55), 1-methyl-3,3-bis(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)ethyl)azetidine (CL-56), 1-methyl-3,3-bis(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)aza Cyclobutane (CL-57), 2-(3,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)azetidin-1-yl)ethane -1-ol (CL-58), 2-(3,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)azetidin-1-yl) Propan-1-ol (CL-59), 3-(bis((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)propan-1-ol (CL-6) , 3-(dimethylamino)propyl 3,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)azetidine-1-carboxylate (CL -60), 2-(bis((Z)-octadec-9-en-1-yl)amino)ethan-1-ol (CL-61), 3-(bis((Z)-octadeca-9-en-1-yl)amino) Carbo-9-en-1-yl)amino)propan-1-ol (CL-62), (11Z,14Z)-2-((dimethylamino)methyl)-2-((9Z, 12Z)-octadeca-9,12-dien-1-yl)eicos-11,14-dien-1-ol (CL-63), (11Z,14Z)-2-(dimethyl Amino)-2-((9Z,12Z)-octadeca-9,12-dien-1-yl)eicosac-11,14-dien-1-ol (CL-64), 3- (Dimethylamino)-2,2-bis((((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)methyl)propan-1-ol (CL -65), (9Z,12Z)-N-(2-(((Z)-octadec-9-en-1-yl)oxy)ethyl)octadec-9,12-diene- 1-amine (CL-7), 1-methyl-3,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)azetidine (CL-8 ), N,2-dimethyl-1,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propan-2-amine (CL-9 ), 3-dimethylamino-2-(cholest-5-ene-3B-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadiene Oxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-oxy)-3′-oxopentyloxy)-3-dimethyl-1-(cis, cis-9′,12′-octadecadienyloxy)propane (CpLinDMA), acetyltrimethylammonium bromide (CTAB), l^-diarachidonyloxy-^TV-di Methyl^-propyl-S-amine (DAraDMA), 0,0'-di-tetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14 ), 3β-[N-(N′,N′-dimethylaminoethane)aminomethanoyl]cholesterol (DC-Chol), dimethyldioctadecyl ammonium (DDA), dimethyl Di-octadecyl ammonium bromide (DDA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), l,2-di-docosahexaene Oxy-(7V,N-dimethyl)-propyl-3-amine (DDocDMA), N-(2-(dimethylamino)ethyl)-4,5-bis(dodecylsulfide) yl) pentylamine (DEDPA), 3-dimethylamino-2-(cholestero-5-ene-3β-oxypentan-3-oxa-an-5-oxy)-1-(cis , cis-9,12-octadecadienyloxy)propane (DEG-CLinDMA), 1,6-dioleyltriethylenetetraamide (dio-TETA), Nl,N19-bis(( S,23E,25E,27E,29E)-16-((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-l-ene -l-yl)nonan-2,4,6,8-tetraenylamide)-24,28-dimethyl-15,22-bisoxy-30-(2,6,6-trimethyl cyclohex-l-en-l-yl)-4,7,10-trioxa-14,21-diazatriaconta-23,25,27,29-tetraen-l-yl)- 4,7,10,13,16-pentaoxanonadecane-1,19-diamide (diVA-PEG-diVA), DiLin-N-methylpiperdine (DL-033), DiLin-N, N-dimethylglycine (DL-036), dioleyl-N,N-dimethylglycine (DL-048), 3-((1,3-bis(((9Z,12Z )-octadeca-9,12-dienyl)oxy)propan-2-yl)amino)propionic acid (DLAPA), 1,2-dilinoxy-3-dimethyl Aminopropane (DLenDMA), 1-linoleyl-2-linoleoxy-3-dimethylaminopropane (DLin-2-DMAP), 3-(N,N-dilinolein methylamino)-1,2-propanediol (DLinAP), 1,2-N,N'-dilinoleylaminemethanoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilincarbDAP Linseed oil acylaminoformyl-3-dimethylaminopropane (DLinCDAP), 1,2-dilinseed oil acylaminoformyloxy-3-dimethylaminopropane (DLin-C- DAP), 1,2-dilinoleoxy-3-(dimethylamino)acetyloxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-dimethyl Aminopropane (DLinDAP), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoxy-3-dimethylaminopropane Propane (DLinDMA 1), 1,2-dilinoleoxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), dilinoleyl -4-Aminobutyric acid (DLinFAB), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin- K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2- Dilinoleoxy-3-morpholinylpropane (DLin-MA), 4-(dimethylamino)butyric acid (6Z, 9Z, 28Z, 31Z) - 37 carbons - 6, 9, 28 , 31-tetraen-19-ester (DLin-MC3-DMA), l,2-dilinoleoxy-3-(N-methylpiperbenzoyl)propane (DLinMPZ), 1,2-dilinoleyl Dilinoleoxy-3-(N-methylpiperidinyl)propane (DLin-MPZ), Dilinoleoxy-3-piperidylpropylamine (DLinPip), 1.2 Dilinoleoxy-3-(3 '-Hydroxypiperidinyl)-propylamine (DLinPip-3OH), 1,2-dilinoleyloxy 3-(4'-hydroxypiperidinyl)-propylamine (DLinPip-4OH), 1,2-dilinoleoxy Oloxy-3-hydroxypropane (DLinPO), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l,2-dilinoleylthiopropane -3-Trimethylaminopropane (DLinTAP), 1,2-Dilinoleyl-3-trimethylaminopropane hydrochloride (DLin-TAP.Cl), 1,2-Dilinolein Oloxy-3-trimethylaminopropane (DLinTMA), 1,2-dilinoleoxy-3-trimethylaminopropane hydrochloride (DLin-TMA.Cl), 3-(( 1,3-bis(((9Z,12Z.15Z)-octadeca-9,12,15-trienyl)oxy)propan-2-yl)amino)propionic acid (DLLAPA), 1, 2 Dilinoleoxy 3-(N,N-dimethyl-propylamine) (DLmDEA), l,2-dilauryl-sn-glycerol-3-phosphoethanolamine (DLPE), l,2-di Lauryl-sn-glycerol-3-glycerol (DLPG), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), dimyristyl phospholipid serine ( DMPS), N-[l-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 1,2 -Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE1), l,2-dimyristyl-3-trimethylammonium propane (DMTAP), 3-(N ,N-dioleylamine)-1,2-propanediol (DOAP), 3-((1,3-bis(oleyloxy)propan-2-yl)amino)propionic acid (DOAPA), 1,2-N,N′-dioleylaminemethyl-3-dimethylaminopropane (DOcarbDAP), 1,2-dioleylaminemethyl-3-dimethylammonium- Propane (DOCDAP), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyl-3-dimethylammonium-propane (DODAP), N ,N-dihydroxyethyl N,N-dioctadecyl ammonium chloride (DODEAC), N,N-dimethyl-2,3-dioleoxypropylamine (DODMA), dioleyl -4-Aminobutyric acid (DOFAB), dioctadecylglycinolspermine (DOGS), 1,2-dioleyl-3-methyl-(methoxycarbonyl- Ethyl ammonium-propane (DOMCAP), 1,2-dioleyl-3-N-pyrrolidine-propane (DOP5P), 1,2-dioleyl-3-N-pyridinium-propane bromide Salt (DOP6P), 1,2-dioleyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl 1,2-dioleyloxypropyl-3-dimethyl-hydroxybutylammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl- 3-dimethyl-hydroxypropylammonium bromide (DORIE-HP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe), 2, 3-Dioleyloxy-N-[2(spermine-formamide)ethyl]-N,N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA), 1,3-di Olyloxy-2-(6-carboxy-sperminyl)-propylamide (DOSPER), N-(1-(2,3-dioleyloxy)propyl)-N,N,N -Trimethylammonium chloride (DOTAP), 1,2-dioleyl-3-trimethylammonium-propane (DOTAP1), N-[5'-(2',3'-dioleyl) Uridine]-N',N',N'-trimethylammonium tosylate (DOTAU), 1-[2-(9(Z)-octadecenyloxy)ethyl]-2- (8(Z)-Heptadecenyl-3-(2-hydroxyethyl)imidazolium chloride (DOTIM), N-(1-(2,3-dioleyloxy)propyl)-N ,N,N-trimethylammonium chloride (DOTMA), dioleyl phospholipid acyluridine phosphatidyl choline (DOUPC), 1,2-diphvtanyloxy (Diphvtanyloxy)-W.N-dimethyl) -Butan-4-amine (DPan-C2-DMA), l,2-diphytanyloxy (Diphytanyloxy)-3-(iV,7V-dimethyl)-propylamine (DPanDMA), (2-(dimethyl 2,3-bis(dodecylthio)propyl amino)ethyl)carbamate (DPDEC), dipalmitoyl-4-aminobutyric acid (DPFAB), 1,2-dipalmitoxy Propyl-3-dimethyl-hydroxyethylammonium bromide (DPRIE), 1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP), 1-[2-(hexadecanoyloxy )ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolium chloride (DPTIM), 3-((1,3-bis(stearyloxy)propan-2-yl) )Amino)propionic acid (DSAPA), distearyldimethylammonium (DSDMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA1), 1, 2-Distearyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE), l,2-distearyloxypropyl-3-trimethylammonium propane (DSTAP), di- Tetradecyltrimethylammonium (DTDTMA), 1,2-dioleyl-sn-glycerol-3-ethylphosphocholine (EDOPC), N2-[N2,N5-bis(3-aminopropane) base)-L-ormithyl]-N,N-di-octadecyl-L-glutamic acid tetrahydrotrifluoroacetate (GC33), cholesterol-5-en-3-ol (3P)-, 3-[(3-Aminopropyl)[4-[(3-aminopropyl)amino]butyl]carbamate] (GL67), glyceryl monooleate (GMO), guanidino- Dialkyl-formic acid (GUADACA), 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2 -Pendant oxyethyl ammonium bromide (HEDC), 2,2'-(tertiary butoxycarbonylureadiyl)bis(ethyl-2,1-diyl)ditetradecanoate (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-Tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yldisulfanyl)ethyl)guanidine ( HGT4002), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-diene-l-yl) tetracos-l5,18-di En-l-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-diene-l-yl) twenty-four Carbon-4,15,18-triene-l-amine (HGT5001), Histamine-Cholesterol Hemisuccinate (HisChol), Histamine-Cholesterol Hemisuccinate (Hist-Chol), HydroSoyPC (HSPC) , imidazole cholesteryl ester (ICE), 3-(di-dodecylamine)-N1,N1,4-tri-dodecyl-1-piperbenzoethylamine (KL10), N1-[2-( Di-dodecylamine)ethyl]-N1,N4,N4-tri-dodecyl-1,4-piperamide diethylamine (KL22), 14,25-di-tridecyl- 15,18,21,24-tetraaza-trioctadecane (KL25), N,N-di-n-tetradecyl, N-methyl-N-(2-guanidino)ethylammonium (lipid 1 ), N,N-di-n-octadecyl, N-methyl-N-(2-guanidino)ethylammonium chloride (lipid 2), (9Z,12Z)-octadecyl-9,12 -Dienoic acid 3-((4,4-bis(octyloxy)butyl)oxy)-2-((((3-(diethylamino)propyloxy)carbonyl)oxy)methyl )propyl ester (lipid A), (9Z,12Z)-octadeca-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyl)oxy)-2-(((( (3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (lipid A1), 2,2-dilinoleyl-4-dimethylaminoethyl-[ l,3]-dioxolane (lipid A2), bis(decanoic acid)((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy) )bis(octadecy-8,l-diyl)ester (lipid B), 9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoic acid) 2-((4 -(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diester (lipid C), 3-octylundecane Acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl ester (lipid D), 5-(dimethylamino) )Valerate (6Z,16Z)-12-((Z)-dec-4-en-1-yl)dococ-6,16-diene-11-ester (lipid I), dioctadecanoate Alkyl-(2-hydroxy-3-propylamino)amino polyionine (lipid T), (3-((6Z,9Z,28Z,31Z)-trioctadecane-6,9,28 ,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), as described in U.S. Provisional Application No. 61/384,050 (MC3 thioester), ( 4-((6Z,9Z,28Z,31Z)-Hexadecanoic-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 Ether), 3-((2-(((9Z,12Z)-octadeca-9,12-dienyl)oxy)ethyl)amino)propionic acid (MLAPA), 3-((2 -(((9Z,12Z,15Z)-octadeca-9,12,15-trienyl)oxy)ethylamino)propionic acid (MLLAPA), monomycolic acid (mon-omycolyl)glycerol (MMG ), 3-((2-(Oleyloxy)ethyl)amino)propionic acid (MOAPA), 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MoChol), 1,2-dioleyl-3-N-morpholine-propane (MoDO), methylpyridinyl-dialkyl-carboxylic acid (MPDACA), monopalmitoyl phosphatidylcholine (MPPC), 3-( (2-(stearyloxy)ethyl)amino)propionic acid (MSAPA), N1-[2-((lS)-1-[(3-aminopropyl)amino]-4-[ Bis(3-amino-propyl)amino]butylformamide)ethyl]-3,4-bis[oleoxy]-benzamide (MVL5), 2-({8-[(3β )-Cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene- 1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy yl)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2R) ), phosphatidylcholine (PC), l,3-bis-(l,2-bis-tetradecyloxy-propyl-3-dimethylethoxylammonium bromide)-propan-2-ol (PCL-2), palmityl-oleyl-n-arginine (PONA), stearylamine (STA), 2-(((tertiary butyldimethylsilyl)oxy)methyl )-2-(hydroxymethyl)propane-1,3-diol (Synthesis Example 1 (A)), (9Z)-tetradec-9-enoic acid 3-((tertiary butyl(dimethyl (Si)oxy)-2,2-bis(((9Z)-tetradec-9-enyloxy)methyl)propyl ester (Synthetic Example 1 (B)), (9Z)- Tetradec-9-enoic acid 3-hydroxy-2,2-bis(((9Z)-tetradec-9-enyloxy)methyl)propyl ester (Synthetic Example 1 (C)), ( 9Z)-tetradec-9-enoic acid 3-((4-(dimethylamino)butyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoic acid) methyl)propyl ester (Synthetic Example 1 (D)), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-(( 2-Hydroxydodecyl)(2-hydroxyundecanyl)amino)pentan-2-yl)-l,4-dimethane-2,5-dione) (Target 24), cocoon honey 6'6'-Dibehenate (TDB), 1,1'-(2-(4-(2-((2-(Bis(2hydroxydodecyl)amino)ethyl))(2 -Hydroxydodecyl)amino)ethyl)piperidine-1-yl)ethylureadiyl)di-dodecan-2-ol (Tech G1), 3-((1,3-bis( ((9Z,12Z)-octadeca-9,12-dienyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienyl)oxy )Methyl)prop-2-yl)amino)propionic acid (TLAPA), (l-(2,3-linseed oiloxypropoxy)-2-(linseed oiloxy)-(7V, Λ/-dimethyl)-propyl-3-amine) (TLinDMA), 3-((1,3-bis(((9Z.12Z.15Z))-octadeca-9.12.15-trienyl) )oxy)-2-((((9Z.12Z.15E)-octadeca-9,12,15-trienyl)oxy)methyl)propan-2-yl)amino)propionic acid (TLLAPA), N-(α-trimethylammonioacetyl)-di-dodecyl-D-glutamic acid chloride (TMAG), 3-((1,3-bis(((Z )-Octadec-9-enyl)oxy)-2-((((Z)-octadec-9-enyl)oxy)methyl)prop-2-yl)amine) Propionic acid (TOAPA), 3-((1,3-bis(stearyloxy)-2-((stearyloxy)methyl)propan-2-yl)amino)propionic acid (TSAPA) , 1,N19-bis((16E,18E,20E,22E)-17,21-dimethyl-15-side oxy-23-(2,6,6-trimethylcyclohex-1-ene- 1-yl)-4,7,10-trioxa-14-aza-16,18,20,22-tetraen-1-yl)-4,7,10,13,16- Pentaoxanonadecane-1,19-diamide (VA-PEG-VA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxy Heterocyclopentane (XTC), disclosed in Non-patent Document 11 (YSK05), 1,2-di-γ-linoleoxy-N,N-dimethylaminopropane (γ-DLenDMA) , a-D-tocopherol hemisuccinyl, bis(octadeca-9,12-dienoic acid)(9Z,9,Z,12Z,12,Z)-2-((2-(((3-( Dimethylamino)propoxy)carbonyl)oxy)tetradecyl)oxy)propan-1,3-diester, dioctanoic acid 2-(((13Z,16Z)-4-(((( 3-(diethylamino)propoxy)carbonyl)oxy)dococanoic-13,16-dienyl)oxy)propan-1,3-diester, dioctanoic acid 2-(( (13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)dococ-13,16-dienyl)oxy)propan-1, 3-Diester, dioctanoic acid 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanyl)oxy)propan-1,3 -Diesester, bis(decanoic acid) 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1 ,3-diester, bis(decanoic acid)2-((4-(((3-(diethylamino)propyloxy)carbonyl)oxy)hexadecanyl)oxy)propyl-1, 3-Diester, dioctanoic acid 2-(10-dodecyl-3-ethyl-8,14-bisoxy-7,9,13-trioxa-3-azaeicosane-20 -yl)propane-1,3-diester, (9Z,9′Z)bis-tetradec-9-enoic acid 2-(((4-(dimethylamino)butyl)oxy)methyl )-2-((octyloxy)methyl)propane-1,3-diester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z) -2-(((1-(cyclopropylmethyl)piperidine-4-carbonyl)oxy)methyl)propan-1,3-diester, bis(decanoic acid)((2-(((1 -Isopropylpiperidine-4-carbonyl)oxy)methyl)-1,4-phenylene)bis(oxy))bis(oct-8,1-diyl)ester, dodecanoic acid 2 -((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diester, dodecanoic acid 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propan-1,3-diester, dodecanoic acid 2- ((4-(((3-(Dimethylamino)propoxy)carbonyl)oxy)hexadecyl)oxy)propan-1,3-diester, ditetradecanoic acid 2- ((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diester, ditetradecane Acid 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diester, ditetradecane Acid 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diester, dioleic acid (Z )-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diester, bis(octadecyl) Carbon-9,12,15-trienoic acid)(9Z,9,Z,12Z,12,Z,15Z,15,Z)-2-((4-(((3-(dimethylamino)) Propoxy)carbonyl)oxy)hexadecyl)oxy)propyl-1,3-diester, bis(octadeca-9,12-dienoic acid)(9Z,9,Z,12Z,12 ,Z)-2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propan-1,3-diester, bis( Octadeca-9,12-dienoic acid)(9Z,9,Z,12Z,12,Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)) Oxy)hexadecanoyl)oxy)propane-1,3-diester, N,N,N-trimethyl-5-side oxy-5-(3-((3-pentyloctanoyl)oxy) base)-2,2-bis(((3-pentyloctyl)oxy)methyl)propoxy)pentane-1-ammonium iodide, 3-pentyloctanoic acid 3-((5-(dimethyl) Amino)pentyl)oxy)-2,2-bis(((3-pentyloctyl)oxy)methyl)propyl ester, 3-dimethylaminopropyl carbonate (9Z,12Z) -Octoc-19,22-diene-11-ester, (9Z,9′Z)bis-tetradec-9-enoic acid 2-(((N,N-dimethyl-β-propylamine acyl)oxy]methyl}-2-[(octyloxy)methyl)propane-1,3-diester, 8-dimethyldiosuberate ΟΊ,O1-(2-(7- Dodecyl-14-methyl-3,9-dioxy-2,4,8,10-tetraoxa-14-azapentadecyl)propan-1,3-diyl) ester , 8-dimethylΟΊ,01-(2-(((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propan-1,3-diyl) diosuberate, 8- Methyl suberic acid 1-(3-((6,6-bis((2-propylpentyl)oxy)hexyl)oxy)-2-(((1,4-dimethylpiperanoic acid) Biridin-4-carbonyl)oxy)methyl)propyl) ester, octadeca-9,12-dienoic acid (9Z,12Z)-5-(((3-(dimethylamino)propyloxy) Carbonyl)oxy)-7-octylpentadecyl ester, 5-(((3-(dimethylamino)propyloxy)carbonyl)oxy)-7-octylpentadecyl octanoate , 10-octylsebacic acid 1-(3-((6,6-bis((2-propylpentyl)oxy)hexyl)oxy)-2-(((1,4-di Methylpiperidine-4-carbonyl)oxy)methyl)propyl)ester, 3-(((3-(dimethylamino)propyloxy)carbonyl)oxy)-5-octyl decanoate Tridecyl ester, 8-methylsuberic acid 1-(16-((4,4-bis(octyloxy)butyl)oxy)methyl)-9-dodecyl-2-methyl -7,13-dilateral oxy-6,8,12,14-tetraoxa-2-azaheptadecane-17-yl) ester, (9Z,12Z)-octadecacarbon-9,12- Dienoic acid 3-((5-(dimethylamino)pentylyl)oxy)-2,2-bis(((9Z)-tetradec-9-enyloxy)methyl)propane Ester, 3-pentyloctanoic acid 3-((5-(dimethylamino)pentylyl)oxy)-2,2-bis((3-pentyloctanoyl)oxy)methyl)propyl ester , bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((3-(diethylamino)propyl)oxy) Methyl)propane-1,3-diester, bis(decanoic acid)((2-(((4-(dimethylamino)butyl)oxy)methyl)-1,4-phenylene) Bis(oxy))bis(oct-8,1-diyl)ester, 8-methylsuberic acid 1-(3-((4,4-bis(octyloxy)butyl)oxy)oxy)-2 -(((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) ester, 1-methylpyrrolidine-3-carboxylic acid 3-((4,4-bis(octyloxy)) Butylyl)oxy)-2-((palmityloxy)methyl)propyl ester, 1-methylpyrrolidine-3-carboxylic acid 3-((4,4-bis(octyloxy)butyl)oxy) -2-((tetradecanoyloxy)methyl)propyl ester, 9-pentyltetradecanoate 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)- 13-(octyloxy)tridecyl ester, 1-methylpyrrolidine-3-carboxylic acid 3-((4,4-bis(octyloxy)butyl)oxy)-2-(dodecane Cyloxy)methyl)propyl ester, 9-pentyltetradecanoate 3-(((3-(dimethylamino)propyloxy)carbonyl)oxy)-13-hydroxytridecyl ester, 7-Hexyltridecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octyloxy)tridecanoic acid ester, dioctanoic acid 2-(5 -(3-((1-methylpyrrolidin-3-carbonyl)oxy)-2-((tetradecanoyloxy)methyl)propoxy)-5-pentyloxypentyl)propyl- 1,3-diester, 5-heptyldodecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octyloxy)tridecane Ester, dioctanoic acid 2-(5-(3-((1-methylpyrrolidine-3-carbonyl)oxy)-2-((palmitoyloxy)methyl)propoxy)-5-side oxygen Pentyl)propyl-1,3-diester, 5-heptyldodecanoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecanoic acid Alkyl ester, bis(6,6-bis(octyloxy)hexanoic acid) 2-(((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propan-1,3-diester, Octacarbon-9,12-dienoic acid (9Z,12Z)-3-(((3-dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecane Ester, (9Z)-Octadec-9-enoic acid 3-((5-(dimethylamino)pentyl)oxy)-2,2-bis((9Z)-tetradecano- 9-enyloxy)methyl)propyl ester, 2-(10-dodecyl-3-ethyl-8,14-bisoxy-7,9,13-trioxa-3 dioctanoate -Azanonadecan-19-yl)propan-1,3-diester, bis(decanoic acid)((2-(((1-methylpiperidin-4-carbonyl)oxy)methyl)- 1,4-phenylene)bis(oxy))bis(oct-8,1-diyl) ester, bis(4,4-bis(octyloxy)butyrate) 2-(((3-( Dimethylamino)propyl)oxy)methyl)propyl-1,3-diester, octadeca-9,12-dienoic acid (9Z,12Z)-2-(((11Z,14Z )-2-((3-(dimethylamino)propyl)oxy)eicosan-11,14-dien-1-yl)oxy)ethyl ester, bis(4,4-bis (Octyloxy)butyrate) 2-(((1,3-dimethylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3-diester, succinic acid (13Z,16Z)- 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)dococane-13,16-diene-1-ester heptadecan-9-ester, 3-(( 3-Ethyl-10-((9Z,12Z)-octadeca-9,12-dien-1-yl)-8,15-bisoxy-7,9,14-trioxa-3 -Azaheptadecan-17-yl)disulfanyl)propionic acid 2,2-bis(heptyloxy)ethyl ester, bis(4,4-bis(octyloxy)butyric acid) 2-(( (1-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3-diester, 10-octylsebacic acid 1-(3-((1,3-dimethylpyrrolidine) -3-carbonyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienyloxy)methyl)propyl) ester, 2,2-bis(heptyloxy) )acetic acid (13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)dococane-13,16-diene-1-ester, 2,2 -Bis(heptyloxy)acetic acid (13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)dococane-13,16-diene-1 -Ester, acetic acid (20,23R)-2-methyl-9-[(9Z,12Z)-octadeca-9,12-dien-1-yl]-7-side oxy-6,8, 11-Trioxa-2-aza-20-ene-23-ester, 3-(dimethylamino)propyl carbonate (11Z,14Z)-1-{[(9Z,12R) -12-hydroxyoctadeca-9-en-1-yl] ester, 3-(dimethylamino)propionic acid (12Z,15Z)-1-(((9Z,12Z)-octadeca- 9,12-diene-1-yloxy)carbonyl)oxy)octadecanoic-12,15-diene-3-ester, octadeca-9,12-dienoic acid (9Z,12Z) -3-((4,4-bis(octyloxy)butyl)oxy)-2-((((3-(dimethylamino)propyl)aminoformyl)oxy)methyl) Propyl ester, 9-pentyltetradecanoate (12Z,15Z)-3-((4-(dimethylamino)butyl)oxy)ecosan-12,15-diene-1-ester , Octadeca-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-((((1,2,2 ,6,6-pentamethylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl ester, 7-hexyltridecanoic acid (12Z,15Z)-3-((4-(di Methylamino)butyl)oxy)octadecanoic-12,15-diene-1-ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-((4,4 -Bis(octyloxy)butyl)oxy)-2-(((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)methyl)propyl ester, 5-heptyl Dodecanoic acid (12Z,15Z)-3-((4-(dimethylamino)butyl)oxy)octadecanoic acid-12,15-diene-1-ester, octadecanoic acid-9, 12-dienoic acid(9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-((((1-ethylpiperidin-4-yl)oxy) base)carbonyl)oxy)methyl)propyl ester, 3-octyl undecanoic acid (12Z,15Z)-3-((4-(dimethylamino)butyl)oxy)octane- 12,15-diene-1-ester, formate, (9Z)-hexadec-9-enoic acid 3-((5-(dimethylamino)pentyl)oxy)-2, 2-bis(((9Z)-tetradec-9-enyloxy)methyl)propyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-((4,4 -Bis(octyloxy)butyl)oxy)-2-(((((1-methylazetidin-3-yl)oxy)carbonyl)oxy)methyl)propyl ester, Carbon-9,12-dienoic acid (9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propyl)oxy)octane-12,15- Diene-1-ester, 4,4-bis((2-ethylhexyl)oxy)butyrate 2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecane Alkyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-((((1-methyl Piperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyl) Oxy)butyl)oxy)-2-(((((1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)methyl)propyl ester, octadeca-9,12-di (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl ester, octadecyl-9,12-dienoic acid (9Z, 12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-((((3-(4-methylpiperidine-1-yl)propoxy)carbonyl)oxy methyl)propyl ester, 3-(dimethylamino)propyltriacontan-11-yl carbonate triacontan-11-ol, octadeca-9,12-dienoic acid (9Z, 12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl )propyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecane Ester, 4-((diethylamino)methyl)benzoic acid 3-((4,4-bis(octyloxy)butyl)oxy)-2-(((9Z,12Z)-octadecane -9,12-Dienyloxy)methyl)propyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy) Carbonyl)oxy)pentadecyl ester, 3-((dimethylamino)methyl)benzoic acid 3-((4,4-bis(octyloxy)butyl)oxy)-2-( ((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-(((3 -(Dimethylamino)propoxy)carbonyl)oxy)pentadecyl ester, 1-methylpiperidine-3-carboxylic acid 3-((4,4-bis(octyloxy)butyl)oxy) )-2-(((9Z,12Z)-octadeca-9,12-dienyloxy)methyl)propyl ester, 1-methylpiperidine-4-carboxylic acid 3-((4,4- Bis(octyloxy)butyloxy)-2-(((9Z,12Z)-octadeca-9,12-dienyloxy)methyl)propyl ester, 1,4-dimethylpiper 3-((4,4-bis(octyloxy)butylyl)oxy)-2-(((9Z,12Z)-octadecanoic acid-9,12-dienyloxy)methyl yl)propyl ester, (9Z)-hexadeca-9-enoic acid 3-((4-(dimethylamino)butyl)oxy)-2,2-bis(((9Z)-tetradecacarbonate) -9-enyloxy)methyl)propyl ester, dioctanoic acid 2-(10-dodecyl-3-ethyl-8,14-bisoxy-7,9,13-trioxa- 3-Azahexadecane-16-yl)propane-1,3-diester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z, 12Z,12'Z)-2 -(((4-(piperidin-1-yl)butyl)oxy)methyl)propane-1,3-diester, 4-methylmorpholine-2-carboxylic acid 3-((4,4-bis (Octyloxy)butyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienyloxy)methyl)propyl ester, 1-methylpyrrolidine-2- Formic acid (2R)-3-((4,4-bis(octyloxy)butylyl)oxy)-2-(((9Z,12Z)-octadecanoic acid-9,12-dienyloxy)methane yl)propyl ester, 1-methylpyrrolidine-2-carboxylic acid (2S)-3-((4,4-bis(octyloxy)butyl)oxy)-2-(((9Z,12Z)-ten Octacarbon-9,12-dienoyloxy)methyl)propyl ester, bis(octacarbon-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-( (((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((9Z,12Z)-octadeca-9,12-dienyloxy)methyl base) propane-1,3-diester, octadecanoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2- (((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl ester, 1-(cyclopropylmethyl)piperidine-4-carboxylic acid 3-(( 4,4-bis(octyloxy)butyloxy)-2-(((9Z,12Z)-octadeca-9,12-dienyloxy)methyl)propyl ester, 1-isopropyl Piperidine-4-carboxylic acid 3-((4,4-bis(octyloxy)butyl)oxy)-2-(((9Z,12Z)-octadecanoic acid-9,12-dienyloxy) )Methyl)propyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyl)oxy)-2-(((3 -(Dimethylamino)propyl)oxy)methyl)propyl ester, 4-(dimethylamino)butyl carbonate (6Z, 9Z, 26Z, 29Z) - tripentacarbon-6, 9,26,29-tetraen-18-ester, (9Z)-tetradec-9-enoic acid 3-((6-(dimethylamino)hexyl)oxy)-2,2- Bis(((9Z)-tetradecacarbon-9-enyloxy)methyl)propyl ester, 3-(dimethylamino)propyl carbonate 2,5-bis((9Z,12Z)-octadecanate Carbon-9,12-dieneoxy)benzyl ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((4- (pyrrolidin-1-yl)butyl)oxy)methyl)propyl-1,3-diester, 5-heptyldodecanoic acid 3-(((3-(dimethylamino)propoxy) )carbonyl)oxy)pentadecyl acetate, (7R,9Z)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)octadecane-9-ene- 7-ester, 9-pentyltetradecanoate 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl ester, octadeca-9,12-diene Acid (9Z,12Z)-3-((6,6-bis(octyloxy)hexyl)oxy)-2-(((3-(diethylamino)propoxy)carbonyl) Oxy)methyl)propyl ester, 7-hexyltrideca-6-enoate 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl ester, octadecyl Carbon-9,12-dienoic acid (9Z,12Z)-3-(2,2-bis(heptyloxy)acetyloxy)-2-((((2-(dimethylamino)ethyl) Oxy)carbonyl)oxy)methyl)propyl ester, 3-octyldodeca-2-enoic acid 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)deca Pentaalkyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((( 5-heptyldodecyl)oxy)methyl)propyl ester, 3-octylundecanoate 3-(((3-dimethylamino)propoxy)carbonyl)oxy)deca Pentaalkyl ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((( 9-Pentyltetradecyl)oxy)methyl)propyl ester, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl diacetate }oxy)tripentacarbon-9,26-diene-7,29-diester, 8,8-bis((2-propylpentyl)oxy)octanoic acid 3-(((3-(di Methylamino)propoxy)carbonyl)oxy)pentadecyl ester, octadecyl-9,12-dienoic acid (9Z,12Z)-3-(((3-(diethylamino) Propoxy)carbonyl)oxy)-2-(((7-hexyltridecyl)oxy)methyl)propyl ester, 8,8-bis((2-propylpentyl)oxy) 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl octanoate, octadeca-9,12-dienoic acid (9Z,12Z)-3 -(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3-octylundecanyl)oxy)methyl)propyl ester, 8,8 - 3-(((3-(diethylamino)propyloxy)carbonyl)oxy)pentadecyl bis((2-propylpentyl)oxy)octanoate, 8,8-dibutoxy 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl octanoate, (9Z)-tetradec-9-enoic acid 3-((5-(di Methylamino)pentyl)oxy)-2,2-bis(((9Z)-tetradec-9-enyloxy)methyl)propyl ester, 3-(dimethylamino) Propyl carbonate (6Z, 9Z, 26Z, 29Z)-tripentacarbon-6,9,26,29-tetraene-18-ester, 3-(dimethylamino)propionic acid 2,5-bis( (9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl ester, bis(octadeca-9,12-dienoic acid) (9Z,9'Z,12Z, 12'Z)-2-(((3-(4-methylpiperidine-1-yl)propyl)oxy)methyl)propan-1,3-diester, 8,8-bis(octyl) Oxy)octanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl ester, 3-(dimethylamino)propyloctadecane-11 carbonate -Ester, 2,4-bis((9Z,12Z)-octadeca-9,12-dienyloxy)benzyl 4-(dimethylamino)butyrate, octadeca-9,12 -Dienoic acid (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((2-heptylundecanyl)oxy) methyl)propyl ester, 6,6-bis((2-ethylhexyl)oxy)hexanoic acid 3-(((3-(diethylamino)propoxy)carbonyl)oxy)deca Pentaalkyl ester, bis(2-heptylundecanoic acid) 2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propan-1,3-diester , 6,6-bis(hexyloxy)hexanoate 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl ester, 4-(dimethylamino) 4-Methyl-2,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl butyrate, 6,6-bis(octyloxy) 3-(((3-(diethylamino)propyloxy)carbonyl)oxy)pentadecyl hexanoate, 4-methyl-2,5-bis((9Z,12Z)-octadecane -9,12-Dienyloxy)benzylcarbonate 4-(dimethylamino)butyl ester, 4,4-bis((2-propylpentyl)oxy)butyrate 3-((( 3-(Dimethylamino)propoxy)carbonyl)oxy)pentadecyl ester, 2-(12-dodecyl-3-ethyl-8,14-bis-pentyloxy-7 dioctanoate) ,9,13-trioxa-3-azaoctadecan-18-yl)propan-1,3-diester, dioctanoic acid 2-(5-side oxy-5-((3-((( 3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)pentyl)propan-1,3-diester, 4-methyl-2,5-bis( (9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzylcarbonate 3-(dimethylamino)propyl ester, 4,4-bis((2-propyl) Pentyl)oxy)butyrate 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl ester, 2-(11-dodecyl dioctanoate) -3-Ethyl-9,15-bisoxy-8,10,14-trioxa-3-azanonadecan-19-yl)propan-1,3-diester, dioctanoic acid 2- (10-Dodecyl-3-ethyl-8,15-bisoxy-7,9,14-trioxa-3-azanonadecan-19-yl)propan-1,3- Diester, dioctanoic acid 2-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-side oxygen Pentyl)propane-1,3-diester, dioctanoic acid 2-(5-((4-(((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)hexadecanoic acid Alkyl)oxy)-5-Pendant oxypentyl)propane-1,3-diester, dioctanoic acid 2-(5-((4-((((R)-1-methylpyrrolidine- 3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-pentyloxypentyl)propane-1,3-diester, dioctanoic acid 2-(5-((4-( ((((S)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-pentyloxypentyl)propan-1,3-di Ester, dioctanoic acid 2-(5-side oxy-5-((4-((S)-pyrrolidine-2-carbonyl)oxy)hexadecyl)oxy)pentyl)propyl-1, 3-diester, dioctanoic acid 2-(5-((4-((1,3-dimethylpyrrolidine-3-carbonyl)oxy)hexadecyl)oxy)-5-pentanoxypentanyl yl)propane-1,3-diester, dioctanoic acid 2-(5-((4-((1,4-dimethylpiperidin-4-carbonyl)oxy)hexadecyl)oxy)- 5-Pendantoxypentyl)propyl-1,3-diester, 4,4-bis(octyloxy)butyl(3-(diethylamino)propyl)pentadecan-1 dicarbonate, 3-Diester, 4,4-bis((2-propylpentyl)oxy)butyrate 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecane Ester, bis(decanoic acid)((2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-1,4-phenylene)bis(oxy) ) bis(oct-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)propyloxy)carbonyl)oxy) Hexyl ester, 6,6-bis(octyloxy)hexanoate (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)octane-12 ,15-diene-1-ester, 6,6-bis(octyloxy)hexanoate 3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl ester, 6,6-Bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl ester, 5-(4,6-diheptyl -1,3-Diethane-2-yl)pentanoate 3-(((3-(diethylamino)propyloxy)carbonyl)oxy)pentadecyl ester, 6,6-bis(octyl) Oxy)hexanoate 3-((5-(diethylamino)pentyl)oxy)pentadecyl ester, 1,4-dimethylpiperidine-4-carboxylic acid 1-((6,6 -Bis(octyloxy)hexyl)oxy)pentadecan-3-ester, 6,6-bis(octyloxy)hexanoic acid 3-((3-(1-methylpiperidine-4-) 1-((6,6-bis(octyloxy)hexyl)oxy)pentadecyl ester, 1,3-dimethylpyrrolidine-3-carboxylate Alk-3-ester, 4,4-bis((2-ethylhexyl)oxy)butyrate 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecane Ester, bis(8-(octanoyloxy)octanoic acid) 2-(((1,3-dimethylpyrrolidine-3-carbonyl)oxy)methyl)propan-1,3-diester, bis( Decanoic acid) ((2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)-1,4-phenylene)bis(oxy))bis(octyl) -8,1-diyl) ester, pyrrolidine-2-carboxylic acid (2R)-1-((6,6-bis(octyloxy)hexyl)oxy)pentadecan-3-ester, 1 -Methylpyrrolidine-2-carboxylic acid (2S)-1-((6,6-bis(octyloxy)hexyl)oxy)pentadecan-3-ester, 1-methylpyrrolidine-2 -Formic acid (2R)-1-((6,6-bis(octyloxy)hexyl)oxy)pentadecan-3-ester, 6,6-bis((3-ethylpentyl)oxy 3-(((3-(dimethylamino)propyloxy)carbonyl)oxy)pentadecyl hexanoate, 6,6-bis((2-propylpentyl)oxy)hexyl 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl acid ester, 6,6-bis((2-propylpentyl)oxy)hexanoic acid 3- (((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl ester, 6,6-bis(octyloxy)hexanoate 3-(((2-(diethylamine) ethoxy)carbonyl)oxy)pentadecyl ester, 6,6-bis(octyloxy)hexanoate 3-(((3-morpholinylpropoxy)carbonyl)oxy)pentadecane Ester, 6,6-bis(octyloxy)hexanoate 3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecyl ester, 6,6-bis (Octyloxy)hexanoic acid 3-(((3-(4-methylpiperidine-1-yl)propoxy)carbonyl)oxy)pentadecyl ester, 4,4-bis(octyloxy) 3-(((3-(diethylamino)propyloxy)carbonyl)oxy)pentadecyl butyrate, (9Z,9′Z)bis-tetradec-9-enoic acid 2-( ((4-(dimethylamino)butyl)oxy)methyl)-2-((dodecyloxy)methyl)propan-1,3-diester, bis(octadecanoic-9) ,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((4-(dimethylamino)butyl)oxy)methyl)propan-1,3-di Ester, 6,6-bis(octyloxy)hexanoate 3-(((4-(diethylamino)butoxy)carbonyl)oxy)pentadecyl ester, 6,6-bis(octyloxy) 3-(((3-(pipienyl)-1-yl)propoxy)carbonyl)oxy)pentadecyl hexanoate, 6.6-bis(octyloxy)hexanoic acid 3-(((3- Piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl ester, 4,4-bis(octyloxy)butyrate 3-(((3-(dimethylamino)propoxy) )carbonyl)oxy)pentadecyl ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(9-dodecyl-2 -Methyl-7,12-bisoxy-6,8,13-trioxa-2-azatetradecan-14-yl)propan-1,3-diester, octadecane-9, 12-dienoic acid (9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadecanoic acid-9,12-dienoyloxy)ethyl base)-8,13-dilateral oxy-7,9-dioxa-3,14-diazahexadecane-16-ester, dioctanoic acid 2-((2-(((3-(di Ethylamino)propoxy)carbonyl)oxy)tetradecyl)oxy)propan-1,3-diester, dioctanoic acid 2-(9-dodecyl-2-methyl-7 ,13-dilateral oxy-6,8,12-trioxa-2-azanonadecan-19-yl)propan-1,3-diester, (9Z,9′Z)bis-tetradecane Carbon-9-enoic acid 2-((decyloxy)methyl)-2-(((4-(dimethylamino)butyl)oxy)methyl)propane-1,3-diester, Bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((3-morpholinopropyl)oxy)methyl)propan- 1,3-diester, 3-(dimethylamino)propyl carbonate (6Z, 9Z, 28Z, 31Z)-37-6,9,28,31-tetraene-19-ester, 4 -(Dimethylamino)butyric acid 2,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl ester, dioctanoic acid 2-(10- Dodecyl-3-ethyl-8,14-bisoxy-7,9,13-trioxa-3-azaoctadecan-18-yl)propan-1,3-diester, Bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-2-(((1,3-dimethylpyrrolidine-3-carbonyl)oxy) Methyl)propane-1,3-diester, trioctanoic acid ((5-((dimethylamino)methyl)benzene-1,2,3-triyl)tris(oxy))tris(dec- 10,1-diyl) ester, 9-dioctyldizelaic acid 0',0-(((5-((dimethylamino)methyl)-1,3-phenylene)bis( Oxygen)) bis(prop-3,1-diyl)) ester, octadeca-9,12-dienoic acid (9Z,12Z)-3-(3-((dimethylamino)methyl) )-5-(3-((3-octylundecanyl)oxy)propoxy)phenoxy)propyl ester, bis(decanoic acid)(((5-((dimethylamine base)methyl)-1,3-phenylene)bis(oxy))bis(prop-3,1-diyl))bis(oxy))bis(4-side oxybutyl-4,1 -Diyl) ester, dicaprylic acid (R)-4-(3-((R)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl )phenoxy)butan-1,2-diester, dioctanoic acid (S)-4-(3-((S)-3,4-bis(octanoyloxy)butoxy)-5-(( Dimethylamino)methyl)phenoxy)butan-1,2-diester, (R)-4-(3-((S)-3,4-bis(octyloxy)butanyl dioctanoate) Oxy)-5-((dimethylamino)methyl)phenoxy)butan-1,2-diester, tetraoctanoic acid 4,4'-((5-((dimethylamino)methyl) base)-1,3-phenylene)bis(oxy))bis(but-1,2-diyl)ester, 6,6'-((5-((dimethylamino)methyl) -1,3-phenylene)bis(oxy)dihexanoate di-dodecyl ester, 5,5'-((5-((dimethylamino)methyl)-1,3- Phenylene)bis(oxy)dipentanoate bis((9Z,12Z)-octadeca-9,12-dien-1-yl) ester, bis(decanoic acid)(((5-(( Dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(6-side oxyhexyl-6,1-diyl) ester, bis (Methylene)bis(8-(octanoyloxy)octanoic acid)(5-((dimethylamino)methyl)-1,3-phenylene) ester, bis(methylene)bis( 10-(Octyloxy)decanoic acid)(5-((dimethylamino)methyl)-1,3-phenylene) ester, dioctanoic acid (((5-((dimethylamino) )Methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(6-side oxyhexyl-6,1-diyl) ester, bis(decanoic acid)( ((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(8-pentoxyoctyl-8,1- Diyl) ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-(((5-((dimethylamino)methyl)- 1,3-phenylene)bis(methylene))bis(oxy))bis(4-side oxybut-4,1-diyl) ester, 8-dinonyldiosuberic acid 0' ,0-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)) ester, bis(10-(octyloxy)decyl)bis 0,0'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)) succinate, bis((9Z,12Z)-eightadecane Carbon-9,12-dien-1-yl)disuccinic acid 0,0'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene) )) ester, bis(methylene)bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-(5-((((3-(diethyl Amino)propoxy)carbonyl)oxy)methyl)-1,3-phenylene) ester, octadeca-9,12-dienoic acid (9Z,12Z)-4-(3-( (Dimethylamino)methyl)-5-(4-(oleyloxy)butoxy)phenoxy)butyl ester, bis(octadeca-9,12,15-trienoic acid)( 9Z,9'Z,12Z,12'Z,15Z,15'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis (But-4,1-diyl) ester, ditetradecanoate ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis( Butyl-4,1-diyl) ester, dioleic acid (Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis( Butyl-4,1-diyl) ester, dodecanoic acid ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6 ,1-diyl) ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((((5-((diethylamino) Methyl)-1,3-phenylene)bis(oxy))bis(ethyl-2,1-diyl))bis(oxy))bis(ethyl-2,1-diyl) ester, 8 ,8'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))dioctanoic acid didecyl ester, bis(3-octylundecanoic acid) )((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propan-3,1-diyl) ester, bis(octadeca- 9,12-dienoic acid)(9Z.9'Z.12Z.12'Z)-((5-((diethylamino)methyl-2-methyl-1.3-phenylene)bis( Oxygen)) bis(but-4,1-diyl)ester, dodecanoic acid ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy) ) bis(oct-8,1-diyl)ester, bis(decanoic acid)((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis (Octa-8,1-diyl) ester, bis(octadeca-9,12-dienoic acid) (9Z.9'Z.12Z.12'Z)-((5-((dimethylamine methyl)methyl-2-methyl-1.3-phenyl)bis(oxy))bis(but-4,1-diyl)ester, bis(dodeca-8-enoic acid)(8Z,8 'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hex-6,1-diyl) ester, bis(deca Octacarbon-9,12-dienoic acid)(9Z,9'Z,12Z, 12'Z)-((5-((3-hydroxyazetidin-1-yl)methyl)-1, 3-phenylene)bis(oxy)bis(but-4,1-diyl)ester, dioctanoic acid ((5-((dimethylamino)methyl)-1,3-phenylene) )Bis(oxy))bis(hex-6,1-diyl)ester, bis(decanoic acid)((5-((dimethylamino)methyl)-1,3-phenylene)bis (Oxy)) bis(hexane-6,1-diyl) ester, bis(octadeca-9,12-dienoic acid)(9Z.9'Z.12Z.12'Z)-((5- ((dimethylamino)methyl-1.3-phenylene)bis(oxy))bis(oct-8,1-diyl) ester, bis(octadeca-9,12-dienoic acid) (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6, 1-Diyl) ester, dicaproate ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(dec-10,1-diyl ) ester, dioctanoic acid ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(dec-10,1-diyl) ester, dioctanoic acid ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(oct-8,1-diyl)ester, dicaproic acid ((5- ((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(oct-8,1-diyl) ester, bis(octadeca-9,12-di Enoic acid)(9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethyl) -2,1-diyl) ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-((dimethylamino) Methyl)-1,3-phenylene)bis(oxy))bis(prop-3,1-diyl)ester, bis(octadeca-9,12-dienoic acid)(9Z,9' Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(but-4,1-diyl) Ester, bis(methylene)tridecanoic acid (5-((dimethylamino)methyl)-1,3-phenylene) ester, bis(methylene)bis(octadecanoic acid) -9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-(5-((dimethylamino)methyl)-1,3-phenylene) ester, 3- (Dimethylamino)propionic acid (2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methyl ester, bis( Octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-5-(((3-(dimethylamino)propyl)oxy)methyl) -1,3-phenylene ester, 1-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-N,N dimethyl methylamine, 3-(dimethylamino)propionic acid 3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl ester, 1- (3,5-bis(4,4-bis(octyloxy)butoxy)phenyl)-N,N-dimethylmethylamine, tetraoctanoic acid (((5-((dimethylamino )Methyl)-1,3-phenylene)bis(oxy))bis(but-4,1-diyl))bis(oxy))bis(propylene-3,2,1-triyl) Ester, bis(decanoic acid)((5-(((4-(dimethylamino)butyl)oxy)methyl)-1,3-phenylene)bis(oxy))bis(octyl- 8,1-diyl)ester, bis(decanoic acid)((5-(((3-(dimethylamino)propyl)oxy)methyl)-1,3-phenylene)bis (Oxy)) bis(octyl-8,1-diyl) ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z, 12Z,12'Z)-((5- (3-morpholinopropyl)-1,3-phenylene)bis(oxy))bis(but-4,1-diyl)ester, bis(octadeca-9,12-dienoic acid) )(9Z,9'Z,12Z,12'Z)-((5-(3-(dimethylamino)propyl)-1,3-phenylene)bis(oxy))bis(butyl) -4,1-diyl) ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z)-((5-(3-(piperidine-1) -(yl)propyl)-1,3-phenylene)bis(oxy))bis(but-4,1-diyl)ester, bis(methylene)bis(9-pentyltetradecanoic acid) )(5-((dimethylamino)methyl)-1,3-phenylene) ester, bis(methylene)bis(7-hexyltridecanoic acid)(5-((dimethyl Amino)methyl)-1,3-phenyl)ester, bis(methylene)bis(5-heptyldodecanoic acid)(5-((dimethylamino)methyl)-1 ,3-phenylene) ester, bis(3-octylundecanoic acid)((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy)) Bis(but-4,1-diyl)ester, bis(5-heptyldodecanoic acid)((5-((dimethylamino)methyl)-1,3-phenylene)bis( Oxygen))bis(but-4,1-diyl)ester,bis(9-pentyltetradecanoic acid)((5-((dimethylamino)methyl)-1,3-phenylene Bis(oxy))bis(but-4,1-diyl)ester, bis(7-hexyltridecanoic acid)((5-((dimethylamino)methyl)-1,3 -Phenylene)bis(oxy))bis(but-4,1-diyl)ester, bis(octadeca-9,12-dienoic acid)(9Z,9'Z,12Z,12'Z )-((5-(pyrrolidin-1-ylmethyl)-1,3-phenylene)bis(oxy))bis(but-4,1-diyl)ester, tetracaprylic acid (((5 -((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(methylene))bis(prop-3,2,1-triyl)ester,tetrakis Caprylic acid (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(but-4,1-diyl))bis(propane-3, 2,1-triyl) ester, octadeca-9,12-dienoic acid (9Z.12Z)-4-(3-((dimethylamino)methyl-5-(4-((3 -Octylundecanyl)oxy)butoxy)phenoxy)butyl ester, 0,0'-((5-((dimethylamino)methyl)-1,3-phenylene) Bis(1,3-bis(octyloxy)propan-2-yl)bis(methylene)disuccinate, bis(methylene)bis(6-((nonyloxy) Carbonyl)oxy)hexanoic acid) (5-((dimethylamino)methyl)-1,3-phenylene) ester, dihexanoic acid 2-(3-(4-(5-((dimethylamino)methyl) Methylamino)methyl)-2-methyl-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenoxy)butoxy)-3 -Pendant oxypropyl)propan-1,3-diester, 3-octylundecanoic acid 3-((dimethylamino)methyl)-5-(((8-(octyloxy) )octyl)oxy)methyl)benzyl ester, trioctanoate ((5-((diethylamino)methyl)benzene-1,2,3-triyl)tris(oxy))tris(decane) -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-trimethyldodeca-2.6.10-trien-1-ylpropan-1,3 -Diamine, 4-(dimethylamino)butyric acid 1,17-bis(2-((2-pentylcyclopropyl)methyl)cyclopropyl)heptadecane-9-ester, (7Z )-17-{[4-(Dimethylamino)butyl]oxy}ethyl hexadecanoate, (Z)-6-(2-(dimethylamino)-3 -(Octadec-9-en-1-yloxy)propoxy)hexanoic acid methyl ester, 2-(di-dodecylamine)-1-(4-(N-(2-( Dinonylamino)ethyl)-N-dodecylglycinyl)piperidin-1-yl)ethan-1-one, hexanoic acid 3-((3-(1-(3-((( 2-(Dinonylamino)ethyl)(nonyl)amino)propyl)piperidin-4-yl)propyl)(nonyl)amino)propyl ester, hexanoic acid 3-((3 -(4-(3-((2-(Dinonylamino)ethyl)(nonyl)amino)propyl)piperidine-1-yl)-3-side oxypropyl)(nonyl base)amino)propyl ester, 3-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4-(3-(dinonylamino)propyl) Piperidin-1-yl)propan-1-one, 4-((3-(1-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propanyl) Piperidin-4-yl)propyl)(nonyl)amino)butyric acid pentyl ester, 4-((2-(1-(N-(2-(dinonylamino)ethyl))-N- Nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)butyric acid pentyl ester, 4-(((1-(N-(2-(dinonylamino)ethyl) base)-N-nonylglycinyl)pyrrolidin-3-yl)methyl)(nonyl)amino)butyric acid pentyl ester, 4-((2-(1-(N-(2-( Dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)butyric acid pentyl ester, 4-((2-(1- (N-(2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperidin-3-yl)ethyl)(nonyl)amino)butyric acid pentyl ester, 2- (Di-dodecylamine)-1-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pipienyl-1-yl)ethyl -1-one, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(3-(2-(dinonylamino)ethyl)piperidine- 1-yl)ethan-1-one, 4,4'-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperone) -1-yl)-2-Pendant oxyethyl)ureadiyl)dibutyrate dipyl ester, 4-(nonyl(2-(4-(N-nonyl-N-(2-(nonyl) (4-Pendantoxy-4-(Pentyloxy)butyl)amino)ethyl)glyminyl)piperidine-1-yl)-2-Pendantoxyethyl)amino)butyric acid pentyl Ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(3-((dinonylamino)methyl)pyrrolidin-1-yl)ethyl -1-one, 2-((2-(di-dodecylamine)ethyl)(dodecyl)amine)-1-(4-(dinonylglycinyl)piperaniline) -1-yl)ethan-1-one, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(3-(2-(dinonylamino) Ethyl)pyrrolidin-1-yl)ethan-1-one, 4-((3-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propanyl) Carboxyl)piperidine-1-yl)-3-side oxypropyl)(nonyl)amino)butyric acid pentyl ester, hexanoic acid 3-((2-(1-(N-(2-(di) Nonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)propyl ester, 5-((2-(1-(N- (2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)valerate butyl ester, 2-((2 -(Di-dodecylamine)ethyl)(nonyl)amino)-1-(4-(dinonylglycinyl)piperidin-1-yl)ethan-1-one, 6 -((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amine )Propyl hexanoate, 7-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl )(nonyl)amino)ethyl heptanoate, 8-((2-(1-(N-(2-(dinonylamino)ethyl))-N-nonylglycinyl)piperidine -4-yl)ethyl)(nonyl)amino)octanoic acid methyl ester, hexanoic acid 3-((2-(4-(N-(2-(dinonylamino)ethyl))-N-nonan Glyceryl)piperidin-1-yl)-2-side oxyethyl)(nonyl)amino)propyl ester, 5-((2-(4-(N-(2-(dinonyl) (Amino)ethyl)-N-nonylglycinyl)piperidine-1-yl)-2-side oxyethyl)(nonyl)amino)butyl valerate, 6-((2 -(4-(N-(2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperamide-2-pentoxyethyl)(nonyl)amino)hexanoic acid Propyl ester, 7-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidine-1-yl)-2-side oxygen Ethyl)(nonyl)amino)heptanoate, 3-(dinonylamino)-1-(4-(3-((2-(dinonylamino)ethyl)(nonanyl) Base)amino)propionyl)piperidine-1-yl)propan-1-one, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4 -(Di-tetradecylglycinyl)piperidin-1-yl)ethan-1-one, 2-(dinonylamino)-1-(4-(2-((2-(di Nonylamino)ethyl)(nonyl)amino)ethyl)piperidin-1-yl)ethan-1-one, 2-(dinonylamino)-l-(4-(N-( 2-(Dinonylamino)ethyl)-N-dodecylglycinyl)piperidine-1-yl)ethan-1-one, 2-((2-(Dinonylamino) Ethyl)(nonyl)amino)-1-(4-(2-(dinonylamino)ethyl)piperidin-1-yl)ethan-1-one, 8-((2-(4 -(Dinonylglycinyl)piperidin-1-yl)-2-Pendantoxyethyl)(2-((8-Methoxy-8-Pendantoxyoctyl)(nonyl)amine Methyl)ethyl)amino)octanoate, 8-(((2-(dinonylamino)ethyl)(2-(4-(dinonylglycinyl)piperidine-1-yl) -2-Pendant oxyethyl)amino)octanoic acid methyl ester, 8-((2-((2-(4-(dinonylglycinol))piperidine-1-yl)-2-Pendant oxy Ethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoic acid methyl ester, 4-((2-(4-(N-(2-(dinonylamino)ethyl) -N-Nonylglycinyl)piperidine-2-side oxyethyl)(nonyl)amino)butyric acid pentyl ester, 8-((2-(4-(N-(2-(di Nonylamino)ethyl)-N-nonylglycinyl)piperidine-1-yl)-2-side oxyethyl)(nonyl)amino)octanoic acid methyl ester, 2-((2 -(Di-dodecylamine)ethyl)(dodecyl)amine)-1-(5-(dinonylglycinyl)-2,5-diazabicyclo[2.2. 1]Hept-2-yl)ethan-1-one, 2-(dinonylamino)-1-(5-(N-(2-(dinonylamino)ethyl)-N-nonyl Glycinyl)-2,5-diazabicyclo[2.2.1]hept-2-yl)ethan-1-one, N1,N1,N2-tris((9Z,12Z)-octadecane-9 ,12-dien-1-yl)-N2-(2-(piperidine-1-yl)ethyl)ethyl-1,2-diamine, N1,N1,N2-tris((Z)-eightadecane Carbo-9-en-1-yl)-N2-(2-(piperidine-1-yl)ethyl)ethyl-1,2-diamine, 2-(dinonylamino)-l-(4 -(N-(2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperidine-l-yl)ethan-1-one, N1,N1,N2-tri-dodecane Alkyl-N2-(2-(piperidine-1-yl)ethyl)ethyl-1,2-diamine, N1,N1,N2-trinonyl-N2-(2-(piperidine-1-yl) )ethyl)ethyl)ethyl-1,2-diamine, N1,N1,N2-trihexyl-N2-(2-(piperbenz-1-yl)ethyl)ethyl-1,2-diamine, N1-( 2-(4-(2-(Di-dodecylamine)ethyl)piperidine-1-yl)ethyl)-N1,N2,N2-tris((9Z,12Z)-octadecacarbon- 9,12-dien-1-yl)ethyl-1,2-diamine, N1-(2-(4-(2-(di-dodecylamine)ethyl)piperaniline-1-yl) )ethyl)-N1,N2,N2-tris((Z)-octadec-9-en-1-yl)ethyl-1,2-diamine, N1-(2-(4-(2-( Ditetradecylamine-1,2-diamine, N1-(2-( 4-(2-(di-dodecylamine)ethyl)piperidine-1-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2-diamine, N1-(2-(4-(2-(dinonylamino)ethyl)piperidine-1-yl)ethyl)-N1,N2,N2-tri-tetradecylethyl-1,2- Diamine, 2-(di-dodecylamine)-l-(4-(2-((2-(di-dodecylamine)ethyl)(dodecyl)amine)) Ethyl) piperazine-1-yl) ethan-1-one, N1-(2-(4-(2-(bis((9Z,12Z))-octadeca-9,12-dien-1-yl) )Amino)ethyl)piperidin-1-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2- (Di((Z)-Octadec-9-en-1-yl)amino)ethyl)piperidine-1-yl)ethyl)-N1,N2,N2-tri-dodecylethyl- 1,2-Diamine, N1,N1,N2-tri-dodecyl-N2-(2-(4-(2-(dodecyl((9Z,12Z))-octadecyl-9,12 -Dien-1-yl)amino)ethyl)piperidine-1-yl)ethyl)ethyl-1,2-diamine, N1-(2-(4-(2-(ditetradecane) (Amino)ethyl)piperidin-1-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2- (Di((Z)-dodeca-6-en-l-yl)amino)ethyl)piperidine-l-yl)ethyl)-N1,N2,N-tri-dodecylethyl- 1,2-Diamine, (Z)-N1-(2-(4-(2-dodeca-6-en-l-yl(dodecyl)amino)ethyl)piperamide-l- ethyl)-N,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(dinonylamino)ethyl)piperaniline) -l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(dioctylamine)ethyl) )Piper-1-yl)ethyl)-N1,N2,N2-tri-dodecylethane-1,2-diamine, N1-(2-(4-(2-(dihexylamino) Ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, N1-(2-(4-(2-(二-十) Tetraalkylamino)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-trinonylethane-1,2-diamine, 2-((2-(di-dodecane methylamino)ethyl)(dodecyl)amino)-l-(4-(2-(di-dodecylamine)ethyl)piperamide-l-yl)ethan-1-one , N1-(2-(4-(2-(di-dodecylamine)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-trinonylethyl-1,2 -Diamine, N1-(2-(4-(2-(dinonylamino)ethyl)pipienyl-l-yl)ethyl)-N1,N2,N2-trinonylethyl-1,2 -Diamine, N1-(2-(4-(2-(di-dodecylamine)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-trihexylethane-1 ,2-diamine, 12,12'-((2-(4-(2-((2-(di-dodecylamine)ethyl)(dodecyl)amino)ethyl) Dimethyl piperazine-l-yl)ethyl)ureadiyl)di-dodecanoate, 12-((2-(4-(2-((2-(di-dodecylamine)) Ethyl)(dodecyl)amino)ethyl)piperidine-l-yl)ethyl)(dodecyl)amino)methyl dodecanoate, 6,6'-((2- (4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperidine-l-yl)ethyl)ureadiyl)diyl Dipentyl hexanoate, 6-((2-(4-(2-((2-(di-tetradecylamino)ethyl)(tetradecyl)amino)ethyl)piperidine- 1-yl)ethyl)(dodecyl)amino)pentylhexanoate, 6-((2-(4-(2-((2-(di-dodecyl)amino)ethyl) (Dodecyl)amino)ethyl)piperidine-l-yl)ethyl)(dodecyl)amino)pentylhexanoate, 2-(di-dodecylamine)-l -(4-(N-(2-(di-dodecylamine)ethyl)-N-dodecylglycinyl)piperidine-1-yl)ethan-1-one, 2- (Di-dodecylamine)-1-(4-(N-(2-(di-dodecylamine)ethyl)-N-nonylglycinyl)piperamide-1- ethyl)ethanol-1-one, 2-(di-dodecylamine)-N-(2-(4-(2-(di-dodecylamine)ethyl)ethyl)piperidine-l- ethyl)-N-dodecyl acetamide, (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoic acid)((2-(( 3,S',4R)-3,4-Dihydroxypyrrolidin-l-yl)acetyl)ureadiyl)bis(ethyl-2,1-diyl)ester, 2-amino-N,N -Di-hexadecyl-3-(1H-imidazol-5-yl)propanamide, (2-amino-N,N-di-hexadecyl-3-(1H-imidazol-5-yl) )Propanamide, (9Z)-19-[2-(dimethylamino)ethyl]octadeca-9-enoic acid methyl ester, 8-(2-{9-[2-(dimethyl) methylamino)ethyl]octadecyl}cyclopropyl)octanoic acid methyl ester, (9Z)-19-[2-(dimethylamino)ethyl]octadecyl-9-enoic acid methyl ester , 8-(2-{ll-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid ethyl ester, 8-(2-{ll-[(dimethylamino)methyl] Ethyl]octadecyl}cyclopropyl)octanoate, 3-(((2-(dimethylamino)ethoxy)carbonyl)amino)glutarate di((9Z,12Z)-decanoate Octacarbon-9,12-dien-l-yl) ester, 6-((2-(l-(N-(2-(dinonylamino)ethyl))-N-nonylglycinyl) )piperidin-4-yl)ethyl)(tetradecyl)amino)heptylhexanoate, 8-(2-{ll-[(dimethylamino)methyl]nonadecyl}cyclo Propyl)ethyl octanoate, 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)ethyl ethyl)(tetradecyl)amino)octanoate, 8-(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)octanoate, 8-( 2-{9-[(Dimethylamino)methyl]pentadecyl}cyclopropyl)octanoic acid ethyl ester, decanoic acid 3-((2-(l-(N-(2-(dinonyl) Amino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(tetradecyl)amino)propyl ester, 6-((2-(4-(N- (2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperbenzoic acid Heptyl ester, ethyl 8-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, 8-((2-(4-(N-(2- (Dinonylamino)ethyl)-N-nonylglycinyl)piperidine-2-side oxyethyl)(tetradecyl)amino)pentyl octanoate, 8-(2-{ 9-[(Dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid ethyl ester, 6-(2-(8-(2-(dimethylamino)-3-(nonyloxy) (9Z)-21-(dimethylamino)octyl)cyclopropyl)hexanoic acid methyl ester, (9Z)-21-{ [4-(Dimethylamino)butyl]oxy}hexadeca-9-enoic acid methyl ester, (2R)-N,N-dimethyl-1-[(9Z,12Z)-octadecanoate Carbon-9,12-dien-1-yloxy]dodecane-2-amine, (15Z,18Z)-N,N-dimethyltetradecano-15,18-diene-5- Amine, ethyl 8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate, decanoic acid 3-((2-(4-(N-(2 -(Dinonylamino)ethyl)-N-nonylglycinyl)piperidine-l-yl)-2-side oxyethyl)(tetradecyl)amino)propyl ester, 4 -(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)butyric acid ethyl ester, 8-(2-{7-[(dimethylamino)methyl ]Hexadecyl}cyclopropyl)octanoic acid ethyl ester, hexanoic acid 3-((3-(l-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propanyl) acyl)piperidin-4-yl)propyl)(nonyl)amino)propyl ester, 6-(2-{9-[(dimethylamino)methyl]pentadecyl}cyclopropyl )Ethyl hexanoate, 3-((3-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propyl)piperate-l- base)-3-Pendant oxypropyl)(nonyl)amino)propyl ester, 6-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)hexyl Ethyl acid ester, 3-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(4-(3-(dinonylamino)propyl)piperidine-1 -yl)propan-1-one, 4-((3-(l-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propanyl)piperidine-4 -Ethyl)propyl)(nonyl)amino)butyrate, ethyl 6-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)hexanoate , 4-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl) Amino)butyric acid pentyl ester, 6-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)hexanoic acid ethyl ester, 4-(((l-(N -(2-(Dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)methyl)(nonyl)amino)butyric acid pentyl ester, (9Z)- 21-[(Dimethylamino)methyl]ethyl heptacos-9-enoate, 4-((2-(l-(N-(2-(dinonylamino)ethyl)) -N-Nonylglycinyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)butyric acid pentyl ester, (9Z)-21-[(dimethylamino)methyl]di Ethyl octadeca-9-enoate, (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoic acid) ((2-((3,S', 4R)-3,4-dihydroxypyrrolidin-l-yl)acetyl)ureadiyl)bis(ethyl-2,l-diyl)ester, 4-((2-(l-(N-( 2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperidin-3-yl)ethyl)(nonyl)amino)butyric acid pentyl ester, (9Z)-21- [(Dimethylamino)methyl]ethyl 9-ethyl ester, 6-(2-(8-(2-(dimethylamino))-3-(heptyloxy)propyl Oxy)octyl)cyclopropyl)hexanoic acid methyl ester, (9Z)-21-{[4-(dimethylamino)butyl]oxy}octacarbon-9-enoic acid methyl ester, ( 9Z)-21-(dimethylamino)octadecanoic-9-enoic acid methyl ester, 2-(di-dodecylamine)-1-(4-(N-(2-(di Nonylamino)ethyl)-N-nonylglycinyl)piperidine-1-yl)ethyl)ethyl)-(2S)-N.N-dimethyl-1-[(9Z,12Z)-ten Octacarbon-9,12-diene-1-yloxy]nonan-2-amine, (18Z,21Z)-N,N-dimethylheptacarbon-18,21-diene-10-amine , (9Z)-21-[(dimethylamino)methyl]triaconta-9-enoic acid ethyl ester, (9Z)-19-[(dimethylamino)methyl]pentadecacarbonate -Ethyl 9-enoate, (9Z)-19-[(dimethylamino)methyl]ethyl 9-enoate, (9Z)-19-[(dimethylamino) )methyl]ethyl octadeca-9-enoate, (9Z)-19-[(dimethylamino)methyl]octadeca-9-enoate ethyl ester, (5Z)-17 -[(Dimethylamino)methyl]icosac-5-enoic acid ethyl ester, (9Z)-17-[(dimethylamino)methyl]icosac-9-enoic acid Ethyl ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-l-(3-(2-(dinonylamino)ethyl)piperidine-l- ethyl)ethan-1-one, (7Z)-17-[(dimethylamino)methyl]ethyl tricos-7-enoate, 4,4'-((2-(4-( N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperamide-l-yl)-2-side oxyethyl)ureadiyl)dibutyrate dipentyl Ester, 4-(nonyl(2-(4-(N-nonyl-N-(2-(nonyl(4-sideoxy-4-(pentoxy)butyl)amino)ethyl) Glycinyl) piperazine-l-yl)-2-side oxyethyl) amino) butyric acid pentyl ester, (7Z)-17-[(dimethylamino)methyl] 24-carbon -Ethyl 7-enoate, (7Z)-17-[(dimethylamino)methyl]pentazoic-7-enoic acid ethyl ester, 2-((2-(dinonylamino) Ethyl)(nonyl)amino)-l-(3-((dinonyl)amino)methyl)pyrrolidin-1-yl)ethan-1-one, trans-3-[(3}7 -Dimethyloctyl)oxy]-1-methyl-4~[(9Z,12Z)-octadeca-9512-diene-1-oxypyrrolidine, 6-(2-(8-( 2-(Dimethylamino)-3-(hexyloxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, (9Z)-21-{[4-(dimethylamino) Butyl]oxy}nonadenocarbon-9-enoic acid methyl ester, (9Z)-21-(dimethylamino) nonacos-9-enoic acid methyl ester, (2S)-N,N- Dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]tridecane-2-amine, (15Z,18Z)-N,N-dimethyl Hexacarbon-15,18-diene-7-amine, (7Z)-17-[(dimethylamino)methyl]icosac-7-enoic acid ethyl ester, 2-(( 2-(Dinonylamino)ethyl)(nonyl)amine)-l-(3-(2-(dinonylamino)ethyl)pyrrolidin-1-yl)ethan-1-one , 6-(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)hexanoic acid methyl ester, 10-(2-{7-[(dimethylamino) Methyl]hexadecyl}cyclopropyl)decanoate, methyl 8-(2-{ll-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoate, 8 -(2-{ll-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{ll-[(dimethylamino)methyl] Methyl nonadecyl}cyclopropyl)octanoate, 8-(2-{ll-[(dimethylamino)methyl]eicosyl}cyclopropyl)octanoate, methyl ester, 4-((3 -(4-(3-((2-(Dinonylamino)ethyl)(nonyl)amino)propyl)piperidine-l-yl)-3-side oxypropyl)(nonyl) Amino)butyric acid pentyl ester, 8-(2-{9-[(dimethylamino)methyl]pentadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{9- [(Dimethylamino)methyl]hexadecyl}cyclopropyl)octanoic acid methyl ester, hexanoic acid 3-((2-(l-(N-(2-(dinonylamino)ethyl) )-N-Nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)propyl ester, 8-(2-{9-[(dimethylamino)methyl] Heptadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-(dimethylamino)-3-((6-((2-octylcyclopropyl)methoxy)-6-side Oxyhexyl)oxy)propoxy)octanoic acid methyl ester, 5-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl) Piperidin-4-yl)ethyl)(nonyl)amino)butylvalerate, trans-1-methyl-3-[(12Z)-octadec-12-en-1-yloxy ]-4-(octyloxy)pyrrolidine, (9Z)-21-{[4-(dimethylamino)butyl]oxy}triacontacarbon-9-enoic acid methyl ester, (9Z)-21 -(Dimethylamino)triacontacarbon-9-enoic acid methyl ester, 2-((2-(di-dodecylamine)ethyl)(nonyl)amino)-1-(4 -(Dinonylglycinyl)piperidin-1-yl)ethan-1-one Step 1: N-(2-(di-dodecylamine)ethyl)-N-nonylglyamine Acid methyl ester, 1-((2R,3S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-bisoxy-3, 4-Dihydropyrimidine-1(2H)-yl)tetrahydrofuran methanesulfonate, (Z)-16-(3-(decyloxy)-2-(dimethylamino)propoxy)hexadecane -7-Methyl enoate, (2S)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]nonan-2-amine, (14Z,17Z)- N,N-Dimethyltricoc-14,17-diene-6-amine, 6-((2-(l-(N-(2-(dinonylamino)ethyl))-N -Nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)propylhexanoate, 7-(2-(dimethylamino)-3-((6-( (2-octylcyclopropyl)methoxy)-6-side oxyhexyl)oxy)propoxy)heptanoate methyl ester, (7Z)-19-[(dimethylamino)methyl] Methyl octadecanoate, (HZ)-19-[(dimethylamino)methyl]octadecanoate-ll-enoate, 7-((2-(l- (N-(2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)heptanoate, 6- (2-(dimethylamino)-3-((5-methoxy-5-pentyloxypentyl)oxy)propoxy)hexanoic acid (2-octylcyclopropyl)methyl ester, 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amine methyl)octanoate, (9Z)-21-[(dimethylamino)methyl]heptacos-9-enoic acid methyl ester, 6-(2-(dimethylamino)-3- (2-octylcyclopropyl)methyl (4-methoxy-4-sideoxybutoxy)propoxy)hexanoate, (9Z)-21-[(dimethylamino)methyl ] Methyl octadecanoic acid, 3-((2-(4-(N-(2-(dinonylamino)ethyl))-N-nonylglycinyl) Piperane-1-yl)-2-side oxyethyl)(nonyl)amino)propyl ester, (Z)-8-(2-(dimethylamino)-3-((6-side Oxy-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)octanoic acid methyl ester, (9Z)-21-[(dimethylamino)methyl] Methyl nonaenoate, 5-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperone- l-yl)-2-Pendant oxyethyl)(nonyl)amino)valerate butyl ester, (Z)-7-(2-(dimethylamino)-3-((6-Pendant oxy) Methyl-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)heptanoate, 6-((2-(4-(N-(2-(di Nonylamino)ethyl)-N-nonylglycinyl)piperidine-l-yl)-2-side oxyethyl)(nonyl)amino)propylhexanoate, (9Z)- 21-[(Dimethylamino)methyl]tricoc-9-enoic acid methyl ester, (Z)-6-(2-(dimethylamino)-3-((5-methoxy) -5-Pendant oxypentyl)oxy)propoxy)hexanoate undec-2-en-1-ester, (9Z)-19-[(dimethylamino)methyl]twenty-five Carbon-9-enoic acid methyl ester, 7-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pipienyl)-l-yl )-2-Pendant oxyethyl)(nonyl)amino)ethyl heptanoate, (Z)-6-(2-(dimethylamino)-3-(4-methoxy-4- Pendant oxybutoxy)propoxy)hexanoate undec-2-en-1-ester, 6-(2-(dimethylamino)-3-((6-((2-octyl) Cyclopropyl)methoxy)-6-side oxyhexyl)oxy)propoxy)hexanoic acid methyl ester, (9Z)-19-[(dimethylamino)methyl]hexadecan- 9-Methyl enoate, 3-(dinonylamino)-l-(4-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propyl) Piperan-1-yl)propan-1-one, (9Z)-19-[(dimethylamino)methyl]heptacos-9-enoic acid methyl ester, 2-((2-(di Nonylamino)ethyl)(nonyl)amino)-l-(4-(ditetradecylglycinyl)piperidin-1-yl)ethan-1-one, (Z)- 6-(2-(dimethylamino)-3-((6-sideoxy-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)hexyl Acid methyl ester, 8-(2-(dimethylamino)-3-((8-(2-(6-methoxy-6-side oxyhexyl)cyclopropyl)octyl)oxy) Propoxy)octanoic acid methyl ester, 8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid methyl ester, 2-(dinonylamino)- l-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl)piperidin-1-yl)ethan-1-one, trans-l- Methyl-3-[(9Z)-octadec-9-en-l-yloxy]-4-(octyloxy)pyrrolidine, (9Z)-19-{[4-(dimethylamine methyl)butyl]oxy}pentadeca-9-enoate methyl ester, (9Z)-19-(dimethylamino)pentadeca-9-enoate methyl ester, (Z)-16- (2-(Dimethylamino)-3-(nonyloxy)propoxy)hexadecanoic acid methyl ester, (2S)-1-[(9Z,12Z)-octadecanoic acid- 9,12-diene-1-yloxy]decyl-2-amine, (12Z,15Z)-N,N-dimethyleicosanoid-12,15-diene-4-amine, 7- (2-(dimethylamino)-3-((8-(2-(6-methoxy-6-pentoxyhexyl)cyclopropyl)octyl)oxy)propoxy)heptanoic acid Methyl ester, (9Z)-19-[(dimethylamino)methyl]octacarbon-9-enoic acid methyl ester, 2-((2-(dinonylamino)ethyl)(nonan base)amino)-l-(4-(2-(dinonylamino)ethyl)piperidin-l-yl)ethan-1-one, 8-((2-(4-(dinonyl) Glyminyl)piperidine-l-yl)-2-side oxyethyl) (2-((8-methoxy-8-side oxyoctyl)(nonyl)amino)ethyl) Amino)octanoic acid methyl ester, 6-(2-(8-(2-(dimethylamino)-3-((5-methoxy-5-sideoxypentyl)oxy)propoxy) )octyl)cyclopropyl)hexanoic acid methyl ester, 8-{2-[ll-(dimethylamino)heptadecyl]cyclopropyl}octanoic acid ethyl ester, 8-((2-(dionon methylamino)ethyl)(2-(4-(dinonylglycinyl)piperidine-l-yl)-2-side oxyethyl)amino)octanoate, 6-(2- (8-(2-(Dimethylamino)-3-(4-methoxy-4-sideoxybutoxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, 8- {2-[ll-(dimethylamino)octadecyl]cyclopropyl}octanoic acid ethyl ester, 8-((2-((2-(4-(dinonylglycinyl)piperazol) -l-yl)-2-Pendant oxyethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoic acid methyl ester, 8-{2-[ll-(dimethylamino) Nonadecyl]cyclopropyl}octanoic acid ethyl ester, (Z)-16-(2-(dimethylamino)-3-((8-methoxy-8-side oxyoctyl)oxy) )propoxy)hexadeca-7-enoic acid methyl ester, 4-((2-(4-(N-(2-(dinonylamino)ethyl))-N-nonylglycinyl )Penyl-1-yl)-2-side oxyethyl)(nonyl)amino)butyric acid pentyl ester, 8-{2-[ll-(dimethylamino)eicosanyl] ring Propyl}octanoic acid ethyl ester, (Z)-16-(2-(dimethylamino)-3-((7-methoxy-7-pentyloxyheptyl)oxy)propoxy)ten Hexacarbon-7-enoic acid methyl ester, 8-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperone-l- methyl)-2-Pendant oxyethyl)(nonyl)amino)octanoate, 8-{2-[9-(dimethylamino)pentadecyl]cyclopropyl}octanoic acid ethyl ester, (Z)-16-(2-(dimethylamino)-3-((5-methoxy-5-pentyloxypentyl)oxy)propoxy)hexadec-7-enoic acid Methyl ester, (11E,20Z,23Z)-N,N-dimethylnac-11,20,23-triene-10-amine, N,N-dimethyl-1-[(1S, 2R)-2-octylcyclopropyl]pentadecan-8-amine, 8-{2-[9-(dimethylamino)hexadecyl]cyclopropyl}octanoic acid ethyl ester, 2-( (2-(Di-dodecylamine)ethyl)(dodecyl)amine)-l-(5-(dinonylglycinyl)-2,5-diazabicyclo[ 2.2.1]Hept-2-yl)ethan-1-one 3, (Z)-16-(2-(dimethylamino)-3-(4-methoxy-4-sideoxybutoxy) methyl)propoxy)hexadecyl-7-enoate, 6-(2-(8-(2-(dimethylamino)-3-((6-methoxy-6-side oxygen) Methylhexyl)oxy)propyloxy)octyl)cyclopropyl)hexanoate, ethyl 8-{2-[9-(dimethylamino)heptadecyl]cyclopropyl}octanoate, 2-(Dinonylamino)-l-(5-(N-(2-(Dinonylamino)ethyl)-N-nonylglycinyl)-2,5-diazabicyclo [2.2.1]Hept-2-yl)ethan-1-one, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N1,N1,N2-tris((9Z,12Z)-octadeca-9,12-diene-l-yl)-N2-(2-(piperidine-l-yl)ethyl)ethyl-1, 2-Diamine, 8-{2-[9-(Dimethylamino)octadecyl]cyclopropyl}octanoic acid ethyl ester, 1-[(1R,2S)-2-heptylcyclopropyl] -N,N-dimethyloctadecane-9-amine, (Z)-16-(2-(dimethylamino)-3-((6-methoxy-6-side oxyhexyl)) Oxy)propoxy)hexadec-7-enoic acid methyl ester, N1,N1,N2-tris((Z)-octadec-9-en-l-yl)-N2-(2-(piperate) 𠯤-l-yl)ethyl)ethyl-l,2-diamine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}ten Dialkyl-1-amine, 8-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy methyl)propoxy)octanoate, ethyl 4-{2-[ll-(dimethylamino)eicosyl]cyclopropyl}butyrate, trans-1-methyl-3-[ ((9Z,12Z)-octadecanoic-9,12-dienyl)oxy]-4-octyloxy-pyrrolidine, (9Z)-19-(dimethylamino)octadecanoic- 9-enoic acid methyl ester, (9Z)-19-{[4-(dimethylamino)butyl]oxy}hexadeca-9-enoic acid methyl ester, (Z)-16-(2- (Dimethylamino)-3-(heptyloxy)propoxy)hexadeca-7-enoic acid methyl ester, (2R)-1-[(9Z,12Z)-octadeca-9,12 -Dien-1-yloxy]dodecane-2-amine, (13Z,16Z)-N,N-dimethyldococ-13,16-diene-5-amine, N,N -Dimethyl-1-[(1R,2S)-2-Undecylcyclopropyl]tetradecan-5-amine, 7-(2-(dimethylamino)-3-((8 -(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)heptanoate methyl ester, 8-{2-[7-(dimethylamino )Hexadecyl]cyclopropyl}octanoic acid ethyl ester, 2-(di-dodecylamine)-N-dodecyl-N-(2-(piperidine-l-yl)ethyl) Acetamide, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N1-(2-(piperidine-l-yl) Ethyl)-N1,N2,N2-tri-tetradecylethane-l,2-diamine, 6-(2-(dimethylamino)-3-((8-(2-((2 -Pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propyloxy)hexanoic acid methyl ester, 6-{2-[9-(dimethylamino)pentadecyl]cyclo Propyl}ethyl hexanoate, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl] Nonadecan-10-amine, NN1,N2-tri-dodecyl-N2-(2-(piperidine-l-yl)ethyl)ethyl-l,2-diamine, 5-(2-( Dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)valerate methyl ester, 6- {2-[9-(Dimethylamino)hexadecyl]cyclopropyl}hexanoic acid ethyl ester, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl base] hecosan-10-amine, NNN2-trinonyl-N2-(2-(piperidine-l-yl)ethyl)ethyl-1,2-diamine, 4-(2-(dimethyl methylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)butyric acid methyl ester, 6-{2 -[9-(Dimethylamino)heptadecyl]cyclopropyl}hexanoic acid ethyl ester, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] Nonadecan-10-amine, N1,N1,N2-trihexyl-N2-(2-(piperidine-l-yl)ethyl)ethyl-l,2-diamine, 8-(2-(dimethyl methylamino)-3-((9Z,12Z)-octadeca-9,12-diene-l-yloxy)propoxy)octanoic acid methyl ester, 6-{2-[9-(dimethyl Amino)octadecyl]cyclopropyl}hexanoic acid ethyl ester, N1-(2-(4-(2-(di-dodecylamine)ethyl)piperidine-l-yl)ethyl base)-N1,N2,N2-tri-((9Z,12Z)-octadeca-9,12-diene-l-yl)ethane-1,2-diamine, 7-(2-(dimethyl methylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)heptanoate, (9Z)-21-(dimethyl Amino) Ethyl heptadecanoic acid, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, 9-{ [4-(Dimethylamino)butyl]oxy}octadecanedioic acid 1-methyl ester 18-[(2Z)-non-2-en-1-] ester, N1-(2-(4- (2-(Di-dodecylamine)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-tri-((Z)-octadec-9-ene-l- base)ethyl-l,2-diamine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 6-(2-( Dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)hexanoic acid methyl ester, (9Z)-21-(di Methylamino)octadecanoic-9-enoic acid ethyl ester, (9Z)-19-{[4-(dimethylamino)butyl]oxy}octadecanoic-9-enoic acid di Methyl ester, N1-(2-(4-(2-(di-tetradecylamine)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-tri-tetradecyl Ethyl-1,2-diamine, 5-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,l2-dien-l-yloxy)propoxy ethyl)pentanoate, 8-{[4-(dimethylamino)butyl]oxy}-15-(2-octylcyclopropyl)pentadecanoate ethyl ester, (9Z)-21- (Dimethylamino)icos-9-enoic acid ethyl ester, (13Z,16Z)-N,N-dimethyl-3-nonyldococ-13,16-diene-1 -Amine, N1-(2-(4-(2-(di-dodecylamine)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-tri-tetradecyl Ethyl-1,2-diamine, 9-{[4-(dimethylamino)butyl]oxy}-16-(2-octylcyclopropyl)hexadecanoic acid methyl ester, 4-(2 -(Dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)butyric acid methyl ester, (9Z)-21- (Dimethylamino)triacontacarbon-9-enoic acid ethyl ester, (12Z,15Z)-N,N-dimethyl-2-nonylenoicanoate-12,15-diene-1- Amine, methyl 8-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)octanoate, (9Z)-19- (Dimethylamino)pentadeca-9-enoic acid ethyl ester, (18Z,21Z)-8-{[4-(dimethylamino)butyl]oxy}pentadeca-18, 21-Ethyl dienoate, (16Z)-N,N-dimethylpentadeca-16-en-8-amine, (9Z)-19-{[4-(dimethylamino)butanyl ]Oxy}Heptacos-9-enoic acid methyl ester, (9Z)-19-(dimethylamino) heptacos-9-enoic acid methyl ester, 2-(di-dodecyl Amino)-l-(4-(2-((2-(di-dodecylamino)ethyl)(dodecyl)amino)ethyl)piperidine-l-yl)ethyl- 1-one, (Z)-16-(2-(dimethylamino)-3-(hexyloxy)propoxy)hexadecan-7-enoic acid methyl ester, (2S)-1-[ (9Z,12Z)-Octadecan-9,12-dien-1-yloxy]dodecane-2-amine, (16Z,19Z)-N,N-dimethyloctadecanoic-16 , 19-diene-8-amine, N1-(2-(4-(2-(dinonylamino)ethyl)piperidine-l-yl)ethyl)-N1,N2^V2-tri- Tetradecylethane-l,2-diamine, 7-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy )Heptanoic acid methyl ester, (19Z,22Z)-9-{[4-(dimethylamino)butyl]oxy}octacarbon-19,22-dienoic acid methyl ester, (9Z)-19 -(Dimethylamino)ethyl hexadecanoate, (22Z)-N,N-dimethyltriadenodec-22-en-10-amine, N1-(2-( 4-(2-(bis((Z)-octadec-9-en-l-yl)amino)ethyl)piperidine-l-yl)ethyl)-!^^-tri-dodecane Ethyl-1,2-diamine, 5-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)valeric acid Methyl ester, (9Z)-19-(dimethylamino)heptacos-9-enoic acid ethyl ester, 12-{[4-(dimethylamino)butyl]oxy}hexadecane (2-Butylcyclopropyl)methyl acid ester, (20Z)-N,N-dimethylnonacos-20-en-10-amine, N1,N1,N2-tri-dodecyl- N2-(2-(4-(2-(Dodecyl((9Z,12Z)-octadeca-9,12-dienyl)amino)ethyl)piperidine-1-yl)ethyl )ethyl-1,2-diamine, 4-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)butyric acid Methyl ester, (9Z)-19-(dimethylamino)octadecanoic acid ethyl ester, 8-{[4-(dimethylamino)butyl]oxy}heptadecanoic acid (2-octylcyclopropyl)methyl ester, (24Z)-N,N-dimethyltritriadeca-24-en-10-amine, N1-(2-(4-(2-(di- Tetradecylamine)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, (5Z)-17-(diamine Methylamino)ethyl hexadecanoate, (Z)-8-(2-(dimethylamino)-3-(octadec-9-en-l-yloxy) )propoxy)octanoic acid methyl ester, (2Z)-12-{[4-(dimethylamino)butyl]oxy}heptan-2-en-1-eester, (17Z)- N,N-dimethyldecoc-17-en-10-amine, N1-(2-(4-(2-(di((Z))-dodeca-6-en-l-yl) Amino)ethyl)piperidine-l-yl)ethyl)-N1,N2,N2-tri-dodecylethyl-1,2-diamine, (9Z)-17-(dimethylamino )Ethyl hexadecanoate, (Z)-7-(2-(dimethylamino)-3-(octadec-9-en-1-yloxy)propoxy )Methyl heptanoate, (2Z)-8-{[4-(dimethylamino)butyl]oxy}heptadecanoate undec-2-en-l-ester, (14Z)-N, N-Dimethylococ-14-en-10-amine, (7Z)-17-(dimethylamino)ococ-7-enoic acid ethyl ester, (Z)-N1-( 2-(4-(2-Dodeca-6-en-l-yl(dodecyl)amino)ethyl)piper-N!^^-tri-dodecylethyl-1,2 -Diamine, (Z)-5-(2-(dimethylamino)-3-(octadec-9-en-1-yloxy)propoxy)valerate methyl ester, 10-{ [4-(Dimethylamino)butyl]oxy}nonadecanoate (2-hexylcyclopropyl)methyl ester, (15Z)-N,N-dimethylheptacos-15-ene- 10-amine, (7Z)-17-(dimethylamino)ethyl tetracos-7-enoate, (Z)-4-(2-(dimethylamino)-3-(decaenoate) Methyl octacarbon-9-en-1-yloxy)propoxy)butyrate, (2Z)-10-{[4-(dimethylamino)butyl]oxy}nonadecanoate non- 2-en-l-ester, (20Z)-N,N-dimethyl heptadeca-20-en-10-amine, N1-(2-(4-(2-(dioctylamine)) Ethyl)piperidine-l-yl)ethyl)-N1,N2^V2-tri-dodecylethane-l,2-diamine, 6-(2-(dimethylamino)-3- ((8-(2-octylcyclopropyl)octyl)oxy)propoxy)hexanoic acid methyl ester, 6-[2-(9-{[4-(dimethylamino)butyl]oxy Ethyl}octadecyl)cyclopropyl]hexanoate, (7Z)-17-(dimethylamino)pentadeca-7-enoic acid ethyl ester, 1-[(11Z,14Z)- 1-Nonyleicosac-11,14-dien-1-yl]pyrrolidine, (7Z)-17-(dimethylamino)eicosac-7-enoic acid ethyl ester, (20Z, 23Z)-N-ethyl-N-methylnac-20,23-diene-10-amine, N,N-dimethylheptadecane-10-amine, 6-{2-[ ll-(dimethylamino)eicosyl]cyclopropyl}hexanoic acid methyl ester, 6-[2-(ll-{[4-(dimethylamino)butyl]oxy}eicosane methyl)cyclopropyl]hexanoate, 6-(3-(decyloxy)-2-(dimethylamino)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, 8 -{2-[9-(Dimethylamino)octadecyl]cyclopropyl}octanoic acid methyl ester, 8-[2-(9-{[4-(dimethylamino)butyl]oxy] }Octadecyl)cyclopropyl]octanoic acid methyl ester, 7-(2-(8-(2-(dimethylamino)-3-(octyloxy)propoxy)octyl)cyclopropyl ) Methyl heptanoate, representative 8-((2-hydroxyethyl)(tetradecyl)amino)heptadecan-9-octanoate, 2-((2-(di-dodecylamine) Base)ethyl)(dodecyl)amino)-l-(4-(2-(di-dodecylamine)ethyl)piperidine-1-yl)ethan-1-one, ( 2S)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]undecane-2-amine, (17Z,20Z)-N,N-dimethyl Hexadecanoic-17,20-diene-9-amine, (18Z)-4-(dimethylamino)butyric acid heptococ-18-en-10-ester, (2S)-1- ({6-[3B))-Cholester-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadec-9-ene -1-yloxy]propan-2-amine, 10-{2-[7-(dimethylamino)hexadecyl]cyclopropyl}decanoic acid methyl ester, 10-[2-(7- {[4-(Dimethylamino)butyl]oxy}hexadecyl)cyclopropyl]decanoic acid methyl ester, (2S)-N,N-dimethyl-1-({8-[( lR,2R)-2-{[(lS,2S)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)tridecane-2-amine, 6-(2-( Dimethylamino)-3-(nonyloxy)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, (19Z,22Z)-N,N-dimethyloctadecanoic acid- 19,22-Dien-7-amine, dinonylglycine 4-((N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)oxy)pentyl -2-ester, 3-hydroxybut-2-yl N-(2-(dinonylamino)ethyl)-N-nonyl, 8,8'-(26,28-dimethyl-ll, 24,30,43-tetraoxa-10,25,29,44-tetraoxa-19,35-diazapentatriane-19,35-diyl)dioctanoic acid di(heptadecane-9) -yl) ester, 8,8'-(26,27-dimethyl-ll,24,29,42-tetraoxa-10,25,28,43-tetraoxa-19,34-diaza Pentadecane-19,34-diyl)dioctanoate di(heptadecan-9-yl) ester, 8,8'-(ll,24,29,42-tetraxa-10,25,28, 43-tetraoxa-19,34-diazapentadecane-19,34-diyl)dioctanoic acid di(heptadecyl-9-yl) ester, 8,8'-((piperate-1 ,4-diylbis(5-oxopent-5,l-diyl))bis((8-(nonyloxy)-8-pentoxyoctyl)ureadiyl))dioctanoic acid di(decayl) Heptacan-9-yl) ester, 15,18-dimethyl-9,24-bis(8-(nonyloxy)-8-pentoxyoctyl)-14,19-dimethyl-9 ,15,18,24-tetraazatriaconanedioic acid di(heptadecan-9-yl) ester, 15,19-dimethyl-9,25-bis(8-(nonyloxy)- 8-Pendant oxyoctyl)-14,20-di-Pendant oxy-9,15,19,25-tetraazatriacontandioic acid di(heptadecan-9-yl) ester, 15,18 -Diethyl-9,24-bis(8-(nonyloxy)-8-pentanoxyoctyl)-14,19-bis-bisoxy-9,15,18,24-tetraaza30 Di(heptadecan-9-yl) dialkanedioate, N,N-dimethyl-3-{[(9Z,12Z)-octadecan-9,12-dien-1-yloxy ]Methyl}dodecan-1-amine, 8-[2-(ll-{[4-(dimethylamino)butyl]oxy}octadecyl)cyclopropyl]octanoic acid methyl ester, 8 -{2-[ll-(dimethylamino)heptadecyl]cyclopropyl}octanoic acid methyl ester (compound 18), 8-((2-hydroxyethyl)(8-(nonyloxy)- 8-Pendant oxyoctyl)amino)octanoic acid heptadecan-9-ester, 6-(2-(dimethylamino)-3-(heptyloxy)propoxy)hexanoic acid (2-octanoic acid) cyclopropyl)methyl ester, (17Z)-N,N-dimethylhexadecanoic-17-en-9-amine, N1-(2-(4-(2-(di-dodecyl) Amino)ethyl)piperidine-l-yl)ethyl)-N1^V2,N2-trihexylethane-1,2-diamine, N,N-dimethyl-2-{[(9Z,12Z )-octadeca-9,12-dien-1-yloxy]methyl}undecan-1-amine, 8-{2-[ll-(dimethylamino)octadecyl] Cyclopropyl}octanoic acid methyl ester, 6-(2-(dimethylamino)-3-(hexyloxy)propoxy)hexanoic acid (2-octylcyclopropyl)methyl ester, (18Z)- N,N-dimethylheptadecanoic-18-en-10-amine, 2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl tetradecanoate, Nonanoic acid 2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl ester, N-(2-(dinonylamino)ethyl)-N-nonylglyamine Tetradecyl acid ester, N-(2-(dinonylamino)ethyl)-N-nonylglycinate, 4-(2-((2-(dinonylamino)ethyl) (Nonyl)amino)acetylamino)butylvalerate nonyl ester, l,l'-(piperidine-l,4-diyl)bis(5-(didecylamine)pentan-1- Ketone, 2-((2-(dinonylamino)ethyl)(nonyl)amine)-N-tetradecylacetamide, N-decyl-2-((2-(dinonyl) methylamino)ethyl)(nonyl)amino), N1-(3-(3-(dinonylamino)propoxy)propyl)-N1,N2,N2-tri-nonylethyl- l,2-diamine, N1-(2-(dinonylamino)ethyl)-N\N8,N8-trinonyloctyl-l,8-diamine, 8-[2-(ll-{ [4-(Dimethylamino)butyl]oxy}nonadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[ll-(dimethylamino)nonadecyl]cyclopropyl Methyl}octanoate, (Z)-6-(3-(decyloxy)-2-(dimethylamino)propoxy)hexanoate undec-2-ene-l-ester, (2R ,12Z,15Z)-N,N-dimethyl-1-(undecyloxy)ecosan-12,15-diene-2-amine, (21Z,24Z)-N,N-di Methyl triadecacarbon-21,24-diene-9-amine, 2-(dinonylamino)-N-(4-(2-((2-(dinonylamino)ethyl))( Nonyl)amino)-N-methylacetamide)butyl)-N-methylacetamide, decanoic acid 7,10-dimethyl-13,16-dinonyl-6,ll- Bilateral oxy-4-tetradecyl-4,7,10,13,16-pentaazapentadecyl 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-methylethyl Amide, 2-((2-(bis((Z)-non-3-en-l-yl)amino)ethyl)((Z)-non-3-en-l-yl)amino) -N-(2-(2-(dinonylamino)-N-methylacetamide)ethyl)-N-methylacetamide, 2-(dinonylamino)-N- (2-(2-((2-(Dinonylamino)ethyl)(nonyl)amino)acetamido)ethyl)acetamide, 8,1l-dimethyl-5,14 ,17-trinonyl-7,12-di-pentanoxy-5,8,l1,14,17-pentaazahexadecanoic acid pentyl ester, 2-((2-(dinonylamino) Ethyl)(nonyl)amino)-N-methyl-N-(2-(methylamino)ethyl)acetamide, 2-(dinonylamino)-N-(2-( 2-((2-(Dinonylamino)ethyl)(nonyl)amino)-N-methylacetamide)ethyl)-N-methylacetamide, 2-(dinonyl) methylamino)-N-methyl-N-(2-(methylamino)ethyl)acetamide, glycine 2-((N-(2-(dinonylamino)ethyl) -N-Nonylglycinyl)oxy)ethyldinonyl ester, 2-hydroxyethyldinonyl glycinate, 8-[2-(ll-{[4-(dimethylamino) Butyl]oxy}eicosyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[ll-(dimethylamino)eicosyl]cyclopropyl}octanoic acid methyl ester, (Z)- 6-(2-(dimethylamino)-3-(nonyloxy)propoxy)hexanoate undec-2-en-l-ester, (2R,12Z,15Z)-1-(dec Hexaalkoxy)-N,N-dimethyltriacontan-12,15-diene-2-amine, (22Z,25Z)-N,N-dimethyltriacontan-22,25 -Dien-10-amine, 4-(didecylaminobutyric acid) l,l-(pipiperidine-l,4-diyl)bis(4-(ddecylamino)butan-l-one )Tertiary butyl ester, 5-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperamide-l-yl)-5-side oxygen group Heptyl valerate 5-(heptyloxy)-5-side oxyvalerate, 5-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl) )piperidine-l-yl)-5-pentyloxypentanoic acid heptyl ester 5-(heptyloxy)-5-pentyloxypentanoic acid, nonanoic acid (Z)-4-((2-(4-( N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperamide-l-yl)-2-side oxyethyl)(tetradecyl)amine) But-2-ene-1-ester, nonanoic acid (Z)-4-hydroxybut-2-ene-1-ester, decanoic acid (Z)-3-((2-(4-(N-(2- (Dinonylamino)ethyl)-N-nonylglycininyl)piperidine-1-yl)-2-pentanoxyethyl)(tetradec-9-en-1-yl)amine methyl)propyl ester, (Z)-tetradec-9-ene-l-methanesulfonate, 8-[2-(9-{[4-(dimethylamino)butyl]oxy}pentadecane Alkyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[9-(dimethylamino)pentadecyl]cyclopropyl}octanoic acid methyl ester, 6-(2-(dimethylamino) )-3-(heptyloxy)propoxy)hexanoic acid (Z)-undec-2-en-l-ester, (2R,12Z,15Z)-1-(hexyloxy)-N,N -Dimethyleicos-12,15-diene-2-amine, (16Z,19Z)-N,N-dimethyleicos-16,19-diene-6-amine, 8 -((2-(4-(N-(2-(bis((Z)-non-3-en-l-yl)amino)ethyl))-N-((Z)-non-3-ene -l-yl)glyminyl)piperidine-l-yl)-2-side oxyethyl)(nonyl)amino)octanoic acid methyl ester, 4-(nonylglycinyl)piperidine- 1-tertiary butylcarboxylate, (Z)-dec-3-enoic acid 3-((2-(4-(N-(2-(dinonylamino)ethyl))-N-nonylglyamine Enyl)piperidine-l-yl)-2-side oxyethyl)(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-(di Nonylglycinyl)piperidin-1-yl)ethan-1-one(Z)-1-bromonon-4-ene, octanoic acid 3-((2-(4-(N-(2-(di Nonylamino)ethyl)-N-nonylglycinyl)piperamide-side oxyethyl)(dodecyl)amino)propyl ester, glycine tertiary butyldodecyl ester , S-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pipienyl-l-yl)-2-side oxygen Ethyl)(nonyl)amino)butylthiopentyl ester, 3-methylhexanoic acid 3-((2-(l-(N-(2-(dinonylamino)ethyl)-N -Nonylglycinyl)piperidinyl)ethyl)(nonyl)amino)propyl ester, tertiary butyl 4-(2-((3-((3-methylhexyl)) Oxy)propyl)(nonyl)amino)ethyl)piperidine-l-, hexanoic acid 3-((2-(l-(N-(2-(dinonylamino)ethyl)- N-nonylglycinyl)piperidin-4-yl)ethyl)(nonyl)amino)-2-methylpropyl ester, 3-methylhexanoic acid 3-((2-(4-( N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperamide-pentoxyethyl)(nonyl)amino)propyl ester, hexanoic acid 3-(( 2-(4-(N-(2-(Dinonylamino)ethyl)-N-nonylglycinyl)piperamide-pentoxyethyl)(nonyl)amino)-2- Methyl propyl ester, 8-[2-(9-{[4-(dimethylamino)butyl]oxy}hexadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[9- (Dimethylamino)hexadecyl]cyclopropyl}octanoic acid methyl ester, (Z)-6-(2-(dimethylamino)-3-(hexyloxy)propoxy)hexanoic acid Undec-2-ene-l-ester, (2R,12Z,15Z)-1-(decyloxy)-N,N-dimethyleco-12,15-diene-2-amine , (17Z,20Z)-N,N-dimethylhexadecanoic acid 17,20-diene-7-amine, 1-(dinonylglycinyl)piperidine-4-carboxylic acid 2-( (2-(Dinonylamino)ethyl)(nonyl)amino)ethyl ester, 4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl base) cyclohexane-1,4-dicarboxylic acid l-(2-(dinonylamino)ethyl) ester, 2-(dinonylamino)ethan-1-ol, 12-((2- (l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)dodecyl Methyl alkanoate, tertiary butyl 3-(2-((12-methoxy-12-side oxydodecyl)(tetradecyl)amino)ethyl)pyrrolidine-l-carboxylate , Decanoic acid 3-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)ethyl)(decanoic acid Tetraalkyl)amino)propyl ester, tertiary butyl 3-(2-((3-(decyloxy)propyl)(tetradecyl)amino)ethyl)pyrrolidine-l-carboxylate , 6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)ethyl)(tetradecane Heptyl)amino)hexanoate, 3-(2-((6-(heptyloxy)-6-side oxyhexyl)(tetradecyl)amino)ethyl)pyrrolidine-1-carboxylic acid Tertiary butyl ester, 8-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)ethyl) (Tetradecylamino)octanoic acid amyl ester, 3-(2-(tetradecyllamino)ethyl)pyrrolidine-l-carboxylic acid tertiary butyl ester, 12-((2-(l-( Methyl N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-3-yl)ethyl)(tetradecyl)amino)dodecanoate , 3-(2-((12-methoxy-12-side oxydodecyl)(tetradecyl)amino)ethyl)piperidine-1-carboxylic acid butyl ester, decanoic acid 3-( (2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-3-yl)ethyl)(tetradecyl)amine )propyl ester, 6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-3-yl)ethyl)( Tetradecyl)amino)heptylhexanoate, 8-((2-(l-(N-(2-(dinonylamino)ethyl))-N-nonylglycinyl)piperidine -3-yl)ethyl)(tetradecyl)amino)pentyl octanoate, 6-((2-(4-(2-((2-(di-dodecylamine)ethyl)) (Dodecyl)amino)ethyl)piperidine-l-yl)ethyl)(dodecyl)amino)pentylhexanoate Step 1: 6-bromohexanoic acid pentyl ester, 8-[2 -(9-{[4-(Dimethylamino)butyl]oxy}heptadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[9-(dimethylamino)heptadecanyl) Alkyl]cyclopropyl}octanoic acid methyl ester, (2S,12Z,15Z)-N,N-dimethyl-1-(octyloxy)octanoate-12,15-diene-2-amine, 6-(2-(Dimethylamino)-3-(octyloxy)propoxy)hexanoate (2-octylcyclopropyl)methyl ester, (18Z,21Z)-N,N-dimethyl Hexadecanoic-18,21-diene-8-amine, trans-1-methyl-3,4-bis(((Z)-hexadecanoic-9-enyloxy)methyl) Pyrrolidine, (Z)-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pipero-l-yl)- 2-Pendant oxyethyl)(tetradecyl)amino)butyrate non-2-en-l-ester, trans-1-methyl-3,4-bis(((9Z,12Z)- Octadeca-9,12-dienyloxy)methyl)pyrrolidine, 12-((2-(4-(N-(2-(dinonylamino)ethyl))-N-nonyl Glycinyl) piperazine-l-yl)-2-side oxyethyl) (tetradecyl) amino) methyl dodecanoate, (7Z)-17-[2-(dimethyl Amino)ethyl]ethyl hexadecanoate, trans-1-methyl-3,4-bis(((Z)-octadeca-9-enyloxy)methyl )pyrrolidine, 6-(2-{]11-^2-(dimethylamino)ethyl]eicosyl}cyclopropyl)hexanoic acid methyl ester, 12-((2-(l-( Methyl N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)piperidin-4-yl)ethyl)(tetradecyl)amino)dodecanoate , 10-(2-V-^2-(dimethylamino)ethyl]hexadecyl}cyclopropyl)decanoic acid methyl ester, 8-(2-{111-;2-(dimethyl Amino)ethyl]heptadecyl}cyclopropyl)octanoic acid methyl ester, glycine 2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycine Dihydryl)piperidin-4-yl)ethyldinonyl ester, 4-(2-((dinonylglycinyl)oxy)ethyl)piperidin-1-carboxylic acid tertiary butyl ester, 8- (2-{lLl-;2-(Dimethylamino)ethyl]octadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{l11- "2-(dimethylamino) Ethyl]nonadecyl}cyclopropyl)octanoic acid methyl ester, l,-(piperidine-l,4-diyl)bis(2-(dinonylamino)ethyl-1-one), 8- [2-{]11-^2-(dimethylamino)ethyl]eicosyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{9-[2-(dimethylamino) )ethyl]pentadecyl}cyclopropyl)octanoic acid methyl ester, (7Z)-19-{[4-(dimethylamino)butyl]oxy}octadecanoic acid methyl ester , (7Z)-19-(dimethylamino)octadecanoic acid methyl ester, cis-1-methyl-3-[(9Z,12Z)-octadecacarbon-9,12 -Dien-1-yloxy]-4-(octyloxy)pyrrolidine, 2-(di-dodecylamine)-l-(4-(N-(2-(di-dodecylamine) Alkylamino)ethyl)-N-dodecylglycinyl)piperidine-l-yl)ethan-1-one, (Z)-6-(2-(dimethylamino)- 3-(octyloxy)propoxy)hexanoate undec-2-en-l-ester, (2SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12 -Dien-1-yloxy]dec-2-amine (compound 11), (19Z,22Z)-N,N-dimethyloctadecanoic-19,22-diene-9-amine, 8 -(2-{9-[2-(Dimethylamino)ethyl]hexadecyl}cyclopropyl)octanoic acid methyl ester, 5-(2-(4-(N-(2-( Dinonylamino)ethyl)-N-nonylglycinyl)pipiperidine-side oxyethyl)(nonyl)amino)pentyl methyl ester, 8-(2-{9-[2 -(Dimethylamino)ethyl]heptadecyl}cyclopropyl)octanoic acid methyl ester, (7Z)-19-[2-(dimethylamino)ethyl]octadecanoic acid-7- Methyl alkenoate, (Z)-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pipienyl)-l-yl )-2-Pendant oxyethyl)(nonyl)amino)butyric acid pent-2-en-l-ester, (1lZ)-19-[2-(dimethylamino)ethyl]20 Methyl octa-11-enoate, (9Z)-21-[2-(dimethylamino)ethyl] methyl 27-9-enoate, (9Z)-21-[2- (Dimethylamino)ethyl]octacos-9-enoic acid methyl ester, (9Z)-21-[2-(dimethylamino)ethyl]octacos-9-enoic acid Methyl ester, glycine 2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycinyl)pyrrolidin-3-yl)ethyldinonyl ester, (9Z)-21-[2-(Dimethylamino)ethyl]triacontane-9-enoic acid methyl ester, glycine (l-(N-(2-(dinonylamino)ethyl) (9Z)-19-[2-(dimethylamino)ethyl]pentadecacarbon-9 -Methyl enoate, (9Z)-19-[2-(dimethylamino)ethyl]icosac-9-enoic acid methyl ester, 6-(2-(8-(3-(decane) Oxy)-2-(dimethylamino)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, (1lZ)-19-{[4-(dimethylamino)butyl]oxy } Methyl octadeca-l1-enoate, (1lZ)-19-(dimethylamino) methyl octadeca-l1-enoate, (2S)-N,N-dimethyl- 1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]dodecane-2-amine, (14Z,17Z)-N,N-dimethyltetrasan Carbon-14,17-diene-4-amine, methylbis((9Z,12Z)-octadeca-9,12-dieneyl)amine, (9Z)-19-{[4-(dimethyl methylamino)butyl]oxy}octacarbon-9-enoic acid methyl ester, (9Z)-19-(dimethylamino)octacarbon-9-enoic acid methyl ester, (Z)- 17-(2-(Dimethylamino)-3-(octyloxy)propoxy)heptadecan-8-enoic acid methyl ester, (3R,4R)-3,4-bis((Z) -Hexadecacarbon-9-alkenyloxy)-1-methylpyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadecanoic-9,12-di En-1-yloxy]undecane-2-amine, (20Z,23Z)-4-(dimethylamino)butyric acid 29-20,23-diene-10-ester, (20Z,23Z)-4-(dimethylamino)butyric acid 20Z,23Z)-N,N-dimethyl 29-20,23-diene-10-amine, 3-((6Z,9Z,28Z,31Z)-37-6,9, 28,31-tetraen-19-yloxy)-N,N-dimethylpropan-l-amine, 3-((6Z,9Z,28Z,31Z)-trioctacarbon-6,9,28 ,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine, (6Z,9Z,28Z,31Z)-4-(dimethylamino)butyric acid thirty-seven Carbon-6,9,28,31-tetraene-19-ester, 5-(dimethylamino)valerate (6Z,16Z)-12-((Z)-dec-4-enyl)econyl Dicarbon-6,16-diene-l1-ester, (6Z,16Z)-5-(dimethylamino)pentanoic acid 12-((Z)-dec-4-enyl)icosan- 6,16-diene-l1-ester, (6Z,16Z)-5-(dimethylamino)pentanoate 12-((Z)-dec-4-enyl)dococ-6,16 -Diene-11-ester, L-arginine-α-(2,3-dilauryloxy)propylamide, L-lysine-α-(2,3-dilauryloxy) )propylamide, 2,3-dioleyloxypropylamine, 2,3-distearyloxypropylamine, 2,3-dilauryloxypropylamine, dilinoleylmethyl 4-( Dimethylamino)propyl ether), dilinoleylmethyl 4-(dimethylamino)butyl ether) and 2,2-dilinoleyl-4-(2-dimethylamino) Ethyl)-[l,3]-dioxolane.

In some embodiments, the at least one non-cationic lipid includes 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 of these combination. In some embodiments, the LNP may be substantially free of at least one non-cationic lipid. In some embodiments, the LNP may not contain any amount of at least one non-cationic lipid.

In some embodiments, at least one non-cationic lipid can be selected from, but is not limited to, at least one of the following: 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0 diether PC), DSPC, but each tail has 3 unsaturated double bonds (18:3 PC), acylcarnitine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine ( C16 Lyso PC), N-oleyl-SPM (C18:l), N-tetradecylSPM (C24:0), N-nervacylC (C24:l), aminomethanoyl]cholesterol (Cet- P), Cholesterol hemisuccinate (CHEMS), Cholesterol (Chol), Cholesterol hemidicarboxylic acid (Chol-C12), 12-cholesteryloxycarbonylaminododecanoic acid (Chol-C13N), Cholesterol hemisuccinate Oxalate (Chol-C2), Cholesterol hemimalonate (Chol-C3), N-(cholesteryl-oxycarbonyl)glycine (Chol-C3N), Cholesterol hemiglutarate (Chol-C5 ), Cholesterol hemisuberate (Chol-C6), Cholesterol hemisuberate (Chol-C7), Cholesterol hemisuberate (Chol-C8), Cardiolipid (CL), l,2 -Bis(tricosyl-10,12-dicyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetylphosphate (DCP), dicetylphosphate Dihexadecylphosphate (DCP1), 1,2-dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain dihexadecylphosphatidylcholine (DHPC), dihexadecane Phosphoethanolamine (DHPE), 1,2-dilinoleyl-sn-glycerol-3-phosphocholine (DLPC), l,2-dilauryl-sn-glycerol-3-PE (DLPE ), dimyristyl glyceryl hemisuccinate (DMGS), dimyristyl phosphatidylcholine (DMPC), dimyristyl phosphoethanolamine (DMPE), dimyristyl phospholipidyl glycerol (DMPG) ), dioleoxybenzyl alcohol (DOBA), 1,2-dioleylglyceryl-3-hemisuccinate (DOGHEMS), N-[2~(2-{2-[2-(2, 3-bis-octadecy-9-alkenyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-trihydroxy- 6-Hydroxymethyl-tetrahydropyran-2-ylsulfanyl)-propylamide (DOGP4αMan), dioleylphosphatidylcholine (DOPC), dioleylphosphatidylcholine (DOPE), dioleylphosphatidylethanolamine (DOPE), Oleoyl-phosphatidyl ethanolamine 4-(N-maleyl iminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleyl phospholipid glycerol (DOPG), 1,2-dioleyl-sn-glycerol-3-(phospho-L-serine) (DOPS), non-cell-fusogenic phospholipid (DPhPE), dipalmitoyl phosphatidylcholine (DPPC), Palmitoyl Phosphatidyl Ethanolamine (DPPE), Dipalmityl Phosphatidyl Glycerol (DPPG), Dipalmityl Phosphatidyl Serine (DPPS), Distearyl Phosphatidyl Choline (DSPC), Distearyl Dyl-phosphatidyl-ethanolamine (DSPE), distearyl-phosphoethanolamine imidazole (DSPEI), 1,2-di-undecanoyl-sn-glycerol-phosphocholine (DUPC), egg phosphatidylcholine Alkali (EPC), N-histamine cholesteryl carbamate (HCChol), histamine distearyl glycerol (HDSG), N-histamine acyl cholesterol hemisuccinate (HistChol), 1,2 -Dipalmitoylglycerol-hemisuccinate-Nα-histamine-hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-20 Pentakanediylamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH), Hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dipalmitoylglycerol-Oα-histamine-Nα-hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylglycerolamine ( ManDOG), l,2-dioleyl-sn-glycerol-3-phosphoethanolamine-N-[4-(p-maleyl iminomethyl)cyclohexane-methamide] (MCC- PE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1-myristyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), athiol -Reactive maleimide head-terminal lipids, such as 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine-N-[4-(p-maleimidebenzene (MPB-PE), tetracosenoic acid (NA), sodium cholate (NaChol), l,2-dioleyl-sn-glycerol-3-[phosphoethanolamine-N-dodecane Alkyl group (NC12-DOPE), as defined in the synthesis example (ND98) in WO2008042973A2, "N-glutaryl phosphatidyl ethanolamine of formula 1" (NG-PE), N-hydroxysulfosuccinyl acyl Amine (NHS-'x'), "N~(co)-dicarboxylic acid-derived phosphatidylethanolamines covered by formula 1" (NωPE-'x'), oleic acid (OA), 1-oleyl-2 -Cholesterol semisuccinyl-sn-glycerol-3-phosphocholine (OChemsPC), phosphatidic acid (PA), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol (PEG). An example of PEG-PE may be polyethylene glycol-distearyl phospholipid ethanolamine lipid (PEG-PE), phosphatidylglycerol (PG), partially hydrogenated soybean phosphatidylcholine (PHSPC), phosphatidylcholine Alcohol lipid (PI), phosphatidyl inositol-4-phosphate (PIP), palmityl oleyl phosphatidylcholine (POPC), phospholipid ethanolamine (POPE), palmityl oleyl phospholipid glycerol ( POPG), phosphatidyl serine (PS), lissamine rhodamine (lissamine rhodamine) B-phosphatidyl ethanolamine lipid (Rh-PE), purified soybean-derived mixture of phospholipid (SIOO), phosphatidylcholine (SM), 18-1-trans PE, 1-stearyl-2-oleyl-phosphatidylethanolamine (SOPE), soybean phosphatidylcholine (SPC), neural myelin (SPM), α. α'-Coconidyl 6,6'-disbehenate (TDB), l,2-ditearyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), phosphoric acid ((23S,5R )-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dihydropyrimidine-1(2/-/) -Tetrahydrofuran-2-yl)methyl methyl ester, 1,2-diarachidonyl-sn-glycerol-3-phosphocholine, 1,2-diarachidonyl-sn-glycerol- 3-Phosphoethanolamine, 1,2-di-docosahexaenyl-sn-glycerol-3-phosphocholine, 1,2-di-docosahexaenyl-sn-glycerol-3 -Phosphoethanolamine, 1,2-bislinoleyl-sn-glycerol-3-phosphocholine, 1,2-bislinoleyl-sn-glycerol-3-phosphoethanolamine, 1,2-di Linseed oil acyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearyl-sn-glycerol-3-phosphate Ethanolamine, 16-O-monomethylPE, 16-O-dimethylPE and dioleyl phospholipid ethanolamine.

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 its 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 making 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 LNP is ATX-88. As a non-limiting example, LNP contains about 20 to 60% 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% noncationic lipids; about 25 to 55% sterols; and about 0.5 to 15% PEG-modified Lipids.

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 its 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-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 and ATX-0134. Methods for making 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, the LNP contains about 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, ATX-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; About 25 to 55% sterols; and about 0.5 to 15% PEG-modified lipids.

In some embodiments, the LNP is included in International Patent Publications Nos. WO2012019168, WO2012045082, WO2012135805, WO2012158736, WO2013039857, WO2013039861, WO2013052523, WO2013090186, WO2013090648, WO2013096709, WO2013101690, WO2013106496, WO2013130161, WO2013151663, WO2013151664, WO2013151665, WO2013151666, WO2013151667 , WO2013151668, WO2013151669, WO2013151670, WO2013151671, WO2013151672, WO2013151736, WO2014028429, WO2014081507, WO2014093574, WO2014093924, WO2 014113089, WO2014144039, WO2014144711, WO2014144767, WO2014152027, WO2014152030, WO2014152031, WO2014152211, WO2014152540, WO2014158795, WO201415 9813, WO2014164253, WO2015006747, WO2015034925, WO2015034928 , WO2015038892, WO2015048744, WO2015051169, WO2015051173, WO2015051214, WO2015058069, WO2015085318, WO2015089511, WO2015105926, WO2015164674, WO2 015196118, WO2015196128, WO2015196130, WO2015199952, WO2016011222, WO2016011226, WO2016011306, WO2016014846, WO2016022914, WO2016036902, WO201607 7123, WO2016077125, WO2016100812, WO2016118724, WO2016118725 , WO2016164762, WO2016176330, WO2016201377, WO2017004143, WO2017015457, WO2017015463, WO2017015630, WO2017019935, WO2017020026, WO2017031232, WO2 017049074, WO2017049245, WO2017049275, WO2017049286, WO2017062513, WO2017070601, WO2017070613, WO2017070616, WO2017070618, WO2017070620, WO201707 0622, WO2017070623, WO2017070624, WO2017070626, WO2017075531 , WO2017099823, WO2017106799, WO2017112865, WO2017112943, WO2017117528, WO2017120612, WO2017127750, WO2017180917, WO2017201317, WO2017201325, WO2 017201328, WO2017201332, WO2017201333, WO2017201340, WO2017201342, WO2017201346, WO2017201347, WO2017201348, WO2017201349, WO2017201350, WO201720 1352, WO2017214175, WO2017218704, WO2017223135, WO2017223176 , WO2018053209, WO2018075980, WO2018078053, WO2018081459, WO2018081480, WO2018081638, WO2018089540, WO2018089851, WO2018107026, WO2018107088, WO2 018144082, WO2018144775, WO2018144778, WO2018151816, WO2018157009, WO2018170245, WO2018170256, WO2018170260, WO2018170270, WO2018170306, WO201817 0336, WO2018170347, WO2018175783, WO2018187590, WO2018191657 , WO2018191719, WO2018200737, WO2018200943, WO2018213731, WO2018213789, WO2018231990, WO2018232006, WO2018232120, WO2018232355, WO2018232357, WO2 019018765, WO2019023179, WO2019036000, WO2019036008, WO2019036028, WO2019036030, WO2019036670, WO2019036682, WO2019046809, WO2019055807, WO201908 9818, WO2019089828, WO2019103993, WO2019104152, WO2019104160 , WO2019104195, WO2019136241, WO2019143910, WO2019148101, WO2019152557, WO2019200171, WO2019226650, WO2020023390, WO2020047201, WO2020056147, WO2 020056155, WO2020056239, WO2020056304, WO2020056370, WO2020061284, WO2020061295, WO2020061317, WO2020061332, WO2020061367, WO2020061426, WO202006 1457, WO202006242, WO2020069169, WO2020081938, WO2020097291 , WO2020097409, WO2020146805, WO2020160397, WO2020160430, WO2020185811, WO2020190750, WO2020227510, WO2020227537, WO2020227615, WO2020227642, WO2 020227690, WO2020243561, WO2020263883, WO2020263985, WO2021022173, WO2021026358, WO2021030701, WO2021050864, WO2021050986, WO2021055833, WO202105 5835, WO2021055849, WO2021076805, WO2021076811, WO2021142280 , at least one of the cationic lipids described in WO2021154763, WO2021155243, WO2021155267, WO2021155274, WO2021159040 and WO2021159130. As a non-limiting example, LNPs include about 20 to 60% cationic lipids; about 5 to 25% noncationic lipids; about 25 to 55% sterols; and about 0.5 to 15% PEG-modified lipids.

In some embodiments, the LNPs comprise ionizable lipids or lipid-like materials. 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 the art and can be used herein.

In some embodiments, the LNP includes phospholipids. As non-limiting examples, the phospholipid (helper) may be DOPE, DSPC, DOTAP or DOTMA.

In some embodiments, the LNPs comprise PEG derivatives. As non-limiting examples, PEG derivatives may be lipid-anchored, such as PEGs are 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.

In some embodiments, at least one sterol includes at least one cholesterol or cholesterol derivative. In some implementations, the LNP may be substantially free of at least one sterol. In some embodiments, the LNP may not contain any amount of at least one sterol.

In some embodiments, the at least one additional LNP functional component includes at least one component that reduces particle aggregation, at least one component that reduces clearance of LNP from circulation within the body, and at least one component that increases the ability of LNP to cross the mucus layer at least one component that reduces an individual's immune response to administered LNP, at least one component that modifies the membrane fluidity of the LNP, at least one component that contributes to the stability of the LNP, or any combination thereof. In some implementations, the LNP may be substantially free of 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.

In some embodiments, additional LNP functional components may be composed of polymers. In some embodiments, polymers containing additional LNP functional components can be composed of at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least Composed of a polyamide (ATTA), at least one cationic polymer, or any combination thereof.

In some embodiments, the average molecular weight of the polymer moiety (eg, PEG) can be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer can 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, 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, 11,500-20,000, 12,000-20,000, 12,500-20,000, 13,000-20,000, 13,500-20,000, 14,000-20,000, 14,500-20,000, 15,000-20,000, 15,5 00 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, 2,5 00 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, 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, 12,500 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 19,5 00, 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 1 9,000, 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, 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, 15,5 00 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, 4,5 00 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 18,5 00, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500, 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 18,5 00, 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, 1 2,500 to 18,000, 13,000 to 18,000, 13,500 to 18,000, 14,000 to 18,000, 14,500-18,000, 15,000-18,000, 15,500-18,000, 16,000-18,000, 16,500-18,000, 17,000-18,000, 17,500-18,000, 1,500-17,500, 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, 14,5 00 to 17,500, 15,000 to 17,500, 15,500 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, 5,00 0 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, 9,000 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, 13,500 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 16,50 0, 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 16,5 00, 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, 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, 15,500 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, 15,000 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, 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, 14,5 00 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, 11,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500, 13,000 to 14,500, 13,50 0 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, 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, 12,000 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 13,000, 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, 11,000 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, 2,500 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, 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, 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, 2,0 00 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, 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, 10,500 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, 5,00 0 to 10,500, 5,500 to 10,500, 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 10,00 0, 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, 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 9,5 00, 7,000 to 9,500, 7,500 to 9,500, 8,000 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, 5,0 00 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 8,5 00, 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, 1,500 to 8,000, 2,000 to 8,000, 2,500 to 8,000, 3,0 00 to 8,000, 3,500 to 8,000, 4,000 to 8,000, 4,500 to 8,000, 5,000 to 8,000, 5,500 to 8,000, 6,000 to 8,000, 6,500 to 8,000, 7,000 to 8,000, 7,500 to 8,000, 1,500 to 7,500, 2,000 to 7,500, 2,500 to 7,5 00, 3,000 to 7,500, 3,500 to 7,500, 4,000 to 7,500, 4,500 to 7,500, 5,000 to 7,500, 5,500 to 7,500, 6,000 to 7,500, 6,500 to 7,500, 7,000 to 7,500, 1,500 to 7,000, 2,000 to 7,000, 2,500 to 7,000, 3,0 00 to 7,000, 3,500 to 7,000, 4,000 to 7,000, 4,500 to 7,000, 5,000 to 7,000, 5,500 to 7,000, 6,000 to 7,000, 6,500 to 7,000, 1,500 to 6,500, 2,000 to 6,500, 2,500 to 6,500, 3,000 to 6,500, 3,500 to 6,5 00, 4,000 to 6,500, 4,500 to 6,500, 5,000 to 6,500, 5,500 to 6,500, 6,000 to 6,500, 1,500 to 6,000, 2,000 to 6,000, 2,500 to 6,000, 3,000 to 6,000, 3,500 to 6,000, 4,000 to 6,000, 4,500 to 6,000, 5,0 00 to 6,000, 5,500 to 6,000, 1,500 to 5,500, 2,000 to 5,500, 2,500 to 5,500, 3,000 to 5,500, 3,500 to 5,000, 4,000 to 5,500, 4,500 to 5,500, 5,000 to 5,500, 1,500 to 5,000, 2,000 to 5,000, 2,500 to 5,0 00, 3,000 to 5,000, 3,500 to 5,000, 4,000 to 5,000, 4,500 to 5,000, 1,500 to 4,500, 2,000 to 4,500, 2,500 to 4,500, 3,000 to 4,500, 3,500 to 4,500, 4,000 to 4,500, 1,500 to 4,000, 2,000 to 4,000, 2,5 00 to 4,000, 3,000 to 4,000, 3,500 to 4,000, 1,500 to 3,500, 2,000 to 3,500, 2,500 to 3,500, 3,000 to 3,500, 1,500 to 3,000, 2,000 to 3,000, 2,500 to 3,000, 1,500 to 2,500, 2,000 to 2,500, and 1,500 to 2, 000 daltons.

In some embodiments, a polymer (eg, PEG) is conjugated to at least one lipid. In some embodiments, the lipid conjugated to the polymer is composed of at least one neutral lipid, at least one phospholipid, at least one anionic lipid, at least one cationic lipid, at least one cholesterol, at least one cholesterol derivative, or any combination thereof. composition.

In some embodiments, the lipid conjugated to the polymer may be selected from, but not limited to, at least one of the previously listed cationic, noncationic, or sterol lipids.

In some embodiments, at least one PEG-lipid conjugate can be selected from, but is not limited to, at least one of the following: Siglec-IL-PEG-DSPE, carbamic acid (R)-2,3-bis(octadecane) Oxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propyl ester, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG -DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Brain Amine C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000 -DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG link (Click) DMG C14, PEG link C12, PEG link C10, N(carbonyl-methoxypolyethylene glycol-2000)-l,2-distearyl-sn-glyceryl 3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000 -DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearyl-sn-glycerol-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folic Acid PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000 Maleimide, DSPE -PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE -Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearyl-glycerol-polyethylene glycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 cerebroamide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-dihard Fatty acyl-sn-glycerol-3-phosphoethanolamine-PEG2000, carbamic acid (R)-2,3-bis(octadecyloxy)propyl-1-(methoxy poly(ethylene glycol) 2000) Propyl ester, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

The amounts and proportions of the LNP components may vary depending on any amount of the desired form, structure, function, cargo, target, or any combination thereof. The amount of each component may be expressed in various embodiments as a percentage (mol%) of the total molar mass of all lipids or lipid-conjugated components occupied by the indicated component. The amount of each component may be expressed in various embodiments as the relative proportion of each component based on molar mass (molar ratio). The amount of each component may be expressed in various embodiments as the weight (mg or equivalent) of each component used to formulate the LNP before manufacturing. The amounts of each component may be expressed in various embodiments by any other method known in the art. Any formulation given in one expression of the amount of an ingredient ("unit") is expressly meant to cover any formulation in which the amount of an ingredient is expressed in different units, where such expressions are valid when converted to the same units etc. Effectiveness value. In some implementations, "effectively equivalent values" means two or more values that are within about 10% of each other.

In some embodiments, the LNP includes at least one cationic lipid in an amount from about 0.1 to 100 mole %. In some embodiments, the LNP includes at least one cationic lipid in an amount of about 20 to 60 mole %. In some embodiments, the LNP includes at least one cationic lipid in an amount of about 50 to 85 mol%. In some embodiments, the LNPs comprise at least one cationic lipid in an amount of less than about 20 mole %. In some embodiments, the LNPs comprise at least one cationic lipid in an amount greater than about 60 molar % or about 85 molar %. In some embodiments, the LNPs comprise at least one cationic lipid in an amount of about 95 molar % or less. In some embodiments, the LNPs comprise cationic lipids in an amount less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP includes at least one cationic lipid in an amount greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, and 95 mol%. In some embodiments, the LNP includes at least one cationic lipid in an amount of about 20 to 30 mol%, 20 to 35 mol%, 20 to 40 mol%, 20 to 45 mol%, 20 to 50 mol% %, 20 to 55 mol%, 20 to 60 mol%, 20 to 65 mol%, 20 to 70 mol%, 20 to 75 mol%, 20 to 80 mol%, 20 to 85 mol% %, 20 to 90 mol%, 25 to 35 mol%, 25 to 40 mol%, 25 to 45 mol%, 25 to 50 mol%, 25 to 55 mol%, 25 to 60 mol% , 25 to 65 mol%, 25 to 70 mol%, 25 to 75 mol%, 25 to 80 mol%, 25 to 85 mol%, 25 to 90 mol%, 30 to 40 mol%, 30 to 45 mol%, 30 to 50 mol%, 30 to 55 mol%, 30 to 60 mol%, 30 to 65 mol%, 30 to 70 mol%, 30 to 75 mol%, 30 to 80 mol%, 30 to 85 mol%, 30 to 90 mol%, 35 to 40 mol%, 35 to 45 mol%, 35 to 50 mol%, 35 to 55 mol%, 35 to 60 mol%, 35 to 65 mol%, 35 to 70 mol%, 35 to 75 mol%, 35 to 80 mol%, 35 to 85 mol%, 35 to 90 mol%, 40 to 45 Mol%, 40 to 50 Mol%, 40 to 55 Mol%, 40 to 60 Mol%, 40 to 65 Mol%, 40 to 70 Mol%, 40 to 75 Mol%, 40 to 80 Mol% Ear%, 40 to 85 mol%, 40 to 90 mol%, 45 to 55 mol%, 45 to 60 mol%, 45 to 65 mol%, 45 to 70 mol%, 45 to 75 mol% %, 45 to 80 mol%, 45 to 85 mol%, 45 to 90 mol%, 50 to 60 mol%, 50 to 65 mol%, 50 to 70 mol%, 50 to 75 mol% , 50 to 80 mol%, 50 to 85 mol%, 50 to 90 mol%, 55 to 65 mol%, 55 to 70 mol%, 55 to 75 mol%, 55 to 80 mol%, 55 to 85 mol%, 55 to 90 mol%, 60 to 70 mol%, 60 to 75 mol%, 60 to 80 mol%, 60 to 85 mol%, 60 to 90 mol%, 65 to 75 mol%, 65 to 80 mol%, 65 to 85 mol%, 65 to 90 mol%, 70 to 80 mol%, 70 to 85 mol%, 70 to 90 mol%, 75 to 85 mol%, 75 to 90 mol%, 80 to 90 mol%, or 85 to 95 mol%.

In some embodiments, the LNP includes at least one non-cationic lipid in an amount from about 0.1 to 100 mole %. In some embodiments, the LNP includes at least one non-cationic lipid in an amount of about 5 to 35 mol%. In some embodiments, the LNP includes at least one cationic lipid in an amount of about 5 to 25 mol%. In some embodiments, the LNP includes at least one noncationic lipid in an amount of less than about 5 mole %. In some embodiments, the LNPs comprise at least one noncationic lipid in an amount greater than about 25 molar % or about 35 molar %. In some embodiments, the LNP includes at least one non-cationic lipid in an amount of about 95 molar % or less. In some embodiments, the LNP includes at least one noncationic lipid in an amount less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 , 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP includes at least one noncationic lipid in an amount greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%. In some embodiments, the LNP includes at least one noncationic lipid in an amount of about 5 to 15 mol%, 5 to 25 mol%, 5 to 35 mol%, 5 to 45 mol%, 5 to 55 mol% Mol%, 10 to 20 Mol%, 10 to 30 Mol%, 10 to 40 Mol%, 10 to 50 Mol%, 15 to 25 Mol%, 15 to 35 Mol%, 15 to 45 Mol %, 20 to 30 mol%, 20 to 40 mol%, 20 to 50 mol%, 25 to 35 mol%, 25 to 45 mol%, 30 to 40 mol%, 30 to 50 mol% %, and 35 to 45 mol%.

In some embodiments, the LNP includes at least one sterol in an amount from about 0.1 to 100 mole %. In some embodiments, the LNP includes at least one sterol in an amount of about 20 to 45 mol%. In some embodiments, the LNP includes at least one sterol in an amount of about 25 to 55 mol%. In some embodiments, the LNP includes at least one sterol in an amount of less than about 20 mole %. In some embodiments, the LNP includes at least one sterol in an amount greater than about 45 mol% or about 55 mol%. In some embodiments, the LNP includes at least one sterol in an amount of about 95 mole percent or less. In some embodiments, the LNP includes at least one sterol in an amount less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP includes at least one sterol in an amount greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, and 95 mol%. In some embodiments, the LNP includes at least one sterol in an amount of about 10 to 20 mol%, 10 to 30 mol%, 10 to 40 mol%, 10 to 50 mol%, 10 to 60 mol% 15 to 25 mol%, 15 to 35 mol%, 15 to 45 mol%, 15 to 55 mol%, 15 to 65 mol%, 20 to 30 mol%, 20 to 40 mol% %, 20 to 50 mol%, 20 to 60 mol%, 25 to 35 mol%, 25 to 45 mol%, 25 to 55 mol%, 25 to 65 mol%, 30 to 40 mol% , 30 to 50 mol%, 30 to 60 mol%, 35 to 45 mol%, 35 to 55 mol%, 35 to 65 mol%, 40 to 50 mol%, 40 to 60 mol%, 45 to 55 mol%, 45 to 65 mol%, 50 to 60 mol%, and 55 to 65 mol%.

In some embodiments, the LNP includes at least one additional LNP functional component in an amount from about 0.1 to 100 mole %. In some embodiments, the LNP includes at least one additional LNP functional component in an amount of about 0.5 to 15 mole %. In some embodiments, the LNP includes at least one additional LNP functional component in an amount of about 15 to 40 mole %. In some embodiments, the LNP includes at least one additional LNP functional component in an amount of less than about 0.1 mole %. In some embodiments, the LNP includes at least one additional LNP functional component in an amount greater than about 15 mole % or about 40 mole %. In some embodiments, the LNP includes at least one additional LNP functional component in an amount of about 95 mole % or less. In some embodiments, the LNP includes at least one additional LNP functional component in an amount less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40 , 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP includes at least one additional LNP functional component in an amount greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

In some embodiments, the LNP includes at least one additional LNP functional component in an amount of about 0.1 to 1 mol%, 0.1 to 2 mol%, 0.1 to 3 mol%, 0.1 to 4 mol% , 0.1 to 5 mol%, 0.1 to 6 mol%, 0.1 to 7 mol%, 0.1 to 8 mol%, 0.1 to 9 mol%, 0.1 to 10 mol%, 0.1 to 15 mol%, 0.1 to 20 mol%, 0.1 to 25 mol%, 1 to 2 mol%, 1 to 3 mol%, 1 to 4 mol%, 1 to 5 mol%, 1 to 6 mol%, 1 to 7 mol%, 1 to 8 mol%, 1 to 9 mol%, 1 to 10 mol%, 1 to 15 mol%, 1 to 20 mol%, 1 to 25 mol%, 2 to 3 mol%, 2 to 4 mol%, 2 to 5 mol%, 2 to 6 mol%, 2 to 7 mol%, 2 to 8 mol%, 2 to 9 mol%, 2 to 10 Mol%, 2 to 15 Mol%, 2 to 25 Mol%, 3 to 4 Mol%, 3 to 5 Mol%, 3 to 6 Mol%, 3 to 7 Mol%, 3 to 8 Mol% Ear%, 3 to 9 mol%, 3 to 10 mol%, 3 to 15 mol%, 3 to 20 mol%, 3 to 25 mol%, 4 to 5 mol%, 4 to 6 mol% %, 4 to 7 mol%, 4 to 8 mol%, 4 to 9 mol%, 4 to 10 mol%, 4 to 15 mol%, 4 to 20 mol%, 4 to 25 mol% , 5 to 10 mol%, 5 to 15 mol%, 5 to 20 mol%, 5 to 25 mol%, 10 to 15 mol%, 10 to 20 mol%, 10 to 25 mol%, 15 to 20 mol%, 15 to 25 mol%, and 20 to 25 mol%.

In some embodiments, the LNP is composed of about 30 to 60 mol% of at least one cationic lipid; about 0 to 30 mol% of at least one noncationic lipid (eg, phospholipid); about 18.5 to 48.5 mol% of at least one Consisting of a sterol (eg, cholesterol); and about 0 to 10 mole % of at least one additional LNP functional component (eg, PEGylated lipid).

In some embodiments, the LNP is composed of about 35 to 55 mol% of at least one cationic lipid; about 5 to 25 mol% of at least one noncationic lipid (eg, phospholipid); about 30 to 40 mol% of at least Consisting of a sterol (eg, cholesterol); and about 0 to 10 mole % of at least one additional LNP functional component (eg, PEGylated lipid).

In some embodiments, the LNP is composed of about 35 to 45 mol% of at least one cationic lipid; about 25 to 35 mol% of at least one noncationic lipid (eg, phospholipid); about 20 to 30 mol% of at least one Consisting of a sterol (eg, cholesterol); and about 0 to 10 mole % of at least one additional LNP functional component (eg, PEGylated lipid).

In some embodiments, the LNP is composed of about 45 to 65 mol% of at least one cationic lipid; about 5 to 10 mol% of at least one noncationic lipid (eg, phospholipid); about 25 to 40 mol% of at least A sterol (such as cholesterol); and about 0.5 to 10 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 40 to 60 mol% of at least one cationic lipid; about 5 to 15 mol% of at least one noncationic lipid (eg, phospholipid); about 35 to 45 mol% of at least A sterol (such as cholesterol); and about 0.5 to 3 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 30 to 60 mol% of at least one cationic lipid; about 0 to 30 mol% of at least one noncationic lipid (eg, phospholipid); about 15 to 50 mol% of at least A sterol (such as cholesterol); and about 0.01 to 10 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 10 to 75 mol% of at least one cationic lipid; about 0.5 to 50 mol% of at least one noncationic lipid (eg, phospholipid); about 5 to 60 mol% of at least one A sterol (such as cholesterol); and about 0.1 to 20 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 50 to 65 mol% of at least one cationic lipid; about 3 to 15 mol% of at least one noncationic lipid (eg, phospholipid); about 30 to 40 mol% of at least A sterol (such as cholesterol); and about 0.5 to 2 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 50 to 85 mol% of at least one cationic lipid; about 3 to 15 mol% of at least one noncationic lipid (eg, phospholipid); about 30 to 40 mol% of at least A sterol (such as cholesterol); and about 0.5 to 2 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 25 to 75 mol% of at least one cationic lipid; about 0.1 to 15 mol% of at least one noncationic lipid (eg, phospholipid); about 5 to 50 mol% of at least A sterol (such as cholesterol); and about 0.5 to 20 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 50 to 65 mol% of at least one cationic lipid; about 5 to 10 mol% of at least one noncationic lipid (eg, phospholipid); about 25 to 35 mol% of at least A sterol (such as cholesterol); and about 5 to 10 mole % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the LNP is composed of about 20 to 60 mol% of at least one cationic lipid; about 5 to 25 mol% of at least one noncationic lipid (eg, phospholipid); about 25 to 55 mol% of at least one A sterol (such as cholesterol); and about 0.5 to 15 mol % of at least one additional LNP functional component (such as a pegylated lipid).

In some embodiments, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, phospholipids, sterols, and pegylated lipids as described herein. The LNPs in the formulation may comprise about 20 to 70 molar % cationic lipids, such as about 30 to 60%, or about 30 to 50%, or 50%. The LNP in the formulation may comprise about 5 to 20% (molar) phospholipids, such as 5 to 10%, 5 to 15%, 10 to 15%, or about 10% phospholipids. The LNP in the formulation may comprise about 20 to 45% (mol) sterols, such as 20 to 40%, 30 to 40%, or about 38% sterols. The LNP may include about 0.5 to 5.0% pegylated lipid, such as about 0.5 to 2.0%, 1.0 to 3.0%, 1.5 to 2.0%, or about 2.0% pegylated lipid.

As a non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including 3D-P-DMA, phospholipids, sterols, and pegylated lipids.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, DSPC, sterols, and pegylated lipids.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, phospholipids, sterols, and PEG-DMA.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, phospholipids, cholesterol, and pegylated lipids.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including 3D-P-DMA, DSPC, sterols, and pegylated lipids.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including 3D-P-DMA, phospholipids, cholesterol, and pegylated lipids.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including 3D-P-DMA, phospholipids, sterols, and PEG-DMA.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, phospholipids, cholesterol and PEG-DMA.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, DSPC, sterols, and PEG-DMA.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including cationic lipids, DSPC, cholesterol, and pegylated lipids.

As another non-limiting example, the nucleic acid vaccines described herein are formulated in lipid nanoparticles including 3D-P-DMA, DSPC, cholesterol, and PEG-DMA.

In some embodiments, the nucleic acid vaccines described herein are formulated in lipid nanoparticles, and the lipid nanoparticles in the formulation comprise about 50% (on a molar basis) cationic lipids; about 10% ( (moles) of phospholipids; approximately 38% (moles) of sterols; and 1.6% of pegylated lipids.

As non-limiting examples, cationic lipids, sterols, phospholipids and pegylated lipids are 3D-P-DMA, DSPC, cholesterol and PEG-DMA respectively.

In some embodiments, the nucleic acid vaccines described herein are formulated in lipid nanoparticles, and the lipid nanoparticles in the formulation comprise about 53% (by weight) 3D-P-DMA; about 14 % (by weight) DSPC; about 26% (by weight) cholesterol; and about 7% (by weight) PEG-DMA.

In some embodiments, the nucleic acid vaccine compositions described herein can comprise at least one nucleic acid vaccine comprising the polynucleotide of SEQ ID NO: 27 formulated in LNP comprising about 50% (on a molar basis) Cationic lipids; approximately 10% (on a molar basis) phospholipids; approximately 38% (on a molar basis) sterols; and approximately 2.0% PEGylated lipids. As a non-limiting example, the nucleic acid vaccine compositions described herein may comprise at least one nucleic acid vaccine comprising the polynucleotide of SEQ ID NO: 27 formulated in LNP containing about 50% (on a molar basis) 3D-P-DMA; approximately 10% (on a molar basis) DSPC; approximately 38% (on a molar basis) cholesterol; and approximately 2.0% PEG-DMA.

In some embodiments, the nucleic acid vaccine compositions described herein can comprise at least one nucleic acid vaccine comprising the polynucleotide of SEQ ID NO: 43 formulated in LNP comprising about 50% (on a molar basis) Cationic lipids; approximately 10% (on a molar basis) phospholipids; approximately 38% (on a molar basis) sterols; and approximately 2.0% PEGylated lipids. As a non-limiting example, the nucleic acid vaccine compositions described herein may comprise at least one nucleic acid vaccine comprising the polynucleotide of SEQ ID NO: 43 formulated in LNP containing about 50% (on a molar basis) 3D-P-DMA; approximately 10% (on a molar basis) DSPC; approximately 38% (on a molar basis) cholesterol; and approximately 2.0% PEG-DMA.

In some embodiments, the nucleic acid vaccine compositions described herein can comprise at least one nucleic acid vaccine comprising the polynucleotide of SEQ ID NO: 39 formulated in LNP comprising about 50% (on a molar basis) Cationic lipids; approximately 10% (on a molar basis) phospholipids; approximately 38% (on a molar basis) sterols; and approximately 2.0% PEGylated lipids. As a non-limiting example, the nucleic acid vaccine compositions described herein may comprise at least one nucleic acid vaccine comprising the polynucleotide of SEQ ID NO: 39 or SEQ ID NO: 41 formulated in an LNP comprising about 50% ( on a molar basis) of 3D-P-DMA; approximately 10% (on a molar basis) of DSPC; approximately 38% (on a molar basis) of cholesterol; and approximately 2.0% of PEG-DMA.

In some implementations, LNPs can be characterized by their shape. In some embodiments, the LNPs are substantially spherical. In some embodiments, the LNPs are substantially rod-shaped (ie, cylindrical). In some implementations, the LNP is substantially disk-shaped.

In some implementations, LNPs can be characterized by their size. In some embodiments, the size of the LNP can be defined by the diameter of its largest circular cross-section, referred to herein as its diameter. In some implementations, the LNPs can have a diameter of between 30 nm and about 150 nm. In some embodiments, the LNPs can have a diameter ranging from about 40 to 150 nm, 50 to 150 nm, 60 to 150 nm, about 70 to 150 nm, or 80 to 150 nm, 90 to 150 nm, 100 to 150 nm, Diameter between 110 and 150 nm, 120 and 150 nm, 130 and 150 nm, or 140 and 150 nm.

In some embodiments, a population of LNPs, such as those resulting from the same formulation, can be characterized by measuring uniformity of particle size, shape, or mass within the population. Uniformity may be expressed in some embodiments as the polydispersity index (PI) of a population. In some implementations, the uniformity may be expressed in some implementations as the disparity (Đ) of the population. It is understood that the terms "polydispersity index" and "aberration" are synonymous and used interchangeably herein. In some embodiments, the LNP population resulting from a given formulation has a PI between about 0.1 and 1. In some embodiments, the LNP population resulting from a given formulation has a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, the LNP population resulting from a given formulation has a molecular weight of between about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6 , 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1.

In some implementations, LNPs can fully or partially encapsulate articles, such as the nucleic acid constructs of the present disclosure. In some embodiments, substantially 0% of the items present in the final formulation are exposed to the environment outside the LNP (i.e., the items are completely encapsulated). In some embodiments, the article is associated with the LNP but is at least partially exposed to the environment external to the LNP. In some implementations, the LNP may be characterized by % of the article that is not exposed to the environment outside the LNP, such as encapsulation efficiency. For clarity, an encapsulation efficiency of about 100% refers to an LNP formulation in which substantially all of the items are completely encapsulated by the LNP, while an encapsulation efficiency of about 0% refers to an LNP formulation in which substantially no items are encapsulated in the LNP. LNPs in, such as LNPs in which the article is bonded to the outer surface of the LNP. In some embodiments, the LNP can have less than about 100%, less than about 95%, less than about 85%. Less than about 80%, less than about 75%, less than about 70%, less than about 65% %, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, Less than about 20%, less than about 15%, less than about 10%, or less than 5% encapsulation efficiency. In some embodiments, the LNP can have a polymorphic content of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 10 to 70%, 40 to 50%, 30 to 50%, 20 to 50%, 10 to 50%, 30 to 40%, 20 to 40%, 10 to 40%, 20 to Encapsulation efficiencies between 30%, 10 to 30%, and 10 to 20%.

In some embodiments, at least one targeting agent can be present on the outer surface of the nanoparticle. In some embodiments, at least one targeting agent can be conjugated to the lipid component of the nanoparticle. In some embodiments, at least one targeting agent can be conjugated to the polymer component of the nanoparticle. In some embodiments, at least one targeting agent can be anchored to the nanoparticles via hydrophobic and hydrophilic interactions between the at least one targeting agent, the nanoparticle film, and the aqueous environment inside or outside the nanoparticles. particle. In some embodiments, at least one targeting agent is conjugated to the peptide/protein component of the nanoparticle film. In some embodiments, at least one targeting agent is conjugated to a suitable linker moiety that is conjugated to a component of the nanoparticle film. In some embodiments, any combination of forces and bonds can cause the targeting agent to associate with the nanoparticles.

LNPs described herein can be formed using techniques known in the art. As a non-limiting example, an organic solution containing lipids and an acidic aqueous solution containing nucleic acid compositions are mixed in a microfluidic channel, resulting in the formation of a targeting system (delivery vehicle and nucleic acid vaccine).

In some embodiments, the lipid composition is described in terms of the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the molar % of ionizable lipid may be from about 10 molar % to about 80 molar %. As a non-limiting example, the molar % of ionizable lipid may be from about 20 molar % to about 70 molar %. As a non-limiting example, the molar % of ionizable lipid may be from about 30 molar % to about 60 molar %. As a non-limiting example, the mole % of ionizable lipid may be from about 35 mole % to about 55 mole %. As a non-limiting example, the molar % of ionizable lipid may be from about 40 molar % to about 50 molar %. As non-limiting examples, the ionizable lipid molar % of the transfer vehicle batch is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, ±30%, ±25%, ±20%, ±10%, ±5%, Or ±2.5%. In some embodiments, the transfer vehicle variability from batch to batch is less than 15%, less than 10%, or less than 5%.

In some embodiments, the molar % of the helper lipid can be from about 1 molar % to about 50 molar %. In some embodiments, the molar % of the helper lipid can be from about 2 molar % to about 45 molar %. In some embodiments, the molar % of the helper lipid may be from about 3 molar % to about 40 molar %. In some embodiments, the molar % of the helper lipid can be from about 4 molar % to about 35 molar %. In some embodiments, the molar % of the helper lipid may be from about 5 molar % to about 30 molar %. In some embodiments, the molar % of the helper lipid may be from about 10 molar % to about 20 molar %. In some embodiments, the auxiliary lipid mol% of the transfer vehicle batch is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±30%, ±25%, ±20%, ±10%, ±5%, or ±2.5%.

In some embodiments, the molar % of structural lipids may be from about 10 molar % to about 80 molar %. In some embodiments, the molar % of structural lipids may be from about 20 molar % to about 70 molar %. In some embodiments, the molar % of structural lipids may be from about 30 molar % to about 60 molar %. In some embodiments, the molar % of structural lipids may be from about 35 molar % to about 55 molar %. In some embodiments, the molar % of the structural lipid may be from about 40 molar % to about 50 molar %. In some embodiments, the structural lipid mole % of the transfer vehicle batch is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ± 2.5%.

In some embodiments, the molar % of PEG-modified lipids can be from about 0.1 molar % to about 10 molar %. In some embodiments, the molar % of PEG-modified lipids can be from about 0.2 molar % to about 5 molar %. In some embodiments, the molar % of the PEG-modified lipid can be from about 0.5 molar % to about 3 molar %. In some embodiments, the molar % of PEG-modified lipids can be from about 1 molar % to about 2 molar %. In some embodiments, the molar % of PEG-modified lipids can be about 1.5 molar %. In some embodiments, the PEG-modified lipid mole % of the transfer vehicle batch is ±30%, ±25%, ±20%, ±15%, ±10%, ±5% of the target mole % , or ±2.5%.

In some embodiments, lipid nanoparticle formulations can be prepared as described in International Publication No. WO2011127255 or WO2008103276, each of which is incorporated herein by reference in its entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. WO2019131770, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, lipid nanoparticle formulations can be prepared as described in International Publication No. WO2020237227, the contents of which are incorporated herein by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. WO2020237227, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the lipids may be cleavable lipids, such as those described in PCT Patent Application Publication No. WO2012170889, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the nanoparticles described herein can comprise at least one cationic polymer described herein and/or known in the art.

In some embodiments, cationic lipids can be synthesized by methods known in the art and/or as described in PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865; each of which is incorporated herein by reference in its entirety. in.

In some embodiments, the pharmaceutical composition of the nucleic acid vaccine composition may include at least one of the pegylated lipids described in PCT Patent Application Publication No. WO2012099755, the content of which is incorporated by reference in its entirety. into this article.

In some embodiments, the ratio of PEG in lipid nanoparticle (LNP) formulations can be increased or decreased and/or the carbon chain length of the PEG lipid can be modified from C14 to C18 to alter the pharmacokinetics of the LNP formulation and /or biodistribution. As a non-limiting example, LNP formulations may contain a lipid molar ratio of 1-5% PEG-c-DOMG compared to cationic lipids, DSPC and cholesterol. In some embodiments, the LNP formulation of the nucleic acid vaccine composition may contain 3% lipid molar ratio of PEG-c-DOMG. In some embodiments, the LNP formulation of the nucleic acid vaccine composition may contain 1.5% lipid molar ratio of PEG-c-DOMG.

In some embodiments, PEG-c-DOMG can be used with PEG lipids, such as, but not limited to, PEG-DSG (1,2-distearyl-sn-glycerol, methoxypolyethylene glycol) or PEG- DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol) substitution. The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA.

In some embodiments, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristyl-sn-glyceryl-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000), a cationic lipid known in the art. In some embodiments, LNP formulations can contain PEG-DMG 2000 and at least one other component. In some embodiments, LNP formulations can contain PEG-DMG 2000, DSPC, and cholesterol. As non-limiting examples, LNP formulations may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (see, e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS , 2012, 109(36): 14604-14609; incorporated by reference in its entirety).

As another non-limiting example, the nucleic acid vaccine compositions described herein can be formulated as nanoparticles delivered via parenteral routes, as described in U.S. Patent Application Publication No. US20120207845; the contents of which are incorporated herein by reference in their entirety. Incorporated herein by reference.

In some embodiments, the nucleic acid vaccine composition of the present disclosure can be formulated with a plurality of cationic lipids, such as the first and second cationic lipids described in US Patent Application Publication No. US20130017223 to Hope et al. Its contents are incorporated herein by reference in their entirety. The first cationic lipid may be selected based on a first property and the second cationic lipid may be selected based on a second property, where the properties may be determined as outlined in US20130017223. In some implementations, the first and second characteristics are complementary.

Nucleic acid vaccine compositions described herein may be formulated with lipid particles comprising one or more cationic lipids and one or more second lipids and one or more nucleic acids, wherein the lipid particles comprise as disclosed in U.S. Patent Publication No. US20120276209 to Cullis et al. The solid core described in , the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be complexed with cationic amphiphilic molecules in an oil-in-water (o/w) emulsion, such as described in European Publication No. EP2298358 to Satishchandran et al. Its contents are incorporated herein by reference in their entirety. The cationic amphiphilic molecule can be a cationic lipid, modified or unmodified spermine, bupivacaine or benzalkonium chloride, and the oil can be a vegetable or animal oil. As a non-limiting example, at least 10% of the nucleic acid-cationic amphiphilic molecule complex is in the oil phase of an oil-in-water emulsion (see, for example, the complexes described in EP 2298358 to Satishchandran et al.), the contents of which are The full text is incorporated by reference into this article.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a composition including a mixture of cationic compounds and neutral lipids. As a non-limiting example, the cationic compound may be formula (I) disclosed in PCT Patent Application Publication No. WO 1999010390 to Ansell et al., the contents of which are incorporated herein by reference in their entirety, and neutral The lipid may be selected from the group consisting of diylphospholipid choline, diylphospholipid ethanolamine, ceramide and sphingomyelin. In another non-limiting example, the lipid formulation may comprise a cationic lipid of Formula A disclosed in U.S. Patent Publication No. US 20120101148 to Akinc et al., a neutral lipid, a sterol, and PEG or be modified with PEG Lipids, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, LNP formulations can be formulated by methods described in International Publication No. WO2011127255 or WO2008103276. As a non-limiting example, the nucleic acid vaccine composition of the present disclosure can be encapsulated in any of the lipid nanoparticle (LNP) formulations described in WO2011127255 and/or WO2008103276; each of these contents is referred to as The full text is incorporated by reference into this article.

In some embodiments, LNP formulations described herein can include polycationic compositions. As a non-limiting example, the polycationic composition may be selected from Formulas 1-60 of U.S. Patent Publication No. US20050222064; the contents of which are incorporated herein by reference in their entirety. LNP formulations containing polycationic compositions can be used to deliver nucleic acid vaccine compositions described herein in vivo and/or in vitro.

In some embodiments, the LNP formulations described herein may additionally include permeability enhancer molecules. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; the contents of which are incorporated herein by reference in their entirety.

The nanoparticle formulation can be a carbohydrate nanoparticle that includes a carbohydrate carrier and a nucleic acid vaccine composition (eg, a nucleic acid vaccine for COVID-19). As non-limiting examples, carbohydrate carriers may include, but are not limited to, anhydride-modified plant glycogen or glycogen-based materials, phytoglycogen octenylsuccinate, phytoglycogen beta-dextrin, and anhydride-modified phytoglycogen. Phytoglycogen beta-dextrin. (See, eg, PCT Patent Application Publication No. WO2012109121; the contents of which are incorporated herein by reference in their entirety).

Lipid nanoparticle formulations can be modified by replacing the cationic lipids with biodegradable cationic lipids called rapidly eliminating lipid nanoparticles (reLNPs). Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA have been shown to accumulate in plasma and tissues over time and can be a source of potential toxicity. Rapid metabolism of rapidly eliminated lipids may improve the tolerability and therapeutic index of lipid nanoparticles by doses on the order of 1 mg/kg to 10 mg/kg in rats. Inclusion of enzymatically degraded ester linkages can improve the degradation and metabolism profiles of the cationic components while still maintaining the activity of the reLNP formulation. The ester bond may be located internally within the lipid chain or may be terminally located at the end of the lipid chain. Internal ester bonds can replace any carbon in the aliphatic chain.

In some embodiments, the nucleic acid vaccine composition is formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLN) can be spherical with an average diameter between 10 and 1000 nm. SLN has a solid lipid core matrix that can dissolve lipophilic molecules and can be stabilized with surfactants and/or emulsifiers. The lipid nanoparticles may be self-assembling lipid-polymer nanoparticles (see Zhang et al., ACS Nano , 2008, 2(8):1696-1702; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, formulations containing nucleic acid vaccine compositions described herein can also be constructed or modified to passively or actively target different cell types in vivo, including but not limited to immune cells, endothelial cells , antigen-presenting cells and leukocytes (Akinc et al. Mol Ther. 2010, 18:1357-1364; Song et al. Nat Biotechnol. 2005, 23:709-717; Judge et al. J Clin Invest. 2009, 119:661- 673; Kaufmann et al., Microvasc Res , 2010, 80:286-293; 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; Basha et al., Mol. Ther. 2011, 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008, 5:25-44; Peer et al., Science. 2008, 319:627-630; Peer and Lieberman, Gene Ther . 2011, 18:1127-1133; each such content is incorporated herein by reference in its entirety). An example of a formulation that passively targets hepatocytes includes lipid nanoparticle formulations based on DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA, which have been shown to bind to lipoprotein E and promote These formulations are bound and absorbed into liver cells in vivo (Akinc et al. Mol Ther. 2010, 18:1357-1364; the contents of which are incorporated herein by reference in their entirety). Formulations can also be selectively targeted by expressing different ligands on their surface, as exemplified by, but not limited to, folic acid, transferrin, N-acetylgalactosamine (GalNAc) and antibody targeting methods ( Kolhatkar et al., Curr Drug Discov Technol. 2011, 8:197-206; Musacchio and Torchilin, Front Biosci. 2011, 16:1388-1412; Yu et al., Mol Membr Biol. 2010, 27:286-298; Patil et al. Human, Crit Rev Ther Drug Carrier Syst. 2008, 25:1-61; Benoit et al., Biomacromolecules. 2011, 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008, 5:309-319; Akinc et al. Human, Mol Ther. 2010, 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012, 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012, 757:497-507; Peer J Control Release. 2010, 20:63-68; Peer et al., Proc Natl Acad Sci US A. 2007, 104:4095-4100; Kim et al., Methods Mol Biol. 2011, 721:339-353; Subramanya et al., Mol Ther. 2010, 18:2028-2037; Song et al., Nat Biotechnol. 2005, 23:709-717; Peer et al., Science. 2008, 319:627-630; Peer and Lieberman, Gene Ther. 2011, 18: 1127-1133; each such content is incorporated herein by reference in its entirety).

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated for controlled release and/or targeted delivery. As used herein, "controlled release" refers to the release characteristics of a pharmaceutical composition or compound that conform to a specific release pattern to achieve a therapeutic outcome. In some embodiments, nucleic acid vaccine compositions can be encapsulated in delivery agents described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround or envelop. As encapsulation relates to the formulation of the compositions of the present disclosure, the encapsulation may be substantial, complete, or partial. The term "substantially encapsulates" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9, or greater than 99.999% of the pharmaceutical composition of the present disclosure can be encapsulated , surrounded or coated in the delivery agent. "Partially encapsulated" means that less than 10, 10, 20, 30, 40, 50, or less of the pharmaceutical composition or compound of the present disclosure may be enclosed, surrounded, or coated within the delivery agent. Advantageously, encapsulation can be determined by measuring the escape or activity of the pharmaceutical compositions of the present disclosure using fluorescence and/or electron microscopy. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or greater than 99.99% of the disclosed medicines The composition is encapsulated in a delivery agent.

The nucleic acid vaccine composition can be encapsulated into lipid nanoparticles or rapidly eliminated lipid nanoparticles, and the lipid nanoparticles or rapidly eliminated lipid nanoparticles can then be encapsulated into the technology described herein and/or the technology Among known polymers, hydrogels and/or surgical sealants. As non-limiting examples, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics , San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc., Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc., Deerfield, IL).

In some embodiments, lipid nanoparticles can be encapsulated in any polymer known in the art that can form a gel when injected into an individual. As another non-limiting example, lipid nanoparticles can be encapsulated into a biodegradable polymer matrix.

In some embodiments, formulations comprising nucleic acid vaccine compositions for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as aqueous ethylcellulose dispersions (AQUACOAT® and SURELEASE®).

In some embodiments, controlled release and/or targeted delivery formulations can include at least one degradable polyester, which can contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serinate), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), and combinations thereof. In some embodiments, degradable polyesters may include PEG conjugations to form PEGylated polymers.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a targeting lipid having a targeting moiety, such as that disclosed in U.S. Patent Application Publication No. US20130202652 to Manoharan et al. The content is incorporated herein by reference in its entirety. As a non-limiting example, the targeting moiety of Formula I of US 20130202652 to Manoharan et al. can be selected to favor lipid localization to a desired organ, tissue, cell, cell type or subtype, or organelle. Non-limiting targeting moieties contemplated by the present disclosure include transferrin, p-methoxybenzamide, RGD peptide, prostate-specific membrane antigen (PSMA), fucose, antibodies or aptamers.

In some implementations, the nucleic acid vaccine compositions of the present disclosure can be encapsulated in therapeutic nanoparticles. Therapeutic nanoparticles may be formulated by methods described herein and known in the art, such as, but not limited to, PCT Patent Application Publication Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, and WO2012054923 , U.S. Publication Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286 and US20120288541, and US Patent No. 8,206,747 , No. 8,293,276, No. 8,318,208 and No. 8,318,211 ; Each such content is incorporated herein by reference in its entirety. Therapeutic polymer nanoparticles can be identified by methods described in US Publication No. US20120140790, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, therapeutic nanoparticles can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that is released at a consistent rate over a specified period of time. This period of time may include, but is not limited to, hours, days, weeks, months and years. As non-limiting examples, sustained release nanoparticles may include polymers and therapeutic agents, such as but not limited to the nucleic acid vaccine composition of the present disclosure (see PCT Patent Application Publication No. WO2010075072 and US Publication Nos. US20100216804, US20110217377 and US20120201859, each of which is incorporated herein by reference in its entirety).

In some implementations, therapeutic nanoparticles can be formulated to be target-specific. As a non-limiting example, therapeutic nanoparticles may include corticosteroids (see PCT Patent Application Publication No. WO2011084518; the contents of which are incorporated herein by reference in their entirety). In some implementations, therapeutic nanoparticles can be formulated to be cancer specific. As non-limiting examples, therapeutic nanoparticles may be formulated as nanoparticles described in PCT Patent Application Publication Nos. WO2008121949, WO2010005726, WO2010005725, and WO2011084521, and U.S. Publications No. US20100069426, US20120004293 and US20100104655, each of which is incorporated herein by reference in its entirety.

In some embodiments, the nanoparticles of the present disclosure may include a polymeric matrix. As non-limiting examples, nanoparticles may comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydrides, polyhydroxy acids, polypropyl fumarate, polycaprolactone ester, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphorus zene, polyacrylate, poly Methacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethylenimine), poly(serine), poly(L-lactide-co- L-lysine), poly(4-hydroxy-L-proline), or combinations thereof.

In some embodiments, therapeutic nanoparticles comprise diblock copolymers. In some embodiments, the diblock copolymer may include a combination of PEG and polymers such as, but not limited to, polyethylene, polycarbonate, polyanhydrides, polyhydroxy acids, polypropyl fumarate, Polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphorene, polyacrylic acid ester, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethylenimine), poly(serine ester), poly(L-lactide) -co-L-lysine), poly(4-hydroxy-L-proline), or combinations thereof.

As non-limiting examples, therapeutic nanoparticles include PLGA-PEG block copolymers (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, each of which is incorporated by reference in its entirety). In another non-limiting example, the therapeutic nanoparticles are stealth nanoparticles comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968 and PCT Patent Application Publication No. WO2012166923 , each such content is incorporated herein by reference in its entirety).

In some embodiments, therapeutic nanoparticles may include multi-block copolymers, such as, but not limited to, those described in U.S. Pat. Nos. 8,263,665 and 8,287,910; substituting each of these with It is incorporated by reference in its entirety.

In some embodiments, the block copolymers described herein can be included in polyionic complexes including non-polymeric micelles and block copolymers. (See, eg, U.S. Publication No. US20120076836; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, nanoparticles used to deliver the nucleic acid vaccines described herein include block copolymers. Non-limiting examples of block copolymers include those of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, and Formula VII of PCT Patent Application Publication No. WO2015017519, the contents of which are incorporated herein by reference in their entirety. Incorporated herein by reference.

In some embodiments, therapeutic nanoparticles can include at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymer, ethoxyethyl methacrylate, cyanoethyl methacrylate, amine methacrylate Alkyl ester copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates, and combinations thereof.

In some embodiments, therapeutic nanoparticles can comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethylenimine, poly(amidoamine) dendrimers, poly(beta) -aminoester) (see, e.g., U.S. Patent No. 8,287,849; the contents of which are incorporated herein by reference in its entirety) and combinations thereof.

In some embodiments, therapeutic nanoparticles can include at least one degradable polyester, which can contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serinate), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), and combinations thereof. Degradable polyesters may include PEG conjugations to form pegylated polymers.

In some embodiments, therapeutic nanoparticles may include the binding of at least one targeting ligand. The targeting ligand can be any ligand known in the art, such as, but not limited to, monoclonal antibodies. (Kirpotin et al., Cancer Res . 2006, 66:6732-6740; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, therapeutic nanoparticles can be formulated in an aqueous solution that can be used to target cancer (see PCT Patent Application Publication No. WO2011084513 and U.S. Publication No. US20110294717, each of which is cited in its entirety). incorporated herein by reference).

In some embodiments, nucleic acid vaccine compositions can be encapsulated in, linked to, and/or combined with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in: PCT Patent Application Publication Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, Nos. WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and U.S. Publication No. US201102 No. 62491, No. US20100104645, No. US20100087337 and No. US20120244222 No., each such content is incorporated herein by reference in its entirety. Synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As non-limiting examples, synthetic nanocarriers can be synthesized by PCT Patent Application Publication Nos. WO2010005740, WO2010030763 and WO201213501, and U.S. Publication Nos. US20110262491, US20100104645, US20100087337 and US2012024422. Each such content is incorporated herein by reference in its entirety. Synthetic nanocarrier formulations can be lyophilized by methods described in PCT Patent Application Publication No. WO2011072218 and US Patent No. 8,211,473; each of which is incorporated herein by reference in its entirety.

In some embodiments, synthetic nanocarriers may contain reactive groups to release the nucleic acid vaccine compositions described herein (see PCT Patent Application Publication No. WO20120952552 and U.S. Publication No. US20120171229, each of which content is incorporated into this article by reference in its entirety).

In some embodiments, synthetic nanocarriers can be formulated for targeted release. In some embodiments, synthetic nanocarriers can be formulated to release nucleic acid vaccine compositions at a specified pH and/or after a desired time interval. As non-limiting examples, synthetic nanoparticles can be formulated to release nucleic acid vaccine compositions after 24 hours and/or at a pH of 4.5 (see PCT Patent Application Publication Nos. WO2010138193 and WO2010138194, and U.S. Publications No. US20110020388 and US20110027217, each of which is incorporated herein by reference in its entirety).

In some embodiments, synthetic nanocarriers can be formulated for controlled release and/or sustained release of the nucleic acid vaccine compositions described herein. By way of non-limiting example, synthetic nanocarriers for sustained release may be prepared by methods known in the art, described herein, and/or as described in PCT Patent Application Publication No. WO2010138192 and U.S. Publication No. US20100303850. Each such content is incorporated herein by reference in its entirety.

In some implementations, nanoparticles can be optimized for oral administration. Nanoparticles may comprise at least one cationic biopolymer such as, but not limited to, polyglucosamine or derivatives thereof. As a non-limiting example, nanoparticles can be formulated by the methods described in US Publication No. US20120282343; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in modular compositions such as those described in U.S. Patent No. 8,575,123 to Manoharan et al., the contents of which are incorporated by reference in their entirety. into this article. As a non-limiting example, the modular composition may include a nucleic acid (eg, the nucleic acid vaccine composition of the present disclosure), at least one endosomal cleavage component, and at least one targeting ligand. The modular composition may have a formula, such as any of the formulas described in US 8,575,123 to Manoharan et al.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be encapsulated in lipid formulations to form stable nucleic acid-lipid particles (SNALP), such as those disclosed in U.S. Patent No. 8,546,554 to de Fougerolles et al. The content is incorporated into this article by reference in its entirety. Lipids can be cationic or non-cationic. In a non-limiting example, the lipid:nucleic acid ratio (mass/mass ratio) (e.g., lipid:nucleic acid vaccine composition ratio) will be in the following range: about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1, or 5:1, 6 :1, 7:1, 8:1, 9:1, 10:1, or 11:1. In another example, SNALP includes 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (lipid A), 10% dioleyl Dylphospholipid choline (DSPC), 40% cholesterol, 10% polyethylene glycol (PEG)-C-DOMG (mol%), particle size 63.0±20 nm, and nucleic acid/lipid ratio 0.027.

The nucleic acid vaccine composition of the present disclosure can be formulated with nucleic acid-lipid particles containing endosomal membrane destabilizing agents, as disclosed in U.S. Patent No. 7,189,705 issued to Lam et al., the contents of which are incorporated herein by reference in their entirety. . As a non-limiting example, the endosomal membrane destabilizing agent may be Ca ²⁺ ions.

In some embodiments, the nucleic acid vaccine composition of the present disclosure can be formulated with formulated lipid particles (FLiPs) disclosed in U.S. Patent No. 8,148,344 to Akinc et al., the contents of which are incorporated by reference in their entirety. in this article. Akinc et al. teach that FLiP can comprise at least one of a single-stranded or double-stranded oligonucleotide, wherein the oligonucleotide has been bound to a lipophile and an emulsion that has been aggregated, blended, or combined with the bound oligonucleotide. or at least one of liposomes. As disclosed in US 8,148,344 to Akinc et al., these particles have surprisingly been shown to effectively deliver oligonucleotides to the heart, lungs and muscles.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be delivered to cells using compositions that include expression vectors in lipid formulations, as described in U.S. Patent No. 6,086,913 to Tam et al. The content is incorporated herein by reference in its entirety. The composition disclosed by Tam is serum stable and includes an expression vector that includes first and second inverted repeats from adeno-associated virus (AAV), the rep gene from AAV, and nucleic acid fragments. The expression vector in Tam is complexed with lipids.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with the lipid formulation disclosed in US Publication No. US 20120270921 to de Fougerolles et al., the contents of which are incorporated herein by reference in their entirety. . In one non-limiting example, a lipid formulation may include a cationic lipid of Formula A as described in US 20120270921. In another non-limiting example, the exemplary nucleic acid-lipid particle composition disclosed in Table A of US20120270921 can be used with the nucleic acid vaccine composition of the present disclosure.

In some embodiments, the nucleic acid vaccine composition of the present disclosure can be completely encapsulated in the lipid particles disclosed in US Publication No. US 20120276207 to Maurer et al., the contents of which are incorporated herein by reference in their entirety. middle. The particles may comprise a lipid composition comprising preformed lipid vesicles, a charged therapeutic agent, and a destabilizing agent to form a mixture of preformed vesicles and therapeutic agent in a destabilizing solvent, wherein the destabilizing solvent effectively Destabilizes the membrane of preformed lipid vesicles without damaging the vesicles.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with conjugated lipids. In a non-limiting example, the binding lipid may have a formula such as that described in US Publication No. 20120264810 to Lin et al., the contents of which are incorporated herein by reference in their entirety. Conjugated lipids can form lipid particles, which further include cationic lipids, neutral lipids, and lipids capable of reducing aggregation.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in neutral liposome formulations such as those disclosed in US Publication No. 20120244207 to Fitzgerald et al., the contents of which are incorporated by reference in their entirety. method is incorporated into this article. The phrase "neutral liposome formulation" refers to a liposome formulation that has a near-neutral or neutral surface charge at physiological pH. Physiological pH can be, for example, about 7.0 to about 7.5, or, for example, about 7.5, or, for example, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or, for example, 7.3, or, for example, 7.4. Examples of neutral liposomal formulations are ionizable lipid nanoparticles (iLNPs). Neutral liposome formulations may include ionizable cationic lipids, such as DLin-KC2-DMA.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated with charged lipids or amino lipids. As used herein, the term "charged lipid" is intended to include those lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amine head group. Quaternary amines carry a permanent positive charge. The headgroup optionally includes an ionizable group such as a primary, secondary or tertiary amine that can be protonated at physiological pH. The presence of a quaternary amine can change the pKa of an ionizable group relative to the pKa of a group in a structurally similar compound that does not have a quaternary amine (eg, a quaternary amine replaced by a tertiary amine). In some embodiments, the charged lipids are called "amino lipids." In a non-limiting example, the amino lipid can be any of the amino lipids described in US Publication No. 20110256175 to Hope et al., the contents of which are incorporated herein by reference in their entirety. For example, the amino lipid can have the structures disclosed in Tables 3 to 7 of Hope, such as structure (II), DLin-K-C2-DMA, DLin-K2-DMA, DLin-K6-DMA, etc. The resulting pharmaceutical preparation can be lyophilized according to Hope. In another non-limiting example, the amino lipid can be any of the amino lipids described in US 20110117125 to Hope et al., the contents of which are incorporated herein by reference in their entirety, such as structure (I), DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA and other lipids. In another non-limiting example, the amino lipid may have structure (I), (II), (III) or (IV) as described in PCT Patent Application Publication No. WO2009132131 to Manoharan et al. or 4-(R)-DLin-K-DMA (VI), 4-(S)-DLin-K-DMA (V), the contents of which are incorporated herein by reference in their entirety. In another non-limiting example, the charged lipid used in any formulation described herein can be any of the charged lipids described in EP 2509636 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. .

In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with association complexes. In a non-limiting example, the associative complex includes one or more compounds each having a structure defined by formula (I) as disclosed in U.S. Pat. No. 8,034,376 to Manoharan et al., having a structure defined by formula (I) XV), the contents of which are incorporated herein by reference in their entirety. Nucleic acid vaccine compositions may be formulated with any of the association complexes described in U.S. Patent No. 8,034,376, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated with reverse headgroup lipids. As a non-limiting example, nucleic acid vaccine compositions may be formulated with zwitterionic lipids containing a head group in which positive charges are located near the acyl chain region and negative charges are located distal to the head group, such as disclosed in PCT patent application issued to Leung et al. The lipid of structure (A) or structure (I) described in Case No. WO2011056682 is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in a lipid bilayer carrier. As a non-limiting example, the nucleic acid vaccine composition can be combined with a lipid-detergent mixture to provide a nucleic acid-lipid-detergent mixture that includes an amount of about 5 molar % to about 20 molar %. an aggregating agent, a lipid mixture of cationic lipids and melt-promoting lipids in an amount from about 0.5 molar % to about 50 molar %, and a detergent; and then aspirating the nucleic acid-lipid-detergent mixture with a buffered saline solution to remove the detergent and encapsulating the nucleic acid in a lipid bilayer carrier and providing a lipid bilayer-nucleic acid composition in which the ionic strength of the buffer saline solution is sufficient to encapsulate about 40% to about 80% of the nucleic acid, as awarded to Cullis et al. Described in PCT Patent Application Publication No. WO1999018933, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the nucleic acid vaccine composition of the present disclosure may include (a) nucleic acid; (b) 1.0 mol% to 45 mol% of cationic lipids; (c) 0.0 mol% to 90 mol% of Another lipid; (d) 1.0 mol% to 10 mol% of the bilayer stabilizing component; (e) 0.0 mol% to 60 mol% cholesterol; and (f) 0.0 mol% to 10 mol% Cationic polymeric lipids are described in EP 1328254 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.

In some implementations, nucleic acid vaccines can be delivered using smaller LNPs. Such particles may include diameters from less than 0.1 µm up to 100 nm, such as, but not limited to, less than 0.1 µm, less than 1.0 µm, less than 5 µm, less than 10 µm, less than 15 µm, less than 20 µm, less than 25 µm, less than 30 µm, Less than 35 µm, Less than 40 µm, Less than 50 µm, Less than 55 µm, Less than 60 µm, Less than 65 µm, Less than 70 µm, Less than 75 µm, Less than 80 µm, Less than 85 µm, Less than 90 µm, Less than 95 µm, Less than 100 µm, less than 125 µm, less than 150 µm, less than 175 µm, less than 200 µm, less than 225 µm, less than 250 µm, less than 275 µm, less than 300 µm, less than 325 µm, less than 350 µm, less than 375 µm, less than 400 µm, Less than 425 µm, Less than 450 µm, Less than 475 µm, Less than 500 µm, Less than 525 µm, Less than 550 µm, Less than 575 µm, Less than 600 µm, Less than 625 µm, Less than 650 µm, Less than 675 µm, Less than 700 µm, Less than 725 µm, less than 750 µm, less than 775 µm, less than 800 µm, less than 825 µm, less than 850 µm, less than 875 µm, less than 900 µm, less than 925 um, less than 950 µm, less than 975 µm.

In another embodiment, the nucleic acid vaccine can be delivered using smaller LNPs, and the diameter of the LNPs can be about 1 nm to about 100 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 10 nm to about 50 nm, about 20 nm to about 50 nm , about 30 nm to about 50 nm, about 40 nm to about 50 nm, about 20 nm to about 60 nm, about 30 nm to about 60 nm, about 40 nm to about 60 nm, about 20 nm to about 70 nm, about 30 nm to about 70 nm, about 40 nm to about 70 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 20 nm to about 80 nm, about 30 nm to about 80 nm, about 40 nm to about 80 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 20 nm to about 90 nm, about 30 nm to about 90 nm, about 40 nm to about 90 nm, about 50 nm to about 90 nm, about 60 nm to about 90 nm, and/or about 70 nm to about 90 nm.

In some embodiments, nucleic acid vaccines can be formulated in lipid nanoparticles having a diameter of about 10 nm to about 100 nm, such as, but not limited to, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm , about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm, about 30 nm to about 100 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 40 nm to about 90 nm, about 40 nm to about 100 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm, about 50 nm to about 80 nm, about 50 nm to about 90 nm , about 50 nm to about 100 nm, about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm to about 100 nm, about 80 nm to about 90 nm, about 80 nm to about 100 nm, and/or about 90 nm to about 100 nm.

In some embodiments, nucleic acid vaccines can be formulated in lipid nanoparticles having a diameter of 10 to 1000 nm. Nanoparticles can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 ,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245 ,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370 ,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,475,480,485,490,495 ,500,505,510,515,520,525,530,535,540,545,550,555,560,565,570,575,580,585,590,595,600,605,610,615,620 ,625,630,635,640,645,650,655,660,665,670,675,680,685,690,695,700,705,710,715,720,725,730,735,740,745 ,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825,830,835,840,845,850,855,860,865,870 ,875,880,885,890,895,900,905,910,915,920,925,930,935,940,945,950,955,960,965,970,975,980,985,990,995 , or 1000 nm.

In some implementations, lipid nanoparticles can have a diameter of about 10 to 500 nm.

In some embodiments, the diameter of the lipid nanoparticles can be greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm. , greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm. Polymers, biodegradable nanoparticles and core-shell nanoparticles

The nucleic acid vaccine composition of the present disclosure can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers that can be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA), formulations available from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI) , PHASERX ^™ polymer formulations such as, but not limited to, SMARTT POLYMER TECHNOLOGY™ (PHASERX®, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvants available from Vical (San Diego, CA), Polyglucosamine, cyclodextrin, dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers obtained from Calando Pharmaceuticals (Pasadena, CA), RONDEL ^™ (RNAi/oligonucleotide nanoparticles particle delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH-responsive co-block copolymers such as, but not limited to, PHASERX® (Seattle, WA).

Non-limiting examples of polyglucosamine-based formulations include a positively charged polyglucosamine core and an outer portion of a negatively charged matrix (U.S. Publication No. US20120258176; the contents of which are incorporated herein by reference in their entirety). ). Polyglucosamine includes, but is not limited to, N-trimethylpolyglucosamine, mono-N-carboxymethylpolyglucosamine (MCC), N-palmitoyl polyglucosamine (NPCS), EDTA-polyglucosamine Aminosugar, low molecular weight polyglucosamine, polyglucosamine derivatives or combinations thereof.

In some embodiments, the polymers used in the present disclosure have been treated to reduce and/or inhibit attachment of undesirable species, such as but not limited to bacteria, to the polymer surface. The polymer may be treated by methods known in the art and/or described and/or described in PCT Patent Application Publication No. WO2012150467; the contents of which are incorporated herein by reference in their entirety.

Non-limiting examples of PLGA-based formulations include, but are not limited to, PLGA-based injectable depots (e.g., ELIGARD®, prepared by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and The remainder is formed in aqueous solvent and leuprolide. Once injected, PLGA and leuprolide peptide precipitate into the subcutaneous space. Injectable depots based on PLGA can be long-acting.

Many of these polymeric approaches have demonstrated efficacy in delivering oligonucleotides into the cytoplasm in vivo (reviewed in Fougerolles Hum Gene Ther. 2008, 19:125-132; the contents of which are incorporated herein by reference in their entirety) middle). In the case of small interfering RNA (siRNA), two polymer approaches have resulted in robust in vivo delivery of nucleic acids, namely dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery methods used dynamic polyconjugates and has been shown to efficiently deliver siRNA in vivo in mice and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci US A . 2007, 104:12982-12887; the contents of which are incorporated herein by reference in their entirety). This particular approach is a multi-component polymer system whose main features include a membrane-active polymer covalently coupled to a nucleic acid (siRNA in this case) via disulfide bonds, with PEG (for charge masking) and N The -acetylgalactosamine (for hepatocyte targeting) group is attached via a pH-sensitive bond (see again Rozema et al., Proc Natl Acad Sci USA . 2007, 104:12982-12887). After binding to hepatocytes and entering endosomes, the polymer complex dissociates in a low pH environment, exposing the polymer to its positive charge, resulting in endosomal escape and cytoplasmic release of siRNA from the polymer. By replacing the N -acetylgalactosamine group with a mannose group, targeting from hepatocytes expressing asialoglycoprotein receptors to sinusoidal endothelial cells and Kupffer cells was shown to change. Another polymer approach involves the use of transferrin-targeted cyclodextrin-containing polycationic nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in Ewing's sarcoma tumor cells expressing the transferrin receptor (Hu-Lieskovan et al., Cancer Res. 2005, 65: 8984-8982; cited in full are incorporated herein), and siRNA formulated in these nanoparticles is well tolerated in non-human primates (Heidel et al., Proc Natl Acad Sci USA 2007, 104:5715-21; with The full text is incorporated into this article by reference). Both delivery strategies combine rational approaches using targeted delivery and endosomal escape mechanisms.

Polymeric formulations may allow sustained or delayed release of the nucleic acid vaccine composition (eg, following intramuscular, subcutaneous, intraparenchymal, intrathecal, intracerebroventricular administration). Altered release characteristics of nucleic acid vaccine compositions can result in, for example, translation of the encoded protein or polypeptide or peptide over an extended period of time. Biodegradable polymers have previously been used to protect nucleic acids from degradation and have been shown to induce sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci US A. 2007, 104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 2010, 7:1433-1446; Convertine et al., Biomacromolecules. Oct. 1, 2010; Chu et al., Acc Chem Res. Jan. 13, 2012; Manganiello et al., Biomaterials. 2012, 33: 2301-2309; Benoit et al., Biomacromolecules . 2011, 12:2708-2714; Singha et al., Nucleic Acid Ther. 2011, 2:133-147; de Fougerolles Hum Gene Ther. 2008, 19:125-132; Schaffert and Wagner, Gene Ther. 2008, 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011, 8:1455-1468; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature, 2010, 464 :1067-1070; each such content is incorporated herein by reference in its entirety).

In some implementations, the nucleic acid vaccines of the present disclosure can be formulated for controlled release. One form of controlled release formulation contains the therapeutic compound dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer, such as copolymer (lactic acid/glycolic acid) or a salt thereof, as described by Kent et al., U.S. Patent No. 4,675,189 described in their seminal work, the contents of which are incorporated into this article by reference in their entirety. The compounds or salts thereof may also be formulated as cholesterol or other lipid matrix pellets, or silicone elastomer matrix implants. As a non-limiting example, the nucleic acid vaccine of the present disclosure can be dispersed or encapsulated in the polymer disclosed in US Pat. No. 4,675,189 for controlled release. Another form of controlled release formulations comprise solutions of biodegradable polymers, such as copolymers (lactic acid/glycolic acid) or block copolymers of lactic acid and PEG, which are injected subcutaneously or intramuscularly to obtain a reservoir for controlled release. Library mix.

In some embodiments, the pharmaceutical composition can be a sustained release formulation. In other embodiments, sustained release formulations can be used for subcutaneous delivery. Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical Sealants such as fibrinogen polymer (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).

As a non-limiting example, the nucleic acid vaccine composition can be encapsulated in the PLGA microspheres by preparing PLGA microspheres with an adjustable release rate (e.g., days and weeks) while maintaining the nucleic acid vaccine composition during the encapsulation process. The nucleic acid vaccine composition is formulated in PLGA microspheres based on the integrity of the substance. EVAc is a non-biodegradable biocompatible polymer that is widely used in preclinical sustained-release implant applications. Poloxamer F-407 NF is a hydrophilic, nonionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene, which has low viscosity at temperatures less than 5°C and is A solid gel forms at a temperature of 15°C. PEG-based surgical sealants contain two synthetic PEG components mixed in a delivery device that can be prepared in one minute, seals in 3 minutes, and resorbs in 30 days. GELSITE® and natural polymers gel in situ at the site of administration. It has been shown to interact with protein and peptide therapeutic candidates via ionic interactions to provide a stabilizing effect.

Polymeric formulations can also be selectively targeted through the expression of different ligands, such as exemplified by, but not limited to, folic acid, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al. Human, Biomacromolecules. 2011, 12:2708-2714; Rozema et al., Proc Natl Acad Sci US A. 2007, 104:12982-12887; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature , 2010 464 :1067-1070; each such content is incorporated herein by reference in its entirety).

The nucleic acid vaccine compositions of the present disclosure may be formulated with or in polymeric compounds. The polymeric compound may include at least one polymer such as, but not limited to, polyethylene, polyethylene glycol (PEG), poly(l-lysine) (PLL), PEG grafted to PLL, cationic lipid polymers, biodegradable Degradable cationic lipid polymer, polyethyleneimine (PEI), cross-linked branched poly(alkyleneimine), polyamine derivatives, modified poloxamer, biodegradable polymer, elastic biodegradable polymer material, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multi-block copolymer Segment copolymer, linear biodegradable copolymer, poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymer, polycarbonate, Polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyethylene Alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine acid, poly(ethylenimine) ), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), acrylic polymer, amine-containing polymer, poly Glucose polymers, polydextrose polymer derivatives or combinations thereof.

As a non-limiting example, the nucleic acid vaccine composition of the present disclosure may be formulated with a polymeric compound of PEG grafted with PLL as described in US Pat. No. 6,177,274; this patent is incorporated herein by reference in its entirety. The formulations can be used to transfect cells in vitro or to deliver nucleic acid vaccine compositions in vivo. In another example, the nucleic acid vaccine composition can be suspended in a solution or medium with a cationic polymer, a dry pharmaceutical composition, or a solution that can be dried as described in US Publication Nos. US20090042829 and US20090042825; 1. Such content is incorporated herein by reference in its entirety.

As another non-limiting example, the nucleic acid vaccine composition of the present disclosure may be PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, which are incorporated herein by reference in their entirety) or PLGA-PEG PEG-PLGA block copolymer (see U.S. Patent No. 6,004,573, incorporated herein by reference in its entirety) formulation. As a non-limiting example, the nucleic acid vaccine composition of the present disclosure may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968, which is incorporated herein by reference in its entirety).

In some embodiments, nucleic acid vaccine compositions may be formulated with branched PEG molecules as described in or prepared by methods described in PCT Patent Application Publication No. WO20180126084; the contents of which are incorporated by reference in their entirety. in this article. As a non-limiting example, branched chain PEGs useful in the formulations described herein may have Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI of PCT Publication No. WO20180126084, the contents of which are The full text is incorporated by reference into this article.

Polyamine derivatives may be used to deliver nucleic acids or treat and/or prevent disease or be included in implantable or injectable devices (US Publication No. US20100260817; the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, the nucleic acid vaccine composition of the present disclosure can be formulated using the polyamine derivatives described in US Publication No. US20100260817; the content of which is incorporated herein by reference in its entirety. As another non-limiting example, the nucleic acid vaccine compositions of the present disclosure may be delivered using polyamide polymers such as, but not limited to, polyamide polymers made by combining carbohydrate diazide monomers with oligomers. Polymers of 1,3-dipolar addition polymers prepared by combining diyne units of amines (U.S. Patent No. 8,236,280; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the nucleic acid vaccine composition of the present disclosure may be made of at least one polymer and/or other polymers described in PCT Patent Application Publication Nos. WO2011115862, WO2012082574 and WO2012068187 and US Publication No. US20120283427. Derivative Formulation, each such content is incorporated herein by reference in its entirety. The nucleic acid vaccine composition of the present disclosure may be formulated with a polymer of formula Z as described in WO2011115862; the content of which is incorporated herein by reference in its entirety. Nucleic acid vaccine compositions may be formulated with polymers of formula Z, Z' or Z'' as described in PCT Patent Application Publication No. WO2012082574 or WO2012068187 and US Publication No. US2012028342; each of these contents is It is incorporated by reference in its entirety. The polymer formulated with the nucleic acid vaccine composition of the present disclosure can be synthesized by the method described in PCT Patent Application Publication No. WO2012082574 or WO2012068187, the contents of which are each incorporated herein by reference in their entirety. .

The nucleic acid vaccine compositions of the present disclosure may be formulated with at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymer, ethoxyethyl methacrylate, cyanoethyl methacrylate, amine methacrylate Alkyl ester copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates, and combinations thereof.

Formulations of the nucleic acid vaccine compositions of the present disclosure may include at least one amine-containing polymer, such as, but not limited to, polylysine, polyethylenimine, poly(amidoamine) dendrimers, or combinations thereof.

For example, the nucleic acid vaccine compositions of the present disclosure may be formulated in pharmaceutical compounds including poly(ethyleneimine), biodegradable cationic lipid polymers, biodegradable block copolymers, biodegradable polymers, or Biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, biodegradable polyester random copolymer, linear biodegradable copolymer, PAGA, biodegradable cross Dicationic multi-block copolymers or combinations thereof. Biodegradable cationic lipid polymers can be prepared by methods known in the art and/or described in U.S. Patent No. 6,696,038 and U.S. Publication Nos. US20030073619 and US20040142474; refer to each of these as It is incorporated by reference in its entirety. Poly(alkyleneimines) can be prepared using methods known in the art and/or as described in US Publication No. US20100004315, which is incorporated herein by reference in its entirety. Biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester block copolymers, biodegradable polyester polymers or biodegradable polyester random copolymers can Prepared using methods known in the art and/or as described in U.S. Patent Nos. 6,517,869 and 6,267,987, the contents of each of which are incorporated herein by reference in their entirety. Linear biodegradable copolymers can be prepared using methods known in the art and/or as described in U.S. Patent No. 6,652,886, the contents of which are each incorporated herein by reference in their entirety. PAGA polymers can be prepared using methods known in the art and/or as described in U.S. Patent No. 6,217,912; the disclosure of which is incorporated herein by reference in its entirety. PAGA polymers can be copolymerized with polymers such as, but not limited to, poly-L-lysine, polyarginine, polyornithine, histones, antibiotics, etc. to form copolymers or block copolymers. Protamine, protamine, polylactide and poly(lactide-co-glycolide). The biodegradable cross-linked cationic multi-block copolymer can be prepared by methods known in the art and/or as described in U.S. Patent No. 8,057,821 or U.S. Publication No. US2012009145; each of these contents It is incorporated herein by reference in its entirety. For example, multi-block copolymers can be synthesized using linear polyethyleneimine (LPEI) blocks with a different pattern compared to branched polyethyleneimine. Additionally, the composition or pharmaceutical composition may be prepared by methods known in the art, described herein, or as described in U.S. Publication No. US20100004315 or U.S. Patent Nos. 6,267,987 and 6,217,912; These contents are incorporated herein by reference in their entirety.

The nucleic acid vaccine composition of the present disclosure may be formulated with at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serinate), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), and combinations thereof. In some embodiments, degradable polyesters may include PEG conjugations to form PEGylated polymers.

The nucleic acid vaccine composition of the present disclosure can be formulated with at least one cross-linkable polyester. Cross-linkable polyesters include those known in the art and described in US Publication No. US20120269761; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the polymers described herein can be conjugated to lipid-terminated PEG. As a non-limiting example, PLGA can be conjugated to lipid-terminated PEG, forming PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use in the present disclosure include those described in PCT Patent Application Publication No. WO2008103276; the contents of which are incorporated herein by reference in their entirety. The polymers may be combined using ligand conjugates such as, but not limited to, those described in U.S. Patent No. 8,273,363; the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid vaccine compositions described herein can be combined with another compound. Non-limiting examples of conjugates are described in U.S. Patent Nos. 7,964,578 and 7,833,992; the contents of each of these patents are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid vaccine composition of the present disclosure can be combined with the conjugates of Formulas 1-122 as described in U.S. Patent Nos. 7,964,578 and 7,833,992; the contents of these patents are each cited in their entirety. method is incorporated into this article. The nucleic acid vaccine compositions described herein can be combined with metals, such as, but not limited to, gold. (See, eg, Giljohann et al . Journ. Amer. Chem. Soc. 2009, 131(6):2072-2073; the contents of which are incorporated herein by reference in their entirety). In some embodiments, the nucleic acid vaccine compositions described herein can be combined and/or encapsulated in gold nanoparticles (PCT Application Publication No. WO201216269 and US Publication No. US20120302940; refer to each of these incorporated herein by reference in its entirety).

As described in US Publication No. US20100004313, the gene delivery composition may include a nucleotide sequence and a poloxamer. As a non-limiting example, the nucleic acid vaccine composition of the present disclosure can be used in the gene delivery composition with poloxamer described in U.S. Publication No. US20100004313; the contents of which are each incorporated herein by reference in their entirety. .

In some embodiments, polymer formulations comprising the nucleic acid vaccines of the present disclosure may be prepared by polymerizing the polymer formulation, which may include a cationic carrier, with a cationic lipid that may be covalently linked to cholesterol and polyethylene glycol groups. Stable by physical contact. The polymer formulation may be contacted with the cationic lipid polymer using the methods described in US Publication No. US20090042829; the contents of which are incorporated herein by reference in their entirety.

Cationic carriers may include, but are not limited to, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-di Amino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine) ), cationized gelatin, dendrimers, polyglucosamine, 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleyl Oxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(Oleyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl) base) imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminemethamide)ethyl]-N,N-dimethyl-1-propylamine tris Fluoroacetate (DOSPA), 3B-[N-(N',N'-dimethylaminoethane)-aminoformyl]cholesterol hydrochloride (DC-cholesterol HCl), heptadecyl acyl Aminoglycinylspermidine (DOGS), N,N-distearyl-N,N-dimethylamine bromide (DDAB), N-(1,2-dimyristyloxypropane- 3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylamine chloride (DODAC) and combinations thereof.

In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in polymeric complexes of one or more polymers (U.S. Publication Nos. US20120237565 and US20120270927; each of these contents is incorporated by reference in its entirety. incorporated herein). In some embodiments, the polymeric complex includes two or more cationic polymers. The cationic polymer may include poly(ethyleneimine) (PEI), such as linear PEI.

The nucleic acid vaccine composition of the present disclosure can also be formulated into nanoparticles using a combination of polymers, lipids and/or other biodegradable agents, such as but not limited to calcium phosphate. Components can be combined in core-shell, hybrid, and/or layer-by-layer structures to allow fine-tuning of the nanoparticles, thereby enhancing delivery of nucleic acid vaccine compositions (Wang et al., Nat Mater. 2006, 5:791-796; Fuller et al., Biomaterials . 2008, 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev. 2011, 63:748-761; Endres et al., Biomaterials . 2011, 32:7721-7731; Su et al., Mol Mol Pharm. 2011;8(3):774-87; each such content is incorporated herein by reference in its entirety). As non-limiting examples, nanoparticles may include a plurality of polymers, such as, but not limited to, hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG), and/or hydrophilic polymers (e.g., PEG-PLGA), PCT Application Publication No. WO20120225129; the contents of which are incorporated herein by reference in their entirety).

The combination of biodegradable calcium phosphate nanoparticles with lipids and/or polymers can be used to deliver nucleic acid vaccine compositions in vivo. In some embodiments, lipid-coated calcium phosphate nanoparticles, which may also contain targeting ligands such as p-methoxybenzamide, may be used to deliver the nucleic acid vaccine compositions of the present disclosure. For example, to effectively deliver siRNA in a mouse metastatic lung model, lipid-coated calcium phosphate nanoparticles were used (Li et al., J Contr Rel. 2010, 142: 416-421; Li et al., J Contr Rel. 2010, 142: 416-421; Li et al., J Contr Rel. 2012, 158:108-114; Yang et al., Mol Ther. 2012, 20:609-615; each such content is incorporated herein by reference in its entirety). This delivery system combines both targeting nanoparticles and calcium phosphate, a component that enhances endosomal escape, to improve delivery of siRNA.

In some embodiments, calcium phosphate with PEG-polyanionic block copolymers can be used to deliver the nucleic acid vaccine compositions of the present disclosure (Kazikawa et al., J Contr Rel. 2004, 97:345-356; Kazikawa et al., J Contr Rel. 2006, 111:368-370; each such content is incorporated herein by reference in its entirety).

In some embodiments, PEG-charge transfer polymers (Pitella et al., Biomaterials. 2011, 32:3106-3114; the contents of which are incorporated by reference in their entirety) can be used to form nanoparticles to deliver the present invention. Disclosed nucleic acid vaccine composition. PEG-charge-switch polymers can improve PEG-polyanionic block copolymers by cleaving to polycations at acidic pH, thereby enhancing endosomal escape.

In some embodiments, core-shell nanoparticles can be used to form nanoparticles to deliver the nucleic acid vaccine compositions of the present disclosure. The use of core-shell nanoparticles has also focused on high-throughput methods to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA . 2011, 108:12996-13001; refer to the content incorporated herein by reference in its entirety). The complexation, delivery and internalization of polymeric nanoparticles can be precisely controlled by changing the chemical composition of both the core and shell components of the nanoparticles. For example, core-shell nanoparticles can effectively deliver nucleic acid vaccine compositions to mouse hepatocytes after covalently linking cholesterol to the nanoparticles.

In some embodiments, the nanoparticles described herein can be nanoparticles including at least one ligand, and the ligand can be a peptide, a nucleic acid aptamer (which has low nanomoles to its target). Binding affinity is small molecular weight (8 to 13 Kda) single-stranded RNA or DNA), peptide aptamers, antibodies, small molecule ligands such as but not limited to folic acid, p-methoxybenzamide and galactose. (Leng et al. Journal of Drug Delivery. 2017, 17, Article No. 6971297; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG can be used to deliver the nucleic acid vaccine compositions of the present disclosure. As a non-limiting example, lipid-polymer-lipid hybrid nanoparticles can be used to deliver the nucleic acid vaccine compositions described herein (Shi et al., Angew Chem Int Ed. 2011, 50:7027-7031; refer to the content as The full text of which is incorporated herein by reference).

Core-shell nanoparticles used in the nucleic acid vaccine composition of the present disclosure can be formed by the method described in U.S. Patent No. 8,313,777; the content of which is incorporated herein by reference in its entirety.

In some embodiments, core-shell nanoparticles can include a core and a polymer shell of a nucleic acid vaccine composition described herein. The polymer shell can be any of the polymers described herein and known in the art. In another embodiment, a polymer shell can be used to protect the nucleic acid vaccine composition in the core. (See, eg, U.S. Publication No. 20120321719; the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the polymer used in the formulations described herein can be a modified polymer (such as, but not limited to, a modified polyacetal) as described in PCT Application Publication No. WO2011120053; The content is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid vaccine composition can be delivered to a target by contacting a cell with a membrane-destabilizing polymer and a conjugate of the nucleic acid vaccine composition, a targeting ligand, and optionally a linker. The cell or cytosol of a cell. Non-limiting examples of membrane destabilizing polymers are taught in International PCT Application Publication No. WO2020093061, the content of which is incorporated herein by reference in its entirety, such as, but not limited to, the membrane destabilizing polymer of Formula XX therein. Excipients

In some embodiments, pharmaceutical formulations may additionally include pharmaceutically acceptable excipients, as used herein, including, but not limited to, any and all solvents, dispersion media, diluents suitable for the particular dosage form desired. or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifiers, preservatives, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, molar osmolality Regulators, pH regulators and the like. Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st ed., A. R. Gennaro (Lippincott, Williams & Wilkins) , Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of conventional excipient media is included within the scope of this disclosure unless any conventional excipient medium is incompatible with the substance or its derivatives, such as The use of any one or more other components of a pharmaceutical composition by producing any undesirable biological effect or otherwise interacting in a harmful manner is within the scope of the present disclosure.

In some embodiments, a pharmaceutically acceptable excipient can be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for human use and veterinary use. In some embodiments, the excipients may be approved by the United States Food and Drug Administration. In some embodiments, the excipients can be pharmaceutical grade. In some embodiments, the excipients can meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrants, binders, and preservatives. agents, buffers, lubricants and/or oils. Such excipients may optionally be included in pharmaceutical compositions. The compositions may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dicalcium phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbose Alcohol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, etc., and/or combinations thereof.

Exemplary granulating agents and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, Natural sponge, cation exchange resin, calcium carbonate, silicate, sodium carbonate, crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose , Croscarmellose sodium (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, carboxymethylcellulose calcium, silicic acid Magnesium aluminum (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.

Exemplary surfactants and/or emulsifiers include, but are not limited to, natural emulsifiers (eg, gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, gum, pectin , gelatin, egg yolk, casein, lanolin, cholesterol, wax and phosphatidylcholine), colloidal clays (such as bentonite (aluminum silicate) and VEEGUM® (magnesium aluminum silicate)), long-chain amino acid derivatives, High molecular weight alcohols (such as stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, poly vinyl alcohol), carbomers (such as carboxypolymethylene, polyacrylic acid, acrylic polymers and carboxyvinyl polymers), carrageenan, cellulose derivatives (such as sodium carboxymethylcellulose, powdered cellulose , hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (such as polyoxyethylene sorbitan monolaurate (TWEEN® 20), polyoxyethylene sorbitan (TWEEN®60), polyoxyethylene sorbitan monooleate (TWEEN®80), sorbitan monopalmitate (SPAN®40), sorbitan Monostearate (SPAN®60), sorbitan tristearate (SPAN®65), glyceryl monooleate, sorbitan monooleate (SPAN®80)), polyoxyethylene Esters (such as polyoxyethylene monostearate (MYRJ®45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid esters, polyethylene Glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers (e.g. polyoxyethylene lauryl ether (BRIJ® 30)), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleic acid Ester, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC® F 68, POLOXAMER® 188, cetyltrimethylammonium bromide, chloride Cetylpyridium, benzalkonium chloride, docusate sodium, etc., and/or combinations thereof.

Exemplary binders include, but are not limited to, starches (such as corn starch and starch paste); gelatin; sugars (such as sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids ( For example, glycine); natural and synthetic gums (such as gum arabic, sodium alginate, carrageenan extract, panwar gum, ghatti gum, isapol husk) Mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(ethylene) Pyrrolidone), magnesium aluminum silicate (VEEGUM®) and larch arabogalactan); alginate; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylic acid Esters; waxes; water; alcohols, etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Oxidation to mRNA is a potential degradation pathway, especially for liquid mRNA formulations. To prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothio Glycerin, sodium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium EDTA, dipotassium EDTA, ethylenediaminetetraacetic acid, fumaric acid, malic acid, Phosphoric acid, sodium EDTA, tartaric acid, and/or trisodium EDTA. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimonium bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, methyl chloride Phenol, chloroxylenol, cresol, ethanol, glycerin, hexetidine, imidazolidinyl urea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, sorbate Potassium acid, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenols, chlorobutanol, parabens, and/or phenethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deferoxamine mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylene glycol Amine, sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, p-hydroxybenzene Methyl formate, GERMALL® 115, GERMABEN®!!, NEOLONE™, KATHON™, and/or EUXYL®.

In some embodiments, the pH of the pharmaceutical solution is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/or sodium malate. In another embodiment, the exemplary buffers listed above may be used with additional monovalent counterions including, but not limited to, potassium. Divalent cations can also be used as buffer counterions; however, these are not preferred due to complex formation and/or mRNA degradation.

Exemplary buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate , calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium acetate propionate, valeric acid, dicalcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, Potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, disodium phosphate Sodium bishydride, sodium phosphate mixture, bradysamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, etc., and/or other combination.

Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, behenyl, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate , sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot stone, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, palm wax, castor oil Hemp, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, vanillyl alcohol, bottle gourd, grape seed, hazelnut, hyssop, myristanoic acid Isopropyl ester, jojoba, Hawaiian walnut, eye-catching lavender, lavender, lemon, litsea cubeba, macadamia nut, mallow, mango seed, white pond flower seed, mink, nutmeg, olive, orange, Atlantic bluegrass, Palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, camellia, savory, sea buckthorn, sesame, shea oil, silicone, soybean, sunflower , tea tree, thistle, toon, vetiver, walnut and wheat germ oil. Exemplary oils include, but are not limited to, butyl stearate, triglyceride caprylate, triglyceride caprate, cyclomethicone, diethyl sebacate, dimethicone 360, isomyristate Propyl ester, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavorings, and/or fragrances may be present in the compositions at the discretion of the formulator.

Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), added chelating agents (such as DTPA or DTPA-diamide) or calcium chelates (e.g., calcium DTPA, CaNaDTPA-diamide), or Calcium or sodium salts (such as calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate) are added as appropriate. In addition, antioxidants and suspending agents can be used.

In some embodiments of the present disclosure, the nucleic acid vaccine compositions described herein may include at least one nucleic acid vaccine formulated in lipid nanoparticles (LNP) and at least one excipient. As a non-limiting example, the excipient may be a sugar, such as sucrose. Adjuvant

Adjuvants may also be administered with or in combination with one or more nucleic acid vaccines described herein, such as an mRNA vaccine. Adjuvants can be used to enhance the immunogenicity of nucleic acid vaccines, alter immune responses, reduce the amount of nucleic acid vaccines required for immunization, reduce the frequency of additional or "booster" immunizations required, or in people with weakened or immunocompromised immune systems or Improved immune response in older adults. The adjuvant can be a component of a formulation containing the nucleic acid vaccine, or it can be co-administered with the nucleic acid vaccine composition. Co-administration of the adjuvant may be by any method known in the art or described herein, such as, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), or intradermal (ID).

In some embodiments, adjuvants are natural or synthetic. Adjuvants may also be organic or inorganic.

In some embodiments, adjuvants for nucleic acid vaccines are from a class of adjuvants such as, but not limited to, carbohydrates, microorganisms, mineral salts (e.g., aluminum hydroxide, aluminum phosphate gel, or calcium phosphate gel), emulsions ( e.g. oil emulsions, surfactant-based emulsions, purified saponins and oil-in-water emulsions), inert vehicles, particulate adjuvants (e.g. unilamellar liposome vehicles such as virions or structured complexes of saponins and lipids such as Polylactide-co-glycolide (PLG)), microbial derivatives, endogenous human immunomodulators and tonicity-active compounds. A list of adjuvants that may be used in the nucleic acid vaccines described herein can be found in the web-based vaccine adjuvant database Vaxjo (see, e.g., violinet.org/vaxjo or Sayers et al., Journal of Biomedicine and Biotechnology . 2012; 2012:831486 . PMID: 22505817; the contents of which are incorporated herein by reference in their entirety).

Adjuvants for nucleic acid vaccines can be selected by those skilled in the art. Adjuvants can be interferon, TNF-α, TNF-β, chemokines (such as CCL21, eosin, HMGB1, SA100-8α, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD40L, CD28 agonist, PD1, soluble PD1, PDL1, PDL2) or interleukins (e.g. IL1, IL2, IL4, IL6, IL7, IL10, IL12, IL13, IL15, IL17, IL18, IL21 and IL23). Non-limiting examples of adjuvants include Abisco-100 vaccine adjuvant, adamantylamide dipeptide vaccine adjuvant, Adjumer™, AF03, albumin-heparin particulate vaccine adjuvant, seaweed dextran, Algammulin, aluminum glue, Aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, aluminum vaccine adjuvant, amorphous aluminum hydroxyphosphorylated aluminum sulfate adjuvant, Arlacel A, AS0, AS04, AS03, AS-2 vaccine adjuvant , Avridine®, B7-2 vaccine adjuvant, Bay R1005, Bordetella pertussis component vaccine adjuvant, bupivacaine vaccine adjuvant, calcium phosphate gel, calcium phosphate vaccine adjuvant, cationic liposome vaccine adjuvant , Cationic liposome-DNA complex JVRS-100, cholera toxin, cholera toxin B subunit, Corynebacterium-derived P40 vaccine adjuvant, CpG DNA vaccine adjuvant, CRL1OO5, CTA1-DD gene fusion protein, DDA adjuvant, DHEA vaccine adjuvant, DL-PGL (polyester poly(DL-lactide-co-glycolide)) vaccine adjuvant, DOC/Alum complex, E. coli heat-labile toxin, Etx B subunit adjuvant, flagella Protein, Freund's complete adjuvant, Freund's incomplete adjuvant, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, imiquimod, immunoliposomes containing costimulatory molecule antibodies, ISCOM(s)™, ISCOMA-TRIX®, inactivated Corynebacterium parvum vaccine adjuvant, lipopolysaccharide, liposome, loxoribine, LTK63 vaccine mutant adjuvant, LTK72 vaccine adjuvant, LTR192G vaccine adjuvant, Matrix-S, MF59, Montanide Incomplete Seppic Adjuvant, Montanide ISA 51, Montanide ISA 720 Adjuvant, MPL-SE Vaccine Adjuvant, MPL™ Adjuvant, MTP-PE Liposome, Cellular Peptide, Cellular Dipeptide Adjuvant, Xupamidine (Murapalmitine), D-mopamidine, NAGO, nanoemulsion vaccine adjuvant, non-ionic surfactant vesicles, cholera toxin non-toxic mutant E112K mCT-E112K, PMMA, Poly(LC), Polygen vaccine adjuvant, Protein lipid volume, QS-21, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, recombinant hlFN-γ/interferon-g, Rehydragel EV, Rehydragel HPA, Resiquimod, Ribi vaccine adjuvant, SAF -1. Saponin vaccine adjuvant, thymosin, Sendai proteoliposome, Sendai-containing Lipid Ultimate, Specol, SPT (antigen formulation), squalene-based adjuvant, stearyl tyrosine, Theramide®, hydroxybutyrate Aminolysyl muramidyl dipeptide (TMDP), Titer-Max Gold adjuvant, Ty Particles vaccine adjuvant, and VSA-3 adjuvant.

In some embodiments, the nucleic acid vaccines described herein can be used as vaccines and can further include adjuvants that can enable the vaccine to elicit a higher immune response. As a non-limiting example, the adjuvant can be a submicron oil-in-water emulsion, which can elicit higher immune responses in the human pediatric population (see, for example, adjuvants described in U.S. Patent Publication No. US20120027813 and U.S. Patent No. 8,506,966 doses of vaccine, each such content is incorporated herein by reference in its entirety). Drug administration and administration

The present disclosure encompasses delivery of nucleic acid vaccine compositions by any appropriate route taking into account possible developments in drug delivery science, including, for example, COVID-19 nucleic acid vaccines for any therapeutic, prophylactic, pharmaceutical, diagnostic or imaging use. Delivery can be naked or formulated.

The nucleic acid vaccine compositions of the present disclosure can be delivered to cells naked. As used herein, "naked" refers to the delivery of a nucleic acid vaccine composition that does not contain agents that facilitate transfection. For example, nucleic acid vaccine compositions delivered to cells may be free of modifications. Naked nucleic acid vaccine compositions can be delivered to cells using administration routes known in the art and described herein.

The nucleic acid vaccine compositions of the present disclosure may be formulated using the formulation components and methods described herein. The formulations may contain nucleic acid vaccine compositions, which may be modified and/or unmodified. The formulations may further include, but are not limited to, cell penetrating agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and sustained release delivery depots. The formulated nucleic acid vaccine compositions may be delivered to cells using administration routes known in the art and described herein.

Nucleic acid vaccine compositions may also be formulated for direct delivery to organs or tissues in any of several modalities of this technology, including but not limited to: direct immersion or bathing, via catheters, via gels, Powders, ointments, creams, gels, lotions, and/or drops, by using a substrate coated or impregnated with the composition, such as a fabric or biodegradable material, and the like. The nucleic acid vaccine composition of the present disclosure can also be cloned into a retroviral replication vector (RRV) and transduced into cells. Give medication

Also provided herein are methods comprising administering a nucleic acid vaccine described herein to an individual in need thereof. The precise amount required will vary from individual to individual depending on the individual's species, age, health and general condition, severity of disease, the specific composition, its mode of administration, its pattern of activity and similar factors. For ease of administration and uniformity of dosage, the compositions are usually formulated in unit dosage form. However, it should be understood that the total daily dosage of the composition will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound used; the specific composition used; and the age of the patient. , body weight, general health, gender and diet; administration time, route of administration and excretion rate of the specific compound used; duration of treatment; drugs used in combination or concurrently with the specific compound used; and similar factors well known in medical technology.

This disclosure encompasses the following dosage levels: about 0.001 to about 500 mg nucleic acid vaccine (e.g., COVID-19 nucleic acid vaccine, e.g., COVID-19 mRNA vaccine)/kg body weight/day, about 0.001 to about 200 mg/kg, about 0.001 to about 100 mg/kg, 0.01 to about 100 mg/kg, preferably about 0.005 to about 50 mg/kg, 0.01 to about 50 mg/kg, 0.01 to about 40 mg/kg, 0.01 to about 30 mg/kg, 0.01 to about 10 mg/kg, 0.05 to about 50 mg/kg, 0.05 to about 30 mg/kg, 0.05 to about 10 mg/kg, 0.05 to about 5 mg/kg, 0.1 to about 50 mg/kg, 0.1 to about 30 mg /kg, 0.1 to about 10 mg/kg, 0.1 to about 1 mg/kg, 1.0 to about 50 mg/kg, 1.0 to about 40 mg/kg, 1.0 to about 30 mg/kg, 10 to about 50 mg/kg weight. Other embodiments include doses of about 0.001 to 0.010, 0.010 to 0.050, 0.050 to 0.100, 0.1 to 0.5, 0.5 to 1.0, 1.0 to 5.0, 5.0 to 10, 10 to 50 mg/kg, 10 to 100 mg/kg body weight . Doses may be administered approximately hourly, multiple times per day, daily, every other day, weekly, every other week, monthly, every other month, or as needed.

In some embodiments, the composition of the nucleic acid vaccine may be sufficient to deliver about 0.0001 mg/kg to about 100 mg/kg, about 0.001 mg/kg to about 0.05 mg/kg, about 0.005 mg/kg to about 0.05 mg/kg , about 0.001 mg/kg to about 0.005 mg/kg, about 0.05 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 25 mg/kg, about 1 mg /kg to about 50 mg/kg, about 10 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50 mg/k individual body weight/day, administered at dosage levels one or more times a day to obtain Desired therapeutic, diagnostic, preventive or imaging effect. The desired dose may be delivered three times a day, twice a day, once a day, every other day, every third day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, Twelve, thirteen, fourteen or more doses). When multiple dosing is employed, divided dosing regimens such as those described herein may be used.

In some embodiments, the composition of the nucleic acid vaccine described herein can be sufficient to deliver about 1 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 35 µg, 40 µg, 50 µg, 60 dose levels of µg, 70 µg, 80 µg, 90 µg, or 100 µg of the nucleic acid composition.

In some implementations, the nucleic acid vaccine can be administered in a divided dose regimen. As used herein, "fractionated dose" means dividing a single unit dose or total daily dose into two or more doses, such as two or more administrations of a single unit dose. A "single unit dose" as used herein is a dose of any therapeutic agent administered in one dose/single time/single route/single point of contact, ie, a single administration event. As used herein, a "total daily dose" is the amount administered or prescribed in a 24-hour period. It can be administered in a single unit dose. In some embodiments, the nucleic acid vaccines described herein are administered to an individual in divided doses. Nucleic acid vaccines can be formulated in buffer alone or in the formulations described herein.

In some embodiments, the nucleic acid vaccine compositions described herein can be administered to an individual in two separate phases (initial administration phase and maintenance administration phase). The dosing regimen may include an initial higher starting dose of the nucleic acid vaccine first administered to the individual at the beginning of a course of prevention, mitigation, and/or treatment, such as the first dose for prevention of COVID-19, and after the first starting dose lower maintenance dose. In some embodiments, the starting dose and the maintenance dose have the same amount of the nucleic acid vaccine of the present disclosure. In some embodiments, more than one maintenance dose is administered to an individual. Multiple maintenance doses can be administered every two weeks, every three weeks, every four weeks, every month, every two months, every three months, every four months, every five months, or every six months. In the case of vaccination to prevent disease, such as nucleic acid (eg, mRNA) vaccines for COVID-19, the maintenance dose of the nucleic acid vaccine may also be called a booster dose. As used herein, a "booster dose" (or "booster shot") is an additional or supplemental dose of vaccine following the initial primer dose. The amount of the booster dose of nucleic acid vaccine can be the same as the initial starting dose. Alternatively, the amount of the nucleic acid vaccine in the booster dose is less than the original amount of the nucleic acid vaccine in the initial dose. In some embodiments, a subject may receive one, two, three, four, or more booster doses.

Such administration may be used for the chronic or acute treatment or prevention of clinically relevant conditions. The amount of drug that can be combined with the carrier to produce a single dosage form will vary depending on the host treated and the particular mode of administration. A typical formulation will contain from about 5% to about 95% active compound (w/w). Preferably, such formulations contain from about 20% to about 80%, from 30% to about 70%, from 40% to about 60%, or about 50% active compound. In other embodiments, formulations used in the present disclosure will be about 5 to 10%, 10 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60 to 70% , 70 to 80%, 80 to 90%, 90 to 99%, or greater than 99% of active ingredients.

When an individual's condition improves, a maintenance dose of a compound, composition or combination of the present disclosure may be administered if necessary. Subsequently, the dose or frequency of administration, or both, may be reduced with symptoms to a level that maintains improvement when symptoms have alleviated to the desired level, and treatment should be discontinued. However, patients may require long-term intermittent treatment if symptoms of any disease recur.

Those skilled in the art should understand that lower or higher doses than those stated above may be required. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health, sex, diet, timing of administration, excretion rate, drug combination, infection The severity and course of the disease, the patient's management of the infection and the judgment of the treating physician. delivery

In some embodiments, delivery of nucleic acid vaccines can be naked or formulated.

In some embodiments, the nucleic acid vaccines described herein can be delivered to cells naked. As used herein, "naked" refers to the delivery of a nucleic acid vaccine that does not contain agents that facilitate transfection. For example, nucleic acid vaccines delivered to cells may be free of modifications. Naked nucleic acid vaccines can be delivered to cells using administration routes known in the art and described herein.

In some embodiments, the nucleic acid vaccines described herein can be formulated using the methods described herein. The formulations may further include, but are not limited to, cell penetrating agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and sustained release delivery depots. The formulated nucleic acid vaccines can be delivered to cells using administration routes known in the art and described herein.

The compositions may also be formulated for direct delivery to organs or tissues in any of several modalities of this technology, including but not limited to: direct immersion or bathing, via catheters, by gels, powders, Ointments, creams, gels, lotions, and/or drops, by using a substrate coated or impregnated with the composition, such as a fabric or biodegradable material, and the like. invest

In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be administered by any route that produces a preventive or therapeutically effective result. These include, but are not limited to, enteral (into the intestines), gastrointestinal, epidural (into the dura mater), oral (through the mouth), transdermal, epidural, intracerebral (into the brain), intracerebroventricular (into the ventricles of the brain), epidermal (into the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal (through the nose), intravenous (into a vein), intravenous bolus Injection, intravenous drip, intraarterial (into arteries), intramuscular (into muscles), intracardiac (into heart), intraosseous infusion (into bone marrow), intrathecal (into spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous sinus injection (into the pathological cavity), intracavity (into the base of the penis), intravaginal administration, intrauterine, extraamniotic Administer, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), vaginal, insufflation (nasal), sublingual, sublabial, enema, eye drops (to the supraconjunctiva) ), ear drops, ear canal (in or through the ear), intrabuccal (for the cheek), conjunctiva, skin, dental (for one or more teeth), electroosmosis, intracervix, intranasal sinuses, trachea Intra, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracystic, intrachondral (within the cartilage), intracaudal (within the cauda equina), intracisternal ( Within the cistern of the brain), within the cornea (within the cornea), within the dental cornea, within the coronary arteries (within the coronary arteries), intracavernosus (within the expandable space of the corpora cavernosa of the penis), within the intervertebral disc (within the intervertebral disc ), intraductal (inside the glandular duct), intraduodenal (inside the duodenum), intradural (in or below the dura mater), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach intragingival (within the gums), intraileal (within the distal part of the small intestine), intralesional (within or directly introduced into a localized lesion), intraluminal (within the lumen of a tube), intralymphatic ( Intralymph), intramedullary (in the marrow cavity of the bone), intrameningeal (in the meninges), intraocular (in the eye), intraovarian (in the ovary), intrapericardial (in the pericardium), intrapleural (in the pleura), intraprostatic (in the prostate), intrapulmonary (in the lungs or their bronchi), intrasinus (in the paranasal or periorbital sinuses), intraspinal (in the spine), intrasynovial (in Within the synovial cavity of joints), intratendinally (within the tendons), intratesticularly (within the testicles), intrathecally (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the chest cavity), intracanaliculus ( within the small ducts of an organ), intratumoral (within a tumor), intratympanic (within the middle layer of the ear), intravascular (within one or more blood vessels), intraventricular (within the heart of the heart), iontophoresis (with the help of an electric current , in which soluble salt ions migrate into body tissues), lavage (washing or flushing of an open wound or body cavity), larynx (directly in the throat), nasogastric (through the nose and into the stomach), occlusive dressing techniques, trans. Ocular (to the external eye), oropharyngeal (directly to the oropharynx), parenteral, transdermal, periarticular, epidural, perineural, periodontal, transrectal, respiratory (by oral or nasal inhalation into the respiratory tract) , for local or systemic action), retrobulbar (in the pons or behind the eyeball), intramyocardial (into the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, transplacental (across or beyond the placenta), transtracheal (across the tracheal wall), transtympanic (over or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, sacral block, diagnostic, nerve block, bile duct perfusion, cardiac perfusion, light Remove method or spinal cord. In certain embodiments, the composition may be administered in a manner that allows it to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

Delivery of the nucleic acid vaccines described herein to an individual over an extended period of time, such as one week to one year, can be accomplished by a single administration of a controlled release system containing sufficient active ingredient to sustain the desired release period. For this purpose, various controlled release systems can be utilized, such as monolithic or reservoir microcapsules, reservoir implants, polymeric hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches , iontophoresis devices, and alternative injectable dosage forms. Localization to the site where delivery of the active ingredient is desired is an additional feature of some controlled release devices that may prove beneficial in the treatment of certain conditions.

In some embodiments, the nucleic acid vaccines described herein can be administered intranasally similar to the administration of live vaccines. In another aspect, the polynucleotide can be administered intramuscularly or intradermally similar to the administration of inactivated vaccines known in the art.

In some embodiments of transdermal administration, electrodes (e.g., iontophoresis), electroporation, or the application of short high-voltage electrical pulses to the skin, radiofrequency, ultrasound (e.g., sonotophoresis), microsurgery, etc. will be used. Projection (e.g., microneedles), jet injector, thermal ablation, magnetophoresis, laser, velocity, or optomechanical waves to enhance delivery across the skin barrier. Drugs may be contained in a single layer of medicated adhesive, multiple layers of medicated adhesive, a reservoir, a matrix or a vapor-based patch, or patchless technology may be utilized. Encapsulation, skin lipid fluidizers or hollow or solid microstructured transdermal systems (MTS, such as MTS manufactured by 3M), jet injectors may also be used to enhance delivery across the skin barrier. Formulation additives used to aid the passage of therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell-penetrating peptides, penetration enhancers, encapsulation technologies, enzymes, enzyme inhibitors, gels, nanoparticles, and Peptide or protein chaperone.

Other slow release, depot implants or injectable formulations will be apparent to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker Inc., New York, 1978; and Controlled Release of Biologically Active Agents, RW Baker, John Wiley & Sons, New York, 1987. The foregoing is incorporated by reference in its entirety.

Combining the nucleic acid vaccines described herein with polymeric formulations containing biodegradable polymers that form depot formulations upon administration is suitable for obtaining extremely long duration of action formulations.

When formulated for nasal administration, absorption across the nasal mucosa can be further enhanced by surfactants such as glycocholic acid, cholic acid, taurocholic acid, acecholic acid, deoxycholic acid, Chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrins and the like in amounts ranging between about 0.1 and 15% by weight, between about 0.5 and 4% by weight, or About 2% by weight. Another class of absorption enhancers reported to exhibit greater efficacy and reduced irritation are the alkyl maltosides, such as tetradecyl maltoside (Arnold, JJ et al., J Pharm Sci, 2004, 93: 2205-13 ; Ahsan, F et al., Pharm Res , 2001,18:1742-46) and the references therein, which are hereby incorporated by reference in their entirety.

The pharmaceutical compositions may be in the form of sterile injectable preparations, for example, as sterile injectable aqueous or oily suspensions. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are often used as solvents or suspending media. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are suitable for the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. Such oil solutions or suspensions may also contain long chain alcohol diluents or dispersants such as Ph. Helv or similar alcohols.

The pharmaceutical compositions of the present disclosure can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral administration, common carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredients are combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of the present disclosure may also be administered in the form of suppositories for rectal administration. Such compositions may be prepared by mixing the active ingredients of the present disclosure with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release active ingredients. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

Topical administration of the pharmaceutical compositions of the present disclosure is particularly useful when the desired treatment involves areas or organs that are easily accessible by topical administration. For topical application to the skin, the pharmaceutical compositions should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in a vehicle. Carriers for topical administration of compounds of the present disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying waxes, and water. Alternatively, the pharmaceutical compositions may be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a vehicle. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl wax, cetyl stearyl alcohol, 2-octyldodecanol, benzyl alcohol, And water. The pharmaceutical compositions of the present disclosure may also be administered topically to the lower intestinal tract via rectal suppository formulations or in suitable enema formulations. Also included in this disclosure are topical transdermal patches.

The pharmaceutical compositions of the present disclosure may be administered via nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical compounding art, and may employ benzyl alcohol or other suitable preservatives, absorption enhancers that enhance bioavailability, fluorocarbons, and/or other dissolving agents known in the art. Or the dispersant is prepared as a solution in physiological saline.

A variety of formulations offer advantages when formulated for delivery by inhalation. Adsorption of therapeutic agents onto readily dispersible solids such as diketones (e.g., Technosphere particles (Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv 2:1097-1106)) or similar structures results in a structure that allows the therapeutic compound to be Formulations for rapid initial absorption. Lyophilized powders, especially glassy particles, containing therapeutic compounds and excipients can be used for delivery to the lungs with good bioavailability, see for example Exubera ^® (inhaled insulin, Pfizer, Inc. and Aventis Pharmaceuticals Inc. ) and Afrezza ^® (inhaled insulin, Mannkind, Corp.). Dosage Form

The pharmaceutical compositions described herein may be formulated into dosage forms as described herein, such as topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous) dosage forms . liquid dosage form

Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and/or elixirs. In addition to the active ingredients, liquid dosage forms may also contain inert diluents commonly used in the art, including but not limited to water or other solvents, solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil ), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof. In certain embodiments for parenteral administration, the compositions may be mixed with solubilizing agents such as CREMO-PHOR®, alcohols, oils, modified oils, glycols, polysorbates , cyclodextrins, polymers, and/or combinations thereof. injectable form

Injectable preparations, such as sterile injectable aqueous or oily suspensions, may be formulated according to known techniques, and may include suitable dispersing, wetting and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions and/or emulsions in a nontoxic parenterally acceptable diluent and/or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. It is common practice to use sterile fixed oil as the solvent or suspension medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Injectables may be prepared using fatty acids such as oleic acid.

Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, and/or by incorporating a sterilizing agent, in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectables prior to use. media for sterilization.

To prolong the effect of the active ingredient, it may be necessary to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials with low water solubility. The absorption rate of a nucleic acid vaccine depends on its dissolution rate, which in turn depends on the crystal size and crystallization form. Alternatively, absorption of a parenterally administered nucleic acid vaccine can be delayed by dissolving or suspending the nucleic acid vaccine in an oily vehicle. Injectable depot forms are made by forming microencapsule matrices of nucleic acid vaccines in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of nucleic acid vaccine to polymer and the properties of the specific polymer used, the rate of polynucleotide release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Injectable depot formulations can be prepared by encapsulating the nucleic acid vaccine in liposomes or microemulsions that are compatible with body tissue. Lung

Formulations described herein as suitable for transpulmonary delivery may also be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration may be a coarse powder containing the active ingredient and having an average particle size of about 0.2 pm to 500 pm. Such formulations may be administered by nasal inhalation, such as by rapidly inhaling the powder through the nostrils from a container close to the nose.

Formulations suitable for nasal administration may, for example, contain as little as about 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may contain one or more of the additional ingredients described herein . Pharmaceutical compositions may be prepared, packaged and/or sold in formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or buccal tablets prepared using conventional methods, and may, for example, contain from about 0.1% to 20% (w/w) of the active ingredient, the remainder of which may comprise orally soluble and/or degradable compositions, and optionally include one or more of the additional ingredients described herein. Alternatively, formulations suitable for intrabuccal administration may comprise powders and/or aerosolized and/or nebulized solutions and/or suspensions containing the active ingredient. Such powdered, aerosolized and/or aerosolized formulations, when dispersed, may have an average particle size and/or droplet size in the range of about 0.1 nm to about 200 nm, and may further comprise any of the One or more additional ingredients.

General considerations in formulating and/or manufacturing pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy 21st Edition, Lippincott Williams & Wilkins, 2005. Solid dosage form: coating or shell

Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical compounding art. They may optionally contain opacifying agents and may be of such a composition that they release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like. Characteristics of pharmaceutical compositions

Nucleic acid vaccine pharmaceutical compositions described herein may be characterized using one or more of bioavailability, therapeutic window, distribution volume, biological effect, and detection of polynucleotides by mass spectrometry. bioavailability

Nucleic acid vaccines can exhibit increased bioavailability when formulated into compositions with a delivery agent as described herein compared to compositions lacking a delivery agent as described herein. As used herein, the term "bioavailability" refers to the systemic availability of a given amount of a nucleic acid vaccine administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration of the unchanged form of the compound following administration to a mammal. AUC is a measurement of the area under a curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) versus time along the abscissa (X-axis). In general, the AUC of a particular compound can be determined using G. S. Banker, Modem Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, N.Y., Inc., which is known to those of ordinary skill and is incorporated herein by reference in its entirety. Calculated using the method described in 1996.

The Cmax value is the maximum concentration of a compound achieved in the serum or plasma of the mammal after administration of the compound to the mammal. The Cmax value of a particular compound can be measured using methods known to those of ordinary skill. The phrase "increased bioavailability" or "improved pharmacokinetics" as used herein means that when co-administered with a delivery agent as described herein, the first nucleic acid vaccine, measured as AUC, Cmax or Cmin, in breast-feeding Systemic availability in the animal is greater than in the absence of such co-administration. In some embodiments, the bioavailability of the nucleic acid vaccine can be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least About 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least About 85%, at least about 90%, at least about 95%, or about 100%.

In some embodiments, liquid formulations of nucleic acid vaccines may have different in vivo half-lives, requiring dosage adjustments to produce prophylactic or therapeutic effects. To address this issue, in some embodiments, nucleic acid vaccine formulations can be designed to improve bioavailability and/or prophylactic or therapeutic efficacy during repeated administrations. Such formulations may enable sustained release of the nucleic acid vaccine and/or reduce the rate of degradation of the nucleic acid vaccine by nucleases. In some embodiments, suspension formulations are provided that include nucleic acid vaccines, water-immiscible oil depots, surfactants and/or co-surfactants and/or co-solvents. The combination of oil and surfactant enables suspension formulations of nucleic acid vaccines. Delivery of nucleic acid vaccines in water-immiscible reservoirs can be used to improve bioavailability via sustained release of the polynucleotide from the reservoir to the surrounding physiological environment and/or to prevent degradation of the polynucleotide by nucleases.

In some embodiments, cationic nanoparticles containing a combination of divalent and monovalent cations can be formulated with nucleic acid vaccines. Such nanoparticles can form spontaneously in solution over a given period of time (eg, hours, days, etc.). Such nanoparticles do not form in the presence of divalent cations alone or in the presence of monovalent cations alone. Delivering nucleic acid vaccines in cationic nanoparticles or in one or more reservoirs containing cationic nanoparticles may improve nucleic acid vaccine bioavailability by acting as a long-lasting reservoir and/or reducing the rate of nuclease degradation. therapeutic window

The nucleic acid vaccine, when formulated into a composition with a delivery agent as described herein, can exhibit therapeutic efficacy of the administered nucleic acid vaccine composition when compared to the therapeutic window of the administered nucleic acid vaccine composition in the absence of a delivery agent as described herein. The addition of windows. As used herein, the "therapeutic window" refers to the range of plasma concentrations or levels of a therapeutically active substance at the site of action over which there is a higher probability of eliciting a prophylactic or therapeutic effect. In some embodiments, when co-administered with a delivery agent as described herein, the therapeutic window of a nucleic acid vaccine can be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. Distribution volume

Nucleic acid vaccines when formulated into compositions with a delivery agent as described herein may exhibit improved distribution volumes (Vdist), eg, reduced or targeted, compared to compositions lacking a delivery agent as described herein. The volume of distribution (Vdist) relates the amount of a drug (such as the nucleic acid vaccine of the present disclosure) in the body to the concentration of the drug in the blood or plasma. The term "distribution volume" as used herein refers to the volume of liquid required to contain the total amount of drug in the body at the same concentration as in the blood or plasma: Vdist is equal to the amount of drug in the body/drug concentration in the blood or plasma. For example, for a 10 mg dose and a plasma concentration of 10 mg/L, the volume of distribution would be 1 liter. The volume of distribution reflects the extent to which a drug is present in extravascular tissues. The larger distribution volume reflects the tendency of the compound to bind to tissue components compared to plasma protein binding. In a clinical setting, Vdist can be used to determine the starting dose to achieve steady-state concentrations. In some embodiments, when co-administered with a delivery agent as described herein, the volume of distribution of a nucleic acid vaccine can be reduced by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%. Biological effects

In some embodiments, the biological effects of nucleic acid vaccines delivered to animals can be classified by analyzing protein expression in the animals. Protein expression can be determined by analysis of biological samples collected from mammals administered the nucleic acid vaccines described herein. Detection of polynucleotides by mass spectrometry

Mass spectrometry (MS) is an analytical technique that provides information about the structure and molecular mass/concentration of molecules after they are converted into ions. Molecules are first ionized to acquire a positive or negative charge, and then they pass through the mass analyzer and reach different areas of the detector according to their mass-to-charge ratio (m/z).

Mass spectrometry is performed using a mass spectrometer that includes an ion source for ionizing the fractionated sample and producing charged molecules for further analysis. For example, samples can be ionized by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix-assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermal spray/plasma spray ionization, and particle beam ionization. Those skilled in the art will understand that the choice of ionization method may be determined based on the analyte to be measured, sample type, detector type, selection of positive mode versus negative mode, etc.

After a sample has been ionized, the resulting positively or negatively charged ions can be analyzed to determine the mass-to-charge ratio (ie, m/z). Suitable analyzers for determining mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Several detection modes are available for detecting ions. For example, selected ions can be detected (ie using Selective Ion Monitoring Mode (SIM)), or ions can be detected using scanning modes such as Multiple Reaction Monitoring (MRM) or Selected Reaction Monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) combined with stable isotope-labeled peptide standard dilutions has been shown to be an effective method for protein verification (e.g. Keshishian et al., Mol Cell Proteomics, 2009, 8: 2339- 2349; Kuhn et al., Clin Chem 2009, 55:1108-1117; Lopez et al., Clin Chem , 2010, 56:281-290; each of which is incorporated herein by reference in its entirety). Unlike non-targeted mass spectrometry, which is often used in biomarker discovery studies, targeted MS methods are peptide sequence-based MS modes that focus the entire analytical power of the instrument on tens to hundreds of selected peptides in complex mixtures. . By limiting detection and fragmentation to only those peptides from the protein of interest, sensitivity and reproducibility are significantly improved compared to discovery mode MS methods. This mass spectrometry-based multiple reaction monitoring (MRM) protein quantification method can significantly impact the discovery and quantification of biomarkers through rapid, targeted, multiplex protein expression analysis of clinical samples.

In some embodiments, the biological sample, once obtained from the individual, can be subjected to enzymatic digestion. As used herein, the term "digestion" means breaking down into shorter peptides. As used herein, the phrase "treating a sample to digest proteins" means treating the sample in a manner that breaks down the proteins in the sample. Such enzymes include, but are not limited to, trypsin, endoprotease Glu-C, and chymotrypsin.

In some embodiments, electrospray ionization can be used to analyze proteins in biological samples. Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to help transfer ions from solution to the gas phase, where the ions are subsequently analyzed by mass spectrometry. Samples can be analyzed using methods known in the art (eg, Ho et al., Clin Biochem Rev. 2003, 24(1):3-12; incorporated herein by reference in its entirety). Ionic species contained in the solution can be transferred to the gas phase by dispersing a fine spray of charged droplets, evaporating the solvent, and ejecting ions from the charged droplets to create a mist of highly charged droplets. The highly charged liquid droplet mist may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers, such as, but not limited to, quadrupole mass analyzers. Additionally, mass spectrometry can include purification steps. As a non-limiting example, the first quadrupole can be set up to select a single m/z ratio, so it can filter out other molecular ions with different m/z ratios. This can eliminate complex and time-consuming sample purification procedures prior to MS analysis. .

In some embodiments, biological samples can be analyzed for proteins in a tandem ESIMS system (eg, MS/MS). As non-limiting examples, droplets can be analyzed using product scans (or sub-scans), precursor scans (parent scans), neutral loss, or multiple reaction monitoring.

In some implementations, biological samples can be analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides non-destructive vaporization and ionization of large and small molecules such as proteins. In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, weak organic acids that absorb UV light. Non-limiting examples of bases used in MALDI are a-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. Laser radiation of the analyte-matrix mixture can cause the matrix and analyte to evaporate. Laser-induced desorption provides high ion yields of intact analytes and allows compounds to be measured with high precision. Samples can be analyzed using methods known in the art (eg, Lewis, Wei, and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; the contents of which are incorporated herein by reference in their entirety). As non-limiting examples, mass analyzers used in MALDI analysis may include linear time-of-flight (TOF), TOF reflector, or Fourier transform mass analyzers. performance system

In some embodiments, the nucleic acid vaccines described herein can be operably linked to one or more regulatory nucleotide sequences and encoded in an expression construct. The regulatory nucleotide sequence will generally be appropriate for the host cell used for expression. Many types of suitable expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Generally, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, transcription initiation and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated. The promoter may be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct may be present on an episome in the cell, such as a plastid, or the expression construct may be inserted into the chromosome. In certain embodiments, the expression vector includes a selectable marker gene that allows selection of transformed host cells. Certain embodiments include expression vectors encoding a nucleic acid vaccine of a COVID-19 sequence operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers and other expression control elements. In certain embodiments, consideration is given to the selection of host cells to be transformed, the specific nucleic acid vaccine sequences to be expressed, the number of copies of the vector, the ability to control the number of copies, or other proteins encoded by the vector, such as expression of antibiotic markers. Design expression vehicle.

In some embodiments, the nucleic acids described herein can be expressed in microorganisms. As non-limiting examples, the nucleic acid may be in a bacterial system, such as in Bacillus brevis , Bacillus megaterium , Bacillus subtilis , Caulobacter crescentus , Escherichia coli ( Escherichia coli ) and its derivatives. Exemplary promoters include l-arabinose-inducible araBAD promoter (PBAD), lac promoter, l-rhamnose-inducible rhaP BAD promoter, T7 RNA polymerase promoter, trc and tac promoters, lambda phage promoter Pl and anhydrotetracycline-inducible tetA promoter/operator.

In some embodiments, the nucleic acids described herein can be expressed in a yeast expression system. Non-limiting examples of promoters useful in yeast vectors include the promoter of 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem . 255:2073 (1980)); the promoters of other glycolytic enzymes (Hess et al., J. Adv. Enzyme Res . 7:149 (1968); Holland et al., Biochemistry 17:4900 (1978). Other promoters are derived from, for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, Hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase Alcohol oxidase I (AOX1), alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes related to nitrogen metabolism and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and those responsible for the utilization of maltose and galactose enzyme. Any plasmid vector containing a yeast-compatible promoter and termination sequence, with or without an origin of replication, is suitable. Certain yeast expression systems are commercially available, e.g., from Clontech Laboratories, Inc. (Palo Alto, Calif., such as the Pyex 4T vector family for Saccharomyces cerevisiae), Invitrogen (Carlsbad, Calif., such as the Ppicz Series Easy Select Pichia Expression Kit), and Stratagene (La Jolla, Calif., such as the ESP for Schizosaccharomyces pombe) .TM. Yeast Protein Expression and Purification System and Pesc vector for Saccharomyces cerevisiae).

In some embodiments, the nucleic acids described herein can be expressed in mammalian expression systems. Non-limiting examples of mammalian promoters include, for example, promoters from genes such as: hamster ubiquitin/S27a promoter (WO 97/15664), simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter promoter, mouse metallothionein-I promoter, long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus (Moloney murine leukemia virus) long terminal repeat region and the early promoter of human cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock promoters. In certain embodiments, a yeast alcohol oxidase promoter is used.

In some embodiments, promoters for use in mammalian host cells can be obtained from the genome of viruses such as polyomavirus, fowlpox virus (UK 2,211,504, published July 5, 1989), bovine papillomavirus parvovirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40). In other embodiments, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters, and heat shock promoters. The early and late promoters of SV40 are preferably obtained in the form of SV40 restriction fragments that also contain the SV40 viral origin of replication. Fiers et al., Nature 273: 113-120 (1978). The human cytomegalovirus immediate early promoter is suitably obtained as a HindIII E restriction fragment. Greenaway, PJ et al. Gene 18: 355-360 (1982). The foregoing references are incorporated herein by reference in their entirety.

In some embodiments, the nucleic acids described herein can be expressed in insect cell expression systems. Eukaryotic expression systems using insect cell hosts can rely on plastid or baculovirus expression systems. A typical insect host cell is derived from the Spodoptera frugiperda ( Spodoptera frugiperda ). For expression of heterogeneous proteins, the cells were infected with a recombinant version of the baculovirus Autographa californica nuclear polyhedrosis virus that has the gene of interest expressed under the control of the viral polyhedrin promoter. . Other insects infected by this virus include a cell strain commercially known as "High 5" (Invitrogen), which is derived from the cabbage exigua moth ( Trichoplusia ni ). Another baculovirus sometimes used is Bombyx mori nuclear polyhedrosis virus, which infects silkworms (Bombyx mori). Many baculovirus expression systems are commercially available, for example from Thermo Fisher (Bac-N-Blue ^™ k or BAC-TO-BAC ^™ system), Clontech (BacPAK ^™ Baculovirus Expression System), Novagen (Bac Vector System ^TM ) or others obtained from Pharmingen or Quantum Biotechnologies. Another insect cell host is the common fruit fly Drosophila melanogaster , for which Thermo Fisher (The DES ^TM System) commercially provides a transient or stable plastid-based transfection kit.

In some embodiments, cells are transformed with vectors expressing nucleic acids described herein. Transformation techniques for inserting new genetic material into eukaryotic cells, including animal and plant cells, are well known. Viral vectors can be used to insert expression cassettes into the host cell genome. Alternatively, the vector can be transfected into host cells. Transfection can be accomplished by methods as described in the art, such as, but not limited to, calcium phosphate precipitation, electroporation, optical transfection, protoplast fusion, punch infection, and hydrodynamic delivery. IV. How to use

One aspect of the present disclosure provides methods of using the nucleic acid vaccines of the present disclosure and pharmaceutical compositions and formulations comprising the nucleic acid vaccines and at least one pharmaceutically acceptable carrier. Provided herein are compositions, methods, kits and reagents for diagnosing, treating, alleviating or preventing diseases or conditions in humans or other mammals, wherein the active agent is a nucleic acid vaccine, a cell containing a nucleic acid vaccine or a polynucleoside derived from a nucleic acid vaccine Acid-translated peptides.

In some embodiments, any endpoint indicating benefit to an individual may be used to evaluate the use, including but not limited to (1) inhibiting disease progression to a certain extent, including stabilization, slowing, and complete containment; (2) reducing disease exacerbations and/or the number of symptoms; (3) inhibit (i.e., reduce, slow down, or completely stop) the infiltration of disease cells into adjacent peripheral organs and/or tissues; (4) inhibit (i.e., reduce, slow down, or completely stop) the disease Transmission; (5) Alleviation of autoimmune symptoms; (6) Favorable changes in the expression of biomarkers related to the disease; (7) Alleviation to a certain extent of one or more symptoms related to the disease; (8) Increased treatment the duration of disease-free manifestations; or (9) reducing mortality at a given time point after treatment. Therapeutic or prophylactic use

The nucleic acid vaccines described herein can be used to protect, treat or cure infections caused by contact with infectious agents such as, but not limited to, viruses, bacteria, fungi, parasites and protozoa. As a non-limiting example, the infectious agent is a virus and the virus is SARS-CoV-2 and/or variants thereof. In some implementations, the variants of SARS-CoV-2 are VOI, VOC, and VOHC variants.

The nucleic acid vaccines described herein can be used as prophylactics, wherein the nucleic acid vaccine is administered to an individual, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 , for the prevention of COVID-19.

The nucleic acid vaccines described herein can be used as therapeutics, wherein the nucleic acid vaccine is administered to an individual, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 , used to relieve one or more symptoms of COVID-19.

In some embodiments, methods are provided for treating or preventing viral infections and/or diseases, disorders, or conditions associated with viral infections or symptoms thereof in an individual by administering a virus containing one or more encoded viruses Polypeptide-polynucleotide nucleic acid vaccine. Administration may be in combination with antiviral or antibacterial agents or small molecule compounds described herein or known in the art.

In some embodiments, the nucleic acid vaccines described herein may be used to protect against and/or prevent the spread of emerging or engineered threats that may be known or unknown.

In some embodiments, provided herein are the use of nucleic acid polynucleotides described herein to induce translation of one or more proteins, peptides, fragments or variants thereof of a polypeptide (e.g., SARS-CoV-2) in a cell, tissue, or organism. Methods. The translated polypeptides can be used to prevent, mitigate, and/or treat COVID-19. Such translation can be in vitro, in vivo, ex vivo, or in culture. Cells, tissues, or organisms can contain an effective amount of Contact with a composition or pharmaceutical composition of a nucleic acid vaccine that includes a polynucleotide having at least one region encoding a polypeptide of interest (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 , for the treatment and/or prevention of COVID-19.

In some embodiments, an effective amount of a nucleic acid vaccine described herein provided to a cell, tissue, or individual may be sufficient for immunoprophylaxis.

An "effective amount" of a composition of a nucleic acid vaccine is provided based at least in part on: the target tissue, the target cell type, the mode of administration, the physical characteristics of the polynucleotide (e.g., the size of the unmodified and modified nucleosides and number) and other components of the nucleic acid vaccine. An effective amount of a composition containing a nucleic acid vaccine described herein is provided as a function of one or more proteins, polypeptides, peptides, fragments or variants of SARS-CoV-2 produced in the cell compared to untreated cells. An amount that induces or enhances an immune response. Increased yield may be evidenced by increased cell transfection (ie, the percentage of cells transfected with the nucleic acid vaccine), increased protein translation from the polynucleotide, or altered host cell innate immune response.

Provided herein are methods for inducing in vivo translation of one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 in a mammalian subject in need thereof. An effective amount of a nucleic acid vaccine composition containing one or more proteins, polypeptides, peptides, fragments thereof, or variant) of the translatable region of the polynucleotide. The polynucleotide is provided in an amount and under other conditions such that the polynucleotide is translated in the cell. The cell in which the polynucleotide is located, or the tissue in which the cell resides, can be targeted with one or more rounds of nucleic acid vaccine administration.

In certain embodiments, administration of a nucleic acid vaccine comprising a polynucleotide directs the production of one or more polypeptides that provide a function that is substantially absent in the cell, tissue, or organism that translates the polypeptide. Sexual immune system related activity. For example, the missing functional activity may be enzymatic, structural, or gene-regulated in nature. In related embodiments, the polynucleotide administered directs the production of one or more polypeptides that increase functional activity associated with the immune system present but substantially lacking in cells that translate the polypeptides.

Additionally, polypeptides translated from nucleic acid vaccines may directly or indirectly antagonize the activity of biological moieties present in, on the surface of, or secreted from cells. Non-limiting examples of biological moieties that can be antagonized include nucleic acids, carbohydrates, protein toxins (such as Shiga and tetanus toxins), lipids (such as cholesterol), lipoproteins (such as low-density lipoprotein), or small molecule toxins (such as cholera, botulinum toxin and diphtheria toxin). In some embodiments, biomolecules that can be antagonized can be endogenous proteins, which may have undesirable activities, such as, but not limited to, cytotoxic or cytostatic activity. The proteins described herein can be engineered to localize within a cell, possibly within a specific compartment, such as the cytoplasm or nucleus, or engineered to be secreted from the cell or translocated to the cell's plasma membrane.

In some embodiments, the polynucleotides of nucleic acid vaccines and the polypeptides they encode can be used to treat any of a variety of diseases, disorders, and/or conditions, including but not limited to viral infections (e.g., caused by SARS-CoV-2 and/or infections caused by variants thereof).

Individuals to whom nucleic acid vaccines may be administered suffer or may be at risk of contracting a disease, illness or harmful condition. Methods for identifying, diagnosing, and classifying individuals on these basis are provided, which may include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.

The agents (eg, nucleic acid vaccine and any additional part of the composition) may be administered simultaneously, for example, in combined unit doses (eg, providing for simultaneous delivery of both agents). Agents may also be administered at specified intervals, such as, but not limited to, minutes, hours, days, or weeks. Generally speaking, an agent is simultaneously bioavailable, eg detectable, in an individual. In some embodiments, the agents may be administered substantially simultaneously, such as two unit doses administered simultaneously, or a combined unit dose of two agents. In other embodiments, the agent may be delivered in divided unit doses. The agents may be administered in any order, or as one or more formulations including two or more agents. In a preferred embodiment, at least one administration of one of the agents (e.g., the first agent) may occur within a few minutes, one hour, two hours, three hours, or four hours of the other agent (e.g., the second agent). Within hours, or even within a day or two. In some embodiments, the combination can achieve a synergistic result, for example, greater than the additive result, for example, at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than the additive result.

In some embodiments, the nucleic acid vaccines described herein can be administered with other prophylactic or therapeutic compounds. As non-limiting examples, prophylactic or therapeutic compounds may be adjuvants or potentiators. As used herein, when referring to a prophylactic composition, such as a vaccine, the term "booster" refers to the additional administration of the prophylactic composition. A booster dose (or booster vaccine) can be administered after an earlier administration of the prophylactic composition. The administration time between the initial administration of the preventive composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours , 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 4 months, 5 Month, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years , 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years.

In some embodiments, nucleic acid vaccines can be formulated by the methods described herein. In one aspect, the formulation may include a nucleic acid vaccine or polynucleotide that may have therapeutic and/or preventive effects on more than one disease, disorder or condition. As a non-limiting example, a formulation may include a polynucleotide encoding one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-19.

In some embodiments, the nucleic acid vaccines described herein can be used for research in many applications, such as, but not limited to, identification and localization of intracellular and extracellular proteins, protein interactions, signaling pathways, and cell biology. immune response modulation

In some embodiments, a nucleic acid vaccine comprising a polynucleotide described herein may serve as a single component in a vaccine form. As used herein, "vaccine" refers to a composition, substance or preparation that stimulates, induces, induces or enhances immunity in an organism, such as an animal organism, such as a mammalian organism (eg, a human). Preferably, the vaccine provides immunity against one or more diseases or conditions of the organism, including prophylactic and/or therapeutic immunity. Exemplary vaccines include one or more agents that resemble the infectious agent (e.g., a pathogenic microorganism) and may be made from, for example, live, attenuated, modified, attenuated, or inactivated forms of the pathogenic microorganism or antigens derived therefrom, including antigens combination of ingredients. In exemplary embodiments, a vaccine stimulates, induces, induces or enhances immunity in an organism or causes or simulates an infection in an organism without inducing any disease or disorder. Vaccines introduce antigens into the tissues, extracellular space, or cells of an individual and trigger an immune response, thereby protecting the individual from infection by a specific disease or pathogen. The nucleic acid vaccines described herein can encode an antigen and achieve a desired immune response when the polynucleotide is expressed in cells. As a non-limiting example, the nucleic acid vaccine described herein may encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2, and when the polynucleotide is expressed in cells, it can achieve the goal of targeting SARS-CoV-2. Required immune response to CoV-2 to treat and/or prevent COVID-19.

Nucleic acid vaccines may be administered prophylactically or therapeutically as part of an active immunization regimen in healthy individuals, or such nucleic acid vaccines may be administered early in the infection during the incubation phase or during active infection after the onset of symptoms.

The nucleic acid vaccines described herein may also be administered as second-line treatment after standard first-line treatments, such as antibiotics and antiviral agents, fail to induce passive immunity. In this regard, the nucleic acid vaccines described herein may be used in situations where resistance to first-line treatments has developed and disease persists and induces chronic disease.

Nucleic acid vaccines can be administered as part of a treatment regimen for latent viral infections, such as SARS-CoV-2 infection. In this embodiment, one or more polynucleotides are administered to treat and/or prevent COVID-19, which ultimately produce proteins that achieve the desired immune response against SARS-CoV-2.

The use of RNA in or as a vaccine overcomes the shortcomings (in terms of safety, feasibility, applicability and effectiveness in generating an immune response) of conventional genetic vaccinations involving the incorporation of DNA into cells. RNA molecules are considered much safer than DNA vaccines because RNA degrades more easily. It is rapidly cleared from the organism, cannot be integrated into the genome and affects the gene expression of cells in an uncontrollable manner. RNA vaccines are also less likely to cause serious side effects, such as the development of autoimmune diseases or anti-DNA antibodies ( Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), Vol. 2010, Article No. 623687). Transfection with RNA requires only insertion into the cytoplasm of the cell, which is easier to achieve than insertion into the nucleus. However, RNA is susceptible to RNase degradation and other natural breakdowns in the cell's cytoplasm.

Various attempts to increase the stability and shelf life of RNA vaccines. US Publication No. US 20050032730 issued to Von Der Mulbe et al. discloses improving the stability of the mRNA vaccine composition by increasing the G (guanosine)/C (cytosine) content of the mRNA molecule. US Patent No. 5,580,859 to Feigner et al. teaches the incorporation of polynucleotide sequences encoding regulatory proteins that bind to mRNA and modulate mRNA stability. While not wishing to be bound by theory, it is believed that the nucleic acid vaccines described herein may have improved stability and therapeutic efficacy due at least in part to the specificity, purity, and selectivity of the construct design. Additionally, modified nucleosides or combinations thereof can be introduced into the nucleic acid vaccines described herein to activate the innate immune response. Such activated molecules are suitable as adjuvants when combined with polypeptides and/or other vaccines. In certain embodiments, the activation molecule contains a translatable region encoding a polypeptide sequence useful as a vaccine, thereby providing the ability to act as a self-adjuvant.

In some embodiments, the nucleic acid vaccines described herein can be used to prevent, treat and diagnose diseases and diseases caused by infectious agents, such as but not limited to VOCs, VOIs or VOHCs of SARS-CoV-2 or SARS-CovV-2. obstacles. The nucleic acid vaccines described herein may encode at least one polypeptide of interest (eg, one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2) and may be provided to an individual to stimulate the immune system to protect against the pathogen. As non-limiting examples, biological activities and/or effects from infectious agents can be inhibited and/or eliminated by providing: neutralizing antibodies with the ability to bind and neutralize infectious agents; stimulation of polypeptides translated from nucleic acid vaccines Neutralizing antibodies produced by the immune system.

As non-limiting examples, polynucleotides encoding immunogens can be delivered to cells to trigger a variety of innate response pathways (see PCT Patent Application Publication No. WO2012006377 and United States Patent Publication No. US20130177639; the contents of each of these patents incorporated herein by reference in its entirety). As another non-limiting example, the nucleic acid vaccines described herein can be delivered to a vertebrate animal in a dose sufficient to be immunogenic for the vertebrate animal (see PCT Patent Application Publication Nos. WO2012006372 and WO2012006369 and U.S. Publication No. US20130149375 and US20130177640; each of which is incorporated herein by reference in its entirety).

In some embodiments, the nucleic acid vaccines described herein can be delivered to a mammal (eg, a human) in a dose large enough to be immunogenic for stimulating an immune response in the mammal. The immune response protects against viral infection, thereby preventing and/or treating disease. As non-limiting examples, the nucleic acid vaccines described herein can treat and/or prevent infectious diseases, including viral infectious diseases, such as COVID caused by SARS-CoV-2 or VOCs, VOIs or VOHCs of SARS-CoV-2. -19.

The nucleic acid vaccines described herein may be used in a variety of settings depending on the prevalence of infection or the extent or level of unmet medical need. As non-limiting examples, the nucleic acid vaccines described herein may be used to treat and/or prevent COVID-19 infections, including diseases associated with COVID-19 infections, including infections caused by original and mutant forms of SARS-CoV-2. and symptoms.

Symptoms of COVID-19 infection are changing as the disease is better understood, but current symptoms include fever or chills, cough, shortness of breath or difficulty breathing, fatigue, body aches, muscle aches, headache, sore throat , congestion or runny nose, nausea and/or vomiting, diarrhea, and new loss of taste or smell.

In some embodiments, the nucleic acid vaccines described herein may be better designed to produce the proper protein conformation upon translation than current antiviral treatments because the nucleic acid vaccines co-opt native cellular machinery. Unlike traditional vaccines, which are manufactured ex vivo and can trigger undesirable cellular responses, nucleic acid vaccines are presented to cellular systems in a more natural way. In some embodiments, the nucleic acid vaccine described herein is an active vaccine customized for COVID-19, which can not only prevent SARS-CoV-2 infection, but also limit the spread of SARS-CoV-2.

In some embodiments, the nucleic acid vaccines described herein may be used to prevent pandemic COVID-19 by responding to emerging viral strains with an extremely rapid nucleic acid-based vaccine production process.

In some implementations, a single injection of a nucleic acid vaccine can provide protection for an entire season.

In some embodiments, the nucleic acid vaccines described herein can be immunostimulatory. The polynucleotide sequence of the nucleic acid vaccine may further comprise a sequence region encoding an interleukin that promotes an immune response, such as a mononuclear hormone, a lymphokine, an interleukin or a chemokine, such as IL-1, IL- 2. IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-γ, GM-CFS, LT-a, or growth factors such as hGH. COVID-19 Treatment and/or Prevention

In some embodiments, the nucleic acid vaccines described herein encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 and can be used to treat and/or prevent COVID-19.

In some embodiments, the nucleic acid vaccines described herein can produce much higher titers of neutralizing antibodies and can produce responses earlier than commercially available antiviral agents. As a non-limiting example, the nucleic acid vaccines described herein can produce 10 times, or 9 times, or 8 times, or 7 times, or 6 times, or 5 times, or 4 times, or 3 times more neutralization than other vaccines. and antibody titers.

In some embodiments, the nucleic acid vaccines described herein co-opt native cellular machinery to produce the appropriate protein conformation upon translation. Unlike traditional vaccines, which are manufactured ex vivo and can trigger undesirable cellular responses, the nucleic acid vaccines described herein are introduced into cellular systems in a manner that is closer to nature or how normal cellular processing occurs. Additionally, the formulations can be used to protect or target the delivery of nucleic acid vaccines to specific cells or tissues of an individual.

In some embodiments, the nucleic acid vaccines described herein represent a targeted active vaccine that not only prevents infection but also limits the spread of COVID-19.

In some implementations, nucleic acid vaccines can be used to prevent pandemic SARS-CoV-2 infection or COVID-19 by responding to emerging viral strains with an extremely rapid vaccine production process.

In some embodiments, a single injection of a nucleic acid vaccine encoding one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 can provide protection for at least 6 months, at least 1 year, at least 2 years, At least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, at least 13 years, at least 15 years, or more 15 years.

The nucleic acid vaccines described herein may also be used to maintain or restore antigenic memory in individuals or populations as part of a vaccination program against COVID-19 or other diseases caused by SARS-CoV-2.

In some embodiments, nucleic acid vaccine compositions can be produced that include polynucleosides encoding one or more proteins, polypeptides, peptides, fragments, or variants thereof of SARS-CoV-2 that exhibit increased prevalence of year-round infection rates. acid. The protein sequence of SARS-CoV-2 has been shown to change or mutate over time, with some mutations showing increased infection rates. As a non-limiting example, nucleic acid vaccine compositions can be produced that include polynucleotides encoding one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 that exhibit increased prevalence of infection throughout the year. , such as but not limited to the D614G mutation in the spike protein.

In some embodiments, nucleic acid vaccines can be used to induce neutralizing antibodies in individuals. The neutralizing activity of the neutralizing antibodies induced by the nucleic acid vaccines of the invention can be correlated with the resulting effectiveness (eg, immune protection) of the vaccines described herein. In some aspects, the immune protection provided by the nucleic acid vaccines of the present invention may be greater than the immune protection provided in convalescent individuals. The neutralizing activity induced by the nucleic acid vaccine of the present invention can improve the recovery rate of individuals exposed to SARS-CoV-2 or in the recovery phase. Compared to other available mRNA vaccines, such as BNT162b2 and mRNA-1273, the nucleic acid vaccines of the present invention induce higher neutralizing antibody titers in treated individuals. In some embodiments, the nucleic acid vaccine includes a neutralizing antibody titer that is at least 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, or 4 times higher than other COVID-19 vaccines.

In some implementation aspects, vaccination protocols or plans are developed that not only allow for vaccination in the current year, but also allow for memory booster vaccination across years, strains, or groups thereof to establish and maintain within the population. memory. In this way, groups are less likely to succumb to any pandemic or outbreak involving the resurgence of old strains of the virus. Memory booster vaccines can be created or designed using any combination of previous vaccine component strains.

In some implementations, the nucleic acid vaccine as a memory booster vaccine is administered at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 years, or more than 50 years to enhance memory.

In some implementations, the nucleic acid vaccine is administered as a memory booster vaccine to enhance memory of alternate historical years, including every other year of past vaccine component strains relative to the current year. In some embodiments, the selection of vaccine components may be from every 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more years.

In some implementations, a nucleic acid vaccine is administered as a memory booster vaccine to enhance memory over a ten-year period.

In some implementations, nucleic acid booster vaccines can be administered to a population once or regularly to generate herd immunity, which means that greater than 30% of the population is protected.

In some implementations, a nucleic acid booster vaccine can be administered to a population once or regularly to generate herd immunity against COVID-19, meaning that greater than 30% of the population is protected.

In some embodiments, nucleic acid vaccines are used to target risk groups for COVID-19, such as those with pre-existing conditions including, but not limited to, cancer; chronic kidney disease; chronic obstructive pulmonary disease (COPD); from solid organs Transplant, blood or bone marrow transplant, immunodeficiency, HIV, and immunocompromised states (weakened immune system) using corticosteroids or other immune-weakening drugs; obesity (body mass index (BMI) 30 or higher); heart disease symptoms, Such conditions as heart failure, coronary artery disease, or cardiomyopathy; sickle cell disease; type 1 or type 2 diabetes; asthma (moderate to severe); cerebrovascular disease; cystic fibrosis; hypertension or hypertension; neuropathy, Such as dementia, liver disease, pregnancy, pulmonary fibrosis, smoking, and thalassemia.

In some implementations, nucleic acid vaccines are used to protect health care workers at risk of exposure to SARS-CoV-2.

As a non-limiting example, the nucleic acid vaccine of the present disclosure includes an LNP-formulated polynucleotide encoding a full-length S protein (SEQ ID NO: 27) with D614G (referred to as "PTX-B"). Methods of inducing a protective immune response in an individual using a PTX-B vaccine are provided. A protective immune response protects an individual from viral infection, such as that of the original strain of SAR-CoV-2 and its variants. SARS-CoV-2 variants can be any VOC, VOI and/or VOHC strains. As non-limiting examples, the variants are alpha, beta or delta variants.

In some embodiments, the nucleic acid vaccine PTX-B is used to provide protection against alpha variants.

In some embodiments, the nucleic acid vaccine PTX-B is used to provide protection against beta variants.

In some embodiments, the nucleic acid vaccine PTX-B is used to provide protection against delta variants.

In some embodiments, the nucleic acid vaccine PTX-B is used to induce neutralizing antibodies in an individual. The nucleic acid vaccine PTX-B induces antibodies with high neutralizing titers. For example, the neutralizing titer of the induced antibody is at least 100 times, 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, the titer of serum from an infected individual or a convalescent individual. 25x, 20x, 15x, 10x, or 5x.

In some embodiments, dosing regimens for the nucleic acid vaccine PTX-B are provided. Doses of PTX-B range from 1 µg to 500 µg, 1 µg to 1 mg, 1 mg to 10 mg, 1 mg to 100 mg, or 10 mg to 100 mg per kg of individual body weight. In some examples, the dose of PTX-B can be about 10 µg, 20 µg, 25 µg, 30 µg, 35 µg, 40 µg, 45 µg, 50 µg, 60 µg, 70 µg, 80 µg, 90 µg, or Dose level of 100 µg nucleic acid vaccine. Administer at least one dose of the nucleic acid vaccine PTX-B. In some embodiments, at least one booster dose of PTX-B is administered. A booster dose can be administered to the subject one month, two months, three months, four months, six months, or one year or more after the subject receives the first dose of PTX-B. In some cases, more than one, such as two, three, four, or more than four booster doses are administered to an individual.

In another embodiment, the nucleic acid vaccine of the present disclosure includes an LNP-formulated polynucleotide encoding the full-length S of SARS-CoV-2 variant B.1.351 (South African variant (SEQ ID NO: 43)). protein. V. Kits and Devices set

The present disclosure provides various sets of methods for carrying out the present disclosure conveniently and/or efficiently. Typically, a kit will contain an amount and/or number of components sufficient to allow the user to perform multiple treatments on an individual and/or conduct multiple experiments.

In some embodiments, the disclosure provides a kit for modulating gene expression in vitro or in vivo, which includes a nucleic acid vaccine composition of the disclosure or a nucleic acid vaccine composition of the disclosure, modulating other genes, siRNA, miRNA, or Combinations of nucleic acid vaccine compositions with other oligonucleotide molecules.

The kit may further comprise packaging and instructions and/or delivery agents to form a formulation, eg, for administration to an individual in need of treatment with a nucleic acid vaccine composition described herein. Delivery agents may include physiological saline, buffered solutions, lipidoids, dendrimers, or any suitable delivery agent.

In one non-limiting example, the buffer solution may include sodium chloride, calcium chloride, phosphate, and/or EDTA. In another non-limiting example, the buffer solution may include, but is not limited to, normal saline, normal saline with 2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% mannitol, 2 mM calcium Calcium 5% mannitol, Ringer's lactate, sodium chloride, sodium chloride with 2 mM calcium, and mannose (see U.S. Publication No. 20120258046; incorporated herein by reference in its entirety) middle). In another non-limiting example, the buffer solution can be precipitated or it can be lyophilized. The amounts of each component can be varied to achieve consistent, reproducible higher concentration saline or single buffer formulations. The components may also be altered to increase the stability of the nucleic acid vaccine composition in buffer solutions over time and/or under various conditions. device

The present disclosure provides devices that may incorporate the nucleic acid vaccine compositions of the present disclosure. Such devices may contain stable formulations for immediate delivery to individuals in need, such as human patients.

Non-limiting examples of devices include pumps, catheters, needles, transdermal patches, pressurized olfactory delivery devices, electroporation devices, iontophoresis devices, multilayer microfluidic devices. The device can be used to deliver the nucleic acid vaccine compositions of the present disclosure according to a single, multiple or divided dosing regimen. Such devices can be used to deliver the nucleic acid vaccine compositions of the present disclosure to biological tissue, intradermally, subcutaneously or intramuscularly. Further examples of devices suitable for delivering oligonucleotides are disclosed in International Publication WO 2013/090648, the contents of which are incorporated herein by reference in their entirety. VI. Definition

At various locations throughout this specification, substituents of the compounds of the present disclosure are disclosed in groups or ranges. In particular, this disclosure includes each individual subgroup of members of such groups and ranges.

Approximately: As used herein, the term "approximately" means +/-10% of the stated value.

Administering in Combination: As used herein, the term "administering in combination" or "administering in combination" means administering two or more agents to an individual simultaneously or within a time interval such that each agent is effective for the patient Their roles may overlap. In some embodiments, each agent is administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the agents are administered sufficiently closely spaced together to achieve a combined (eg, synergistic) effect.

Adjuvant: As used herein, the term "adjuvant" means a substance that enhances an individual's immune response to an antigen. The nucleic acid vaccines described herein optionally include one or more adjuvants.

Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some implementations, "animal" refers to a human being at any stage of development. In some implementations, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, or a purebred animal.

Antigen: As defined herein, the term "antigen" refers to a composition, such as a substance or agent, that causes an immune response in an organism, such as an immune response in an organism to produce antibodies against the substance or agent, particularly in Causes adaptive immune responses in organisms. An antigen can be any immunogenic substance, including, inter alia, proteins, polypeptides, polysaccharides, nucleic acids, lipids, and the like. Exemplary antigens are derived from infectious agents. Such agents may include parts or subunits of the infectious agent, such as the coat of the infectious agent, such as bacteria, viruses, and other microorganisms; coat components, such as coat proteins or polypeptides; surface components, such as surface proteins or polypeptides; capsular components; cell walls. components; flagella; pili; and/or toxins or toxoids). Certain antigens, such as lipids and/or nucleic acids, are antigenic, preferably when combined with proteins and/or polysaccharides.

Approximately: As used herein, the term "approximately" or "approximately" as applied to the value or values in question means a value similar to the stated reference value. In certain embodiments, unless stated otherwise or otherwise apparent from context, the term "approximately" or "approximately" means in either direction (greater or less) 25%, 20%, 19% of the stated reference value , 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 %, 1%, or a range of values within a smaller percentage (except where such values would exceed 100% of possible values).

Associated with: As used herein, the terms "associated with", "bonded", "connected", "attached" and "tethered" when used with respect to two or more parts mean The moieties are physically associated or linked to each other, either directly or via one or more additional moieties that act as linkers, to form a structure that is sufficiently stable such that the moieties remain under the conditions in which the structure is used (e.g., physiological conditions) Physically associated. "Association" need not be strictly through direct covalent chemical bonding. It may also mean that ionic or hydrogen bonding or hybridization-based connectivity is sufficiently stable such that the "associated" entities remain physically associated.

Bifunctionality: As used herein, the term "bifunctional" refers to any substance, molecule or moiety capable of having or maintaining at least two functions. These features may achieve the same results or different results. The structures producing the functions may be the same or different.

Biocompatibility: As used herein, the term "biocompatibility" means compatible with living cells, tissues, organs or systems without causing virtually any risk of damage, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term "biodegradable" means capable of breaking down into harmless products by the action of living organisms.

Biological Activity: As used herein, the phrase "biological activity" refers to any characteristic of a substance that is active in biological systems and/or organisms. For example, a substance is considered to be biologically active if it has a biological effect on an organism when administered to that organism. In certain embodiments, a polynucleotide described herein is considered biologically active if even a portion of the polynucleotide is biologically active or mimics an activity considered biologically relevant.

Chimera: As used herein, a "chimera" is an entity having two or more incongruous or heterogeneous parts or regions.

Compound: As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers and isotopes of the depicted structure.

The compounds described herein may be asymmetric (eg, have one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods are known in the art for preparing optically active forms from optically active starting materials, such as by resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of alkenes, C=N double bonds, and the like may also exist in the compounds described herein, and all such stable isomers are encompassed by this disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as mixtures of isomers or in separate isomeric forms.

Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the exchange of single bonds with adjacent double bonds and the concomitant migration of protons. Tautomeric forms include proton transfer tautomers in isomeric protonation states with the same empirical formula and overall charge.

Compounds of the present disclosure also include all isotopes of atoms present in intermediates or final compounds. "Isotopes" are atoms with the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared by conventional methods in combination with solvents or water molecules to form solvates and hydrates.

Conserved: As used herein, the term "conserved" means that the nucleotide or amino acid residues of a polynucleotide sequence or a polypeptide sequence, respectively, are unchanged at the same position in two or more sequences being compared. Nucleotide or amino acid residues. Relatively conserved nucleotides or amino acids are conserved nucleotides or amino acids in a sequence that are more closely related to nucleotides or amino acids occurring elsewhere in the sequence.

In some embodiments, two or more sequences are said to be "completely conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. "Highly conservative". In some embodiments, if two or more sequences are at least 30% identical to each other, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical , or at least 95% identical, it is called "conservative". In some embodiments, if two or more sequences are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical to each other, , about 95% identical, about 98% identical, or about 99% identical, it is called "conservative". Sequence conservation may apply to the entire length of a polynucleotide or polypeptide or may apply to a portion, region, or feature thereof.

Controlled release: As used herein, the term "controlled release" refers to the release characteristics of a pharmaceutical composition or compound that conform to a specific release pattern to achieve a therapeutic effect.

Cytostatic: As used herein, "cytostatic" means inhibiting, reducing, inhibiting cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or combinations thereof To grow, divide or add value.

Cytotoxicity: As used herein, "cytotoxicity" means the killing or destruction of cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or combinations thereof Cause harmful, toxic or fatal effects.

Delivery: As used herein, "delivery" means the act or manner of delivering a compound, substance, entity, part, load or payload.

Delivery Agent: As used herein, "delivery agent" refers to any substance that facilitates, at least in part, the delivery of a polynucleotide to a targeted cell in vivo.

Destabilization: As used herein, the term "destabilization," "destabilization," or "destabilized region" means rendering a region or molecule less stable than the starting, wild-type, or native version of the same region or molecule. Small form.

Detectable Marker: As used herein, "detectable marker" means one or more markers, signals, or moieties attached to, incorporated into, or associated with another entity that is An entity is readily detectable by methods known in the art, including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands (such as biotin, avidin, streptavidin, and haptens), quantum dots, and the like things. The detectable label can be located anywhere in the peptides or proteins disclosed herein. It can be within an amino acid, peptide or protein, or located at the N- or C-terminus.

Decompose: As used herein, the term "decompose" means breaking into smaller fragments or components. When referring to polypeptides or proteins, digestion results in the production of the peptide.

Dosage Regimen: As used herein, a "dosage regimen" is a schedule of dosing or a regimen of therapeutic, preventive, or palliative care determined by a physician.

Encapsulate: As used herein, the term "encapsulate" means to enclose, surround or envelop.

Encoded protein cleavage signal: As used herein, "encoded protein cleavage signal" refers to a nucleotide sequence encoding a protein cleavage signal.

Engineered: As used herein, when embodiments of a nucleic acid vaccine are designed to have characteristics or properties (whether structurally or chemically) that are different from the starting point, wild-type, or native molecule, such embodiments are " Engineering".

Effective Amount: As used herein, the term "effective amount" of a pharmaceutical agent is an amount sufficient to achieve a beneficial or desired result, such as a clinical outcome, and thus "effective amount" depends on the situation in which it is used. For example, in the case of administration of an agent to treat cancer, an effective amount of the agent is an amount sufficient to effect treatment of the cancer as defined herein, eg compared to the response obtained without administration of the agent.

Exosomes: As used herein, "exosomes" are vesicles secreted by mammalian cells or complexes involved in RNA degradation.

Representation: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing) , editing, 5' end cap formation and/or 3' end processing); (3) translation of RNA into polypeptides or proteins; and (4) post-translational modification of polypeptides or proteins.

Feature: As used herein, "feature" means a characteristic, characteristic, or unique element.

Formulation: As used herein, "formulation" includes at least one polynucleotide of a nucleic acid vaccine and a delivery agent.

Fragment: As used herein, "fragment" means a portion. For example, fragments of a protein may include polypeptides obtained by cleaving full-length proteins isolated from cultured cells.

Functional: As used herein, a "functional" biomolecule is a form of biomolecule that exhibits properties and/or activities that are characterized by that property and/or activity.

Homology: As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. . In some embodiments, if the sequence of the polymer molecules is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, If they are 85%, 90%, 95%, or 99% identical or similar, they are considered to be "homogeneous" to each other. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). If for at least one extension having at least about 20 amino acids, the polypeptide encoded by the two polynucleotide sequences is at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% If they are consistent, they are considered to have the same origin. In some embodiments, homologous polynucleoside sequences are characterized by encoding a sequence segment having at least 4 to 5 specifically designated amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4 to 5 specifically designated amino acids. Two protein sequences are considered homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, such as between polynucleotide molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules.

For example, calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences). for optimal alignment and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the length of the reference sequence. %, at least 95%, or 100%. The nucleotides at corresponding nucleotide positions are then compared. The molecules are identical at a position in the first sequence when it is occupied by the same nucleotide as the corresponding position in the second sequence. The percent identity between two sequences is related to the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to obtain an optimal alignment of the two sequences. Mathematical algorithms can be used to achieve sequence comparison and determine the percent identity between two sequences. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in: Computational Molecular Biology, Lesk, A. M, ed., Oxford University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, D. W., Academic Press, N.Y., 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M and Griffin, H. G., eds., Humana Press, N.J., 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, N.Y., 1991, each of which is cited in its entirety. method is incorporated into this article. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated using the PAM 120 weighted residue table, In the ALIGN program (version 2.0) with a gap length penalty of 12 and a gap penalty of 4. Alternatively, the GAP program in the GCG suite of software can be used to determine the percent identity between two nucleotide sequences using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated by reference. into this article. The techniques used to determine consistency are encoded in publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG suite (Devereux, J. et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN , and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

Infectious agent: As used herein, the phrase "infectious agent" means an agent capable of producing an infection in an organism, such as an animal. Infectious agent may refer to any microorganism, virus, infectious agent or biological product that may be biotechnologically engineered, or any naturally occurring or bioengineered component of any such microorganism, virus, infectious agent or biological product that may cause disease in humans , new and infectious diseases, death or other biological malfunctions of animals, plants or another living organism.

In vitro: As used herein, the term "ex vivo" refers to an occurrence in an artificial environment (e.g., in a test tube or reaction vessel, in a cell culture, a Petri dish, etc.) rather than in an organism (e.g., animals, plants or microorganisms).

In vivo: As used herein, the term "in vivo" refers to events that occur in an organism, such as an animal, plant, or microorganism, or its cells or tissues.

Isolated: As used herein, the term "isolated" means that a substance or entity has been separated from at least some of the components with which it is associated, whether in nature or in an experimental setting. Isolated substances can have different levels of purity relative to the substances with which they were associated. At least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% of other components with which the isolated substance and/or entity can be originally associated , approximately 90%, or greater separation. In some embodiments, the separated reagents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97 %, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" when it is substantially free of other components. Substantially Isolated: "Substantially isolated" means that a compound is substantially separated from the environment in which it was formed or detected. Partial isolation may include, for example, compositions enriched in compounds disclosed herein. Substantially separating may include containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least A composition of about 99% by weight of a compound of the present disclosure or a salt thereof. Methods for isolating compounds and their salts are known as processes in the art.

Linker: As used herein, "linker" refers to a group of atoms, for example, from about 10 to 1,000 atoms, and may include, but is not limited to, carbon, amine, alkylamino, oxygen, sulfur, trisine, sulfonate Atoms or groups of acyl groups, carbonyl groups, and imines. The linker can be attached at a first end to a modified nucleoside or nucleotide on a nucleobase or sugar moiety and at a second end to a payload, such as a detectable or therapeutic agent. The linker can be of sufficient length so as not to interfere with incorporation into the nucleic acid sequence.

Modified: As used herein, "modified" means changing the state or structure of a molecule described herein. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally.

Mucus: As used herein, "mucus" refers to a naturally occurring substance that is viscous and contains mucin glycoproteins.

Naturally occurring: As used herein, "naturally occurring" means existing in nature without artificial assistance.

Neutralizing Antibody: As used herein, "neutralizing antibody" refers to an antibody that binds to its antigen and protects cells from the antigen or infectious agent by neutralizing or eliminating any biological activity possessed by the antigen or infectious agent.

Non-human vertebrates: As used herein, "non-human vertebrates" includes all vertebrates except Homo sapiens, including wild and domestic species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, banteng, bison, camels, cats, cattle, deer, dogs, donkeys, gayals, goats, guinea pigs, horses, Llama, mule, pig, rabbit, reindeer, sheep, buffalo, and yak.

Nucleic acid vaccine: As used herein, "nucleic acid vaccine" refers to a vaccine or vaccine composition that includes a nucleic acid or nucleic acid molecule (eg, a polynucleotide) encoding an antigen (eg, an antigenic protein or polypeptide). In exemplary embodiments, nucleic acid vaccines include ribonucleic acid ("RNA") polynucleotides, ribonucleic acid ("RNA") or ribonucleic acid ("RNA") molecules. Such implementations may be referred to as ribonucleic acid ("RNA") vaccines.

Off-target: As used herein, "off-target" refers to any unintended effect on any one or more targets, genes, or cellular transcripts.

Open Reading Frame: As used herein, the term "open reading frame" or "ORF" refers to a contiguous polynucleotide sequence, such as a DNA sequence or an RNA sequence (e.g., an mRNA sequence), which contains an initiation codon, contains a plurality of amine groups The subsequent region of the acid-coding codon and the terminal stop codon, and the region containing multiple amino acid-coding codons does not contain a stop codon.

Operably linked: As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, portions, or the like.

Portion: As used herein, a "portion" or "region" of a polynucleotide is defined as any portion of a polynucleotide that is less than the entire length of the polynucleotide.

Peptide: As used herein, a "peptide" is less than or equal to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

Paratope: As used herein, "paratope" refers to the antigen-binding site of an antibody.

Patient: As used herein, "patient" means an individual who may seek or need treatment, request treatment, be receiving treatment, be about to receive treatment, or be cared for by a trained professional for a specific disease or condition.

Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is used herein to mean, within the scope of reasonable medical judgment, suitable for contact with human and animal tissue without undue toxicity, irritation, allergic reaction or other problems or Complications, those compounds, materials, compositions, and/or dosage forms that are matched with a reasonable benefit/risk ratio.

Pharmaceutically acceptable excipient: As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compound described herein (e.g., a vehicle capable of suspending or dissolving the active compound ), and has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example: anti-adhesive agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming agents Or coatings, flavoring agents, fragrances, slip agents (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dicalcium phosphate), calcium stearate, croscarmellose, crospolyvinylpyrrolidone , citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, Methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, Retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A , vitamin E, vitamin C, and xylitol.

Pharmaceutically Acceptable Salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is converted into its salt form by converting an existing acid or base moiety (e.g., by reacting the free base with a suitable organic acid reaction) to modify. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. . Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate , butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, ethane sulfonate, fumarate , Glucoheptonate, Glycerophosphate, Hemisulfate, Enanthate, Caproate, Hydrobromide, Hydrochloride, Hydroiodide, 2-Hydroxy-Ethanesulfonate, Lactonate , lactate, laurate, lauryl sulfate, malate, maleate, malonate, methane sulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oil Acid salt, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, Stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, Methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include, for example, conventional nontoxic salts of the parent compound formed from nontoxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts may be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally speaking, non-aqueous media are preferred, such as diethyl ether, ethyl acetate, ethanol, isopropyl alcohol, or acetonitrile. A list of suitable salts is found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, page 1418; Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley- VCH, 2008, and Beige et al., Journal of Pharmaceutical Science, 66, 1-19 (1977); each of these documents is incorporated by reference in its entirety.

Pharmaceutically acceptable solvate: As used herein, the term "pharmaceutically acceptable solvate" means a compound described herein in a crystal lattice with suitable solvent molecules. Suitable solvents are physiologically tolerable at the doses administered. For example, solvates can be prepared by crystallization, recrystallization, or precipitation from solutions including organic solvents, water, or mixtures thereof. Examples of suitable solvents are ethanol, water (eg monohydrate, dihydrate and trihydrate), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), N,N'-dimethyl Methylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3, 4,5,6-Tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like things. When water is the solvent, the solvate is called a "hydrate".

Pharmacokinetics: As used herein, "pharmacokinetics" refers to any one or more properties of a molecule or compound involved in determining the outcome of a substance administered to a living organism. Pharmacokinetics is divided into several areas, including the extent and rate of absorption, distribution, metabolism, and excretion. This is often called ADME, where: (A) Absorption is the process by which a substance enters the blood circulation; (D) Distribution is the dispersion or diffusion of a substance throughout body fluids and body tissues; (M) Metabolism (or biotransformation) into the parent compound Irreversible transformation into daughter metabolites; and (E) Excretion (or elimination) means the elimination of a substance from the body. In rare cases, some drugs accumulate irreversibly in body tissues.

Physicochemical: As used herein, "physically chemical" means having or relating to physical and/or chemical properties.

Polypeptides per unit drug (PUD): As used herein, PUD or product per unit drug is defined as the total daily dose (usually 1 mg, pg, kg) of a product (such as a peptide) measured in a body fluid or tissue etc.) divided by the measured value in the body fluid.

Prevention: As used herein, the term "prevention" means to partially or completely delay the onset of an infection, disease, disorder and/or condition; to partially or completely delay one or more of a specific infection, disease, disorder and/or condition. The onset of symptoms, characteristics or clinical manifestations; the partial or complete delay in the onset of one or more symptoms, characteristics or manifestations of a particular infection, disease, disorder and/or condition; the partial or complete delay in the onset of an infection, a particular disease, disorder and/or condition or progression of conditions; and/or reduce the risk of developing lesions related to infections, diseases, conditions and/or conditions.

Proliferation: As used herein, the term "proliferation" means to grow, amplify or increase or to cause rapid growth, amplification or increase. "Proliferative" means having the ability to proliferate. "Antiproliferative" means having properties that are opposite or disproportionate to proliferative properties.

Prophylactic: As used herein, "prophylactic" refers to a treatment or course of action used to prevent the spread of disease.

Prevention: As used herein, "prevention" refers to measures taken to maintain health and prevent the spread of disease. "Immunoprophylaxis" refers to measures to generate active or passive immunity to prevent the spread of disease.

Protein cleavage site: As used herein, "protein cleavage site" refers to a site where controlled cleavage of an amino acid chain can be achieved by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein, "protein cleavage signal" refers to at least one amino acid that identifies or labels a polypeptide for cleavage.

Protein of Interest: The term "protein of interest" or "protein of interest" as used herein includes the proteins provided herein and fragments, mutants, variants and altered forms thereof.

Purified: As used herein, "purify," "purified," or "purification" means becoming substantially free from undesired components, material contaminants, contaminants, or defects. pure or clean.

Repeat transfection: As used herein, the term "repeat transfection" refers to transfecting the same cell culture multiple times with a polynucleotide. Cell cultures can be transfected at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, At least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times times, at least 50 times, or more.

Sample: As used herein, the term "sample" or "biological sample" refers to a subgroup of its tissues, cells, or components (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, Amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). Samples may further include homogenates, lysates or extracts prepared from intact organisms or subsets of tissues, cells or component parts thereof, or fractions or portions thereof, including but not limited to, for example, plasma; serum; spinal fluid; lymph fluid; external sections of skin, respiratory tract, intestine and genitourinary tract; tears; saliva; breast milk; blood cells; tumors; organs. A sample also refers to a culture medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

Signal sequence: As used herein, the phrase "signal sequence" refers to a sequence that directs the transport or localization of a protein.

Single Unit Dose: A "single unit dose" as used herein is a dose of any therapeutic agent administered in one dose/single time/single route/single point of contact, ie, a single administration event.

Similarity: As used herein, the term "similarity" refers to the overall relatedness between polymeric molecules, such as between polynucleotide molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. The calculation of the percent similarity of polymer molecules to one another can be performed in the same manner as the calculation of the percent identity, except that the calculation of the percent similarity takes into account conservative substitutions as understood in the art.

Divided dose: As used herein, "fractionated dose" is the division of a single unit dose or total daily dose into two or more doses.

Stable: As used herein, "stable" means a compound that is stable enough to withstand isolation from the reaction mixture to a suitable purity and preferably can be formulated into an effective therapeutic agent.

Stabilization: As used herein, the terms "stabilize," "stabilize," and "stabilize region" mean to stabilize or become stable.

Subject: As used herein, the term "subject" or "patient" refers to any organism to which a composition may be administered, for example, for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical individuals include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans).

Substantially: As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the limits or degrees of the characteristic or characteristic of interest. It is understood by those of ordinary skill in biotechnology that biological and chemical phenomena rarely, if ever, proceed completely and/or continue to completion or that absolute results are rarely achieved or avoided. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein, this term means plus/minus 2% as it relates to the time difference between doses.

Substantially simultaneously: As used herein and when it relates to a plurality of doses, the term means within 2 seconds.

Suffering: An individual "suffering from" a disease, disorder and/or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder and/or condition.

Vulnerable: An individual who is "susceptible" to a disease, disease and/or condition has not yet been diagnosed with the disease, disease and/or condition and/or may not exhibit symptoms of the disease, disease and/or condition, but has a tendency to develop the disease, disease and/or condition or develop its symptoms. . In some embodiments, characteristics of an individual susceptible to a disease, disorder, and/or condition (e.g., cancer) may be one or more of the following: (1) Characteristics associated with developing the disease, disorder, and/or condition; Gene mutations; (2) Genetic polymorphisms associated with the development of diseases, diseases and/or symptoms; (3) The expression and/or activity of proteins and/or nucleic acids associated with the development of diseases, diseases and/or symptoms Increases and/or decreases; (4) Habits and/or lifestyle associated with developing diseases, illnesses and/or conditions; (5) Family history of diseases, illnesses and/or conditions; and (6) Exposure to Microorganisms associated with the development of diseases, illnesses and/or conditions and/or infection by such microorganisms. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Sustained Release: As used herein, the term "sustained release" refers to the release characteristics of a pharmaceutical composition or compound consistent with a release rate over a specified period of time.

Synthetic: The term "synthetic" means produced, prepared and/or manufactured by artificial means. The synthesis of polynucleotides or polypeptides or other molecules described herein may be chemical synthesis or enzymatic synthesis.

Vaccine: As used herein, a vaccine is a compound or composition comprising at least one polynucleotide encoding at least one antigen.

Target Cell: As used herein, "target cell" refers to any relevant cell or cells. These cells can be found in vitro, in vivo, in situ or in tissues or organs of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

Therapeutic Agent: The term "therapeutic agent" refers to any agent that, when administered to an individual, has therapeutic, diagnostic and/or prophylactic effects and/or induces a desired biological and/or pharmacological effect.

Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" means an amount that is sufficient to treat an infection, disease, disorder, and/or condition when administered to an individual suffering from or susceptible to the infection, disease, disorder, and/or condition. The amount of a delivered agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that ameliorates symptoms, diagnoses them, prevents them, and/or delays their onset.

Therapeutically Effective Result: As used herein, the term "therapeutically effective result" means sufficient to treat, ameliorate, or ameliorate an infection, disease, disorder, and/or condition in an individual suffering from, or susceptible to, the infection, disease, disorder, and/or condition. symptoms, diagnosing them, preventing and/or delaying their onset as a result.

Total Daily Dose: As used herein, the "total daily dose" is the amount administered or prescribed within a 24-hour period. It can be administered in a single unit dose.

Transfection: As used herein, the term "transfection" refers to a method of introducing exogenous nucleic acid into a cell. Transfection methods include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

Translation: As used herein, "translation" is the process by which polynucleotide molecules are processed by ribosomes or ribosome-like mechanisms, such as cells or humans, to produce peptides or polypeptides.

Transcription: As used herein, "transcription" is the process whereby a polynucleotide molecule is processed by a polymerase or other enzyme to produce a polynucleotide, such as an RNA polynucleotide.

Treatment: As used herein, the term "treatment" means partial or complete relief, amelioration, amelioration, alleviation, delaying the onset, inhibiting the progression, reducing the severity and/or reducing the severity of a particular infection, disease, disorder and/or condition. The incidence of one or more of its symptoms or characteristics. For the purpose of reducing the risk of developing pathology associated with a disease, infection, illness and/or condition, individuals who do not exhibit symptoms of the disease, infection, illness and/or condition and/or individuals who only exhibit symptoms of the disease, infection, illness and/or condition may be Treatment of individuals with early signs of the disease, infection, disorder and/or condition.

Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule that has not been altered in any way. Unmodified can refer to, but does not always refer to, the wild-type or native form of a biomolecule. Molecules can undergo a series of modifications, whereby each modified molecule can serve as the "unmodified" starting molecule for subsequent modifications.

Vaccine: As used herein, the phrase "vaccine" refers to a biological agent that improves immunity in the context of a specific disease, disorder or condition.

Viral protein: As used herein, the phrase "viral protein" means any protein derived from a virus. Example Example 1. In vivo study of LNP formulated mRNA

Five (5) groups of female C57bl/6 mice, 8 mice/group (6 weeks old) were dosed with the formulations as described in Table 6. Mice were bled on Days 0 and 21 and subsequently received 20 micrograms ("µg") of one of the formulations of Table 6 via intramuscular administration on Days 1 and 22. On day 43, mice were euthanized, and blood was collected by cardiac puncture, spleens were collected, and spleen cells were isolated. Table 7 lists the lipid components of lipid nanoparticles (LNP) used to formulate mRNA. Table 6. Table of preparations group Description of the blend 1 LNP formulated TdTomato mRNA negative control (SEQ ID NO: 46) 2 LNP formulated S protein receptor binding domain (RBD) mRNA (SEQ ID NO: 25) 3 LNP-formulated full-length S protein mRNA ("PTX-B") (SEQ ID NO: 27) 4 LNP formulates full-length S protein with mutated furin site mRNA (SEQ ID NO: 26) 5 Dulbecco's Phosphate-Buffered Saline (DPBS) control Table 7. LNP components Lipids Mol% in formulation Mass % in formulation cationic lipids 49.96 52.26 Phospholipids or DSPC 9.97 14.02 sterol or cholesterol 38.44 26.44 Pegylated lipids or PEG-DMA 1.61 7.26

Clinical isolate virus neutralization analysis was performed on pooled samples from day 21 ex vivo blood draws. At termination (day 43), serum antibody binding to SARS-CoV-2 RBD and S protein, pseudovirus neutralization, clinical isolate virus neutralization, and T cell responses were measured (by enzyme-linked immunospot (ELIspot) and flow cytometry).

There were no obvious adverse reactions in mice. Data from the initial preliminary blood draw showed neutralizing activity for all three constructs. Group 3 formulations (full-length S protein mRNA) were the best, closely followed by Group 4 formulations (full-length S protein with mutated furin sites); Group 2 formulations (S protein RBD domain) performed the worst; and no activity was seen in any of the negative control treatment groups.

Splenocytes were stimulated with SARS-CoV antigen (RBD peptide pool plus S protein), and antigen-specific T cell responses were generated by counting IFN-γ-secreting T cells by ELISpot or by counting Th1 cells by flow cytometry. Interleukins (IFN-γ/TNF-α/IL-2) and Th2 interleukins (IL-4/IL-5) are measured in T cells. Th1 interleukins (IFN-γ/TNF-α/IL-2) and Th2 interleukins (IL-2) were also measured in the supernatants of SARS-CoV antigen-stimulated T cells by multiplex mouse interleukin analysis. -4/IL-5). ELISpot analysis

ELISpot analysis demonstrated that splenocytes from mice treated with the Group 2, 3, and 4 formulations produced T cells that secreted IFN-γ in response to a pool of peptides located between the S protein There are overlapping peptides in the RBD and conserved S2 regions. The responses of Groups 2, 3 and 4 were similar, and neither control group showed a significant response to the peptide pool. Flow cytometry analysis detected no significant Th1 or Th2 responses in the three groups compared with the two control groups.

Direct binding ELISA was used to determine whether any antibodies were primed to bind to the S protein. The RBD domain or full-length S protein was bound to the plate, and different dilutions of serum from treated mice were incubated in the plate, followed by washing and detection by anti-mouse antibodies. Sera from groups 2, 3 and 4 were positive for antibodies that bind both RBD and full-length S protein, and controls were negative for binding. SARS-CoV-2 Neutralization Assay

To determine whether antibodies elicited from Group 2, Group 3 or Group 4 formulations were neutralizing, two different assays were used. The first analysis used SARS-CoV-2 virus isolated from one of the first COVID-19 patients in Ontario and read out different dilutions with live virus and serum from treated mice. Microscopic reading of the health of Vero2 E6 cells cultured together. This assay has been used to characterize sera from convalescent patients, with ID50 values ranging from 1:80 to 1:320, so the dilution series chosen to test these mouse sera was between 1:20 and 1:2560 . Sera from Group 1 mice had minimal detectable neutralizing activity. Group 2 showed some moderate activity, with 6 out of 8 samples having an ID50 between 1:20 and 1:80. Groups 3 and 4 showed strong neutralizing activity. Even at the highest dilution of 1:2560, most serum samples maintained 100% neutralizing activity. The results are provided in Figure 1. Pseudovirus neutralization analysis

The second neutralization assay consists of pseudovirus analysis. This assay utilizes SARS-CoV-2 S protein pseudotyped lentivirus, which encodes the luciferase gene and can infect HEK293T cells expressing hACE2 and TMPRSS2 to obtain better transduction efficiency. This analysis was characterized by the determination of serum titers from approximately 50 convalescent patients, where the mean ID50 of the sera was approximately 1:500, with a range of 1:1 to approximately 1:10,000, but for this study, 1:40 to Dilution range of 1:24,400. Sera from the two control groups (Group 1 and Group 5) had little or no activity. Group 2 had significant but lower neutralizing activity. The activities of Groups 3 and 4 were above the quantitative range of the analysis. Values were extrapolated and the average ID50 values were approximately 1:50,000 (Group 3) and approximately 1:45,000 (Group 4). The results are provided in Figure 2. Conclusion

This study shows that the LNP-formulated mRNA vaccine can induce T cell responses and antibodies that can bind to the S protein of SARS-CoV-2 when injected intramuscularly into mice twice within a three-week period. Treated mice produced antibodies that neutralized SARS-CoV-2 clinical isolates as well as SARS-CoV-2 pseudotyped lentiviruses. This was especially true for the Group 3 and 4 formulations, which resulted in potencies above the quantitation range in each assay. Example 2. Studies on neutralizing antibodies in mice

Vaccine candidate, LNP formulated vaccine encoding the full-length S protein with the D614G mutation (SEQ ID NO: 27; coding region provided as SEQ ID NO: 7) (applicable to vaccine formulations of Group 3 in Table 6, hereafter referred to as ("PTX-B") was selected as a candidate for further research. The ability of PTX-B to generate neutralizing antibodies and T cell responses in mice was evaluated. Three (3) groups of female C57BL/6 mice (10 mice/group) were vaccinated on days 1 and 22 as follows: Group 1: 10 μg LNP-prepared tdTomato mRNA (negative control) Group 2: 1 μg PTX-B Group 3: 10 μg PTX-B

Parameters evaluated in this study included clinically isolated virus neutralization assays and pseudovirus neutralization assays on pooled samples of live blood drawn three weeks after the first immunization (day 22); and the second immunization The following assessments at post-termination (day 43): binding of serum antibodies to the S protein of SARS-CoV-2, neutralization of pseudoviruses, neutralization of clinically isolated viruses, splenocyte T cells by ELISpot and flow cytometry response and cytokine secretion.

The mice showed no obvious adverse reactions during the in vivo phase of the experiment. Data from the initial preliminary blood draw on Day 22 showed that the 10 µg dose level produced neutralizing antibodies, while the 1 µg dose level was only slightly different from the negative control group.

At termination, three weeks after the second immunization (boost), the 1 μg and 10 μg dose groups showed approximately equal T cell responses in the ELISpot assay, but the 10 μg dose level group was inferior in the antibody-based analysis. performed much better, with evidence of higher levels of IgG isotypes (total IgG, IgG1, IgG2b and IgG2c). Mice dosed with 1 µg had higher IgM levels than mice dosed with 10 µg, possibly due to early class switching due to stronger stimulation in the 10 µg group. There was evidence of IgG, especially at the 10 µg dose, but this isotype was not induced to as high a level as the IgG isotype. Both 1 and 10 μg PTX-B elicited extremely strong S-specific IgG, IgG1, IgG2b, and IgG2c (the end-point titers of 1 and 10 μg PTX-B were respectively: 2.7±0.9E6 and 3.0±0.5E7 for IgG; 1.1±0.2E6, 2.8±0.8E6 for IgG1; 9.4±2.0E5, 9.7±3.4E6 for IgG2b; 3.5±1.8E7, 1.95±0.0E8 for IgG2c). Both 1 and 10 μg PTX-B also elicited strong S-specific IgA (the end-point titers of 1 and 10 μg PTX-B were: 3.3±3.1E4, 1.7±0.6E7 respectively), but the titers were lower than those of IgG. . A dose of 10 μg PTX-B generally induced higher S-specific binding antibodies than a dose of 1 μg. The superiority of Th1 antibodies (IgG2b and IgG2c) to Th2 antibodies (IgG1) also indicates that PTX-B induces a Th1-biased antibody response. Very low or minimal S-specific binding antibodies were detected in the serum of control mice receiving tdTomato mRNA.

As in Example 1, the first neutralization assay used SARS-CoV-2 virus isolated from one of the first COVID-19 patients in Ontario, and the second assay used SARS-CoV-2 S protein pseudotyped lentivirus Pseudovirus neutralization analysis. In both antibody neutralization assays, the 10 µg dose group significantly outperformed the 1 µg dose group, although this dose group did show considerable neutralizing activity (comparable to that seen in sera from convalescent patients). Compare). Figure 3 shows the ID50 (dilution at which 50% inhibition of infectivity is seen) of SARS-CoV-2 clinical isolates and pseudovirus neutralization assays. Serum from the negative control group showed no activity in either assay. Serum from the 10 µg group had a dose-responsive effect, showing significantly higher neutralizing activity, especially in the analysis of SARS-CoV-2 clinical isolates. Statistics were performed by Kruskal-Wallis test using multiple comparisons; in Figure 3, **=P<0.01, ***=P<0.001, ****=P<0.0001 . In the pseudovirus analysis, the negative control group had no significant activity, the 1 µg dose group had moderate activity, and the 10 µg group had extremely strong neutralizing activity, with an ID50 of at most 1:90,000.

IFN-γ analysis by ELISpot to determine T cell responses to vaccine immunization. Spleen cells from mice were stimulated with a peptide pool of the SARS-CoV-2 S protein (315 15-mer peptides with 11-mer overlap). IFN-γ-producing T cells were measured by ELISpot analysis. T cells from PTX-B-immunized mice produced IFN-γ more frequently than T cells from mice vaccinated with negative controls (Fig. 4). Mice were vaccinated with PTX-B on the 1st and 22nd days at the beginning and additional injections. Mice were sacrificed on day 43, and splenocytes were stimulated overnight in 96-well ELISpot plates precoated with anti-IFN-γ antibody in the presence of a pool of SARS-CoV-2 peptides. After incubation, plates were washed, stained and treated with anti-IFN-γ HRP antibody and read on an ELISpot reader. Statistics were performed using the Kraska-Wallis test and multiple comparison analysis.

Cytokine analysis by Luminex showed that mice immunized with PTX-B produced high levels of IL-2, IFN-γ, and GM-CSF in a dose-dependent manner, but low levels of IL-4 and IL -10 (Figure 5). Mice were vaccinated with PTX-B on the 1st and 22nd days at the beginning and additional injections. Mice were sacrificed on day 43, and splenocytes were stimulated overnight in the presence of a pool of SARS-CoV-2 peptides. Supernatants were analyzed by Luminex for the presence of IL-2, IFN-γ, GM-CSF, IL-4, IL-5, and IL-10. Statistics were performed by Kraska-Wallis test with multiple comparisons. TNF-α levels were undetectable in analysis of mice vaccinated with PTX-B or control. Interestingly, IL-5 levels were detectable in PTX-B-immunized mice but did not increase with vaccination. cellular immune response

PTX-B also triggers a strong cellular immune response. Mouse splenocytes were prepared 3 weeks after booster vaccination, stimulated with S-peptide pool, and analyzed by flow cytometry of interleukin production by IFN-γ/IL-4 ELISpot, CD4+ and CD8+ T cells, and Multiplex immunoassay detecting interleukins in stimulated splenocyte supernatants to measure S-specific cellular responses. These analyzes showed that both 1 μg and 10 μg PTX-B induced robust S-specific cellular immune responses that were Th1-biased, as shown by the main Th1 interleukin (IFN-γ/TNF- α/IL-2) exceeds Th2 interleukins (IL-4/IL-5) as indicated. Notably, a large number of S-specific CD8+ T cells were induced by PTX-B. Compared with humoral responses, especially nAb responses, cellular responses elicited by 1 μg and 10 μg PTX-B were generally comparable. Interleukin analysis by flow cytometry showed that significant proportions of IL-2- and IFN-γ-producing CD4+ (Figure 6A) and CD8+ (Figure 6B) cells were detected in PTX-B-immunized mice , especially cells that produce CD8+ IFN-γ. In contrast, there were no significant differences in IL-4- and IL-5-producing cells in immunized mice compared with control mice. Mice were vaccinated with PTX-B on the 1st and 22nd days at the beginning and additional injections. Mice were sacrificed on day 43, and splenocytes were stimulated overnight in the presence of a pool of SARS-CoV-2 peptides. After overnight stimulation, cells were surface stained for anti-CD3, anti-CD4, and anti-CD8 antibodies. Cells were then fixed and permeabilized and stained for IL-2, IFN-γ, TNF-α, IL-4, and IL-5. Cells were evaluated using flow cytometry. Figures 6A and 6B show that TNF-α-producing cells were slightly higher than in control mice, but not always in a dose-dependent manner. These results indicate that vaccination with PTX-B induced an S protein-specific Th1 response.

It was determined that immunization with 1 μg or 10 μg of PTX-B produced similar T cell responses, both well above background in the negative control group. For antibody-based assays (antibody levels and neutralizing capacity), the 10 µg dose outperforms the 1 µg dose. Example 3. Mouse AAV6-hACE2 challenge model

A non-GLP challenge study was conducted in C57BL/6 mice transfected with AAV6-hACE2 (the receptor for SARS-CoV-2) to investigate the protective efficacy of PTX-B. Four groups of female C57BL/6 mice (12 mice/group) were vaccinated with PTX-B on day 1 and day 22 as follows: Group 1: Formulation Buffer (Negative Control) Group 2: 20 μg PTX-B Group 3: 4 μg PTX-B Group 4: 1 μg PTX-B

On day 29, animals were transduced with 1×10 ¹¹ copies of AAV-hACE2 vector gene copies per mouse, and then on day 38 with 2.5× ¹⁰ TCID50 per mouse with SARS-CoV-2 Intranasal attack. The study was terminated on day 42. Parameters evaluated in this study included the infectivity of lung homogenates, viral RNA levels in the lungs, and lung histopathology. Mice were euthanized and one lung removed for histological examination, while the second lung was bisected and homogenized in culture medium for infectivity testing and in RNA extraction buffer for For viral load determination.

The mice showed no obvious adverse reactions during the in vivo phase of the experiment. On day 38, body weights were measured at the time of challenge, and then again on day 42, just before sacrifice. Statistically significant weight loss was observed in the 1 μg PTX-B vaccinated group (20.39 vs. 18.54, 9.07% p=0.0016) (Figure 7). C57BL/6 mice were prime-boost immunized with PTX-B and transduced with AAV6-hACE2. Body weights were measured at the time of challenge (day 38) and immediately before sacrifice (day 42). Analysis was performed by two-way analysis of variation and multiple comparisons. In Figure 7, **p<0.01. No significant weight loss was observed in the 20 µg or 4 µg groups or the formulation control group.

As shown in Figure 8, PTX-B provided protective efficacy at all three dose levels tested. No infectious virus was found in mice immunized with 20 or 4 µg vaccine (TCID50=0), and 10 of 12 mice immunized with 1 µg were also free of infectious virus (mean TCID50=1.25±2.93 ), while infectious SARS-CoV-2 could be easily detected in 11 out of 12 mice in the formulation buffer negative control group. In Figure 8, TCID50 means the tissue culture 50% infectious dose. As shown, PTX-B neutralizes SARS-CoV-2. TCID50 was measured in AAV6-hACE2 transduced C57BL/6 mice immunized by prime-boost with 3 different doses of PTX-B or a formulation buffer negative control. Mice were transduced with AAV6-hACE2 7 days after the boost and challenged 9 days later. All mice were sacrificed 4 days after challenge with SARS-CoV-2, and virus was measured in lung homogenates (n=12 per group) (**** p<0.0001).

Furthermore, detection of viral RNA in the lungs by PCR demonstrated reduced dose responsiveness, with a more than 100-fold difference between the means of the high-dose group and the negative control group. Lung histopathology was graded on one lung section from an infected mouse. All mice exhibited significant histopathology. It is unclear how much of the pathology is attributable to SARS-CoV-2 and how much to the AAV6 virus used to express hACE2; however, there was histopathology in groups of mice treated with two higher dose levels of PTX-B There is a downward trend in scores (Figure 9). In summary, mice were immunized with indicated amounts of PTX-B, transduced with AAV6-hACE2, and challenged with SARS-CoV-2 nine days later. Four days after challenge, mice were immunized and left lungs were fixed in formalin, processed for histology and examined microscopically by a certified pathologist blinded to treatment conditions. Each sample is assigned a histological score from 1 to 5, with the lowest score being normal. A trend toward decreased pathology was seen with increasing vaccine doses.

In conclusion, using an AAV6-hACE2 transduced mouse model, administration of PTX-B (1, 4, and 20 μg) conferred protection against SARS-CoV-2 infection and had a positive impact on lung pathology, indicating that PTX-B has Protective or damage prevention characteristics. The total amount of SARS-CoV-2 mRNA in the lungs was also reduced at the time of euthanasia. Postchallenge weight loss was observed in the low-dose (1 µg) vaccination group. Example 4. Hamster attack model

A challenge study was conducted in 6- to 8-week-old male Syrian golden hamsters challenged with SARS-CoV-2 to determine whether the vaccine protects against infection.

Syrian golden hamsters are susceptible to SARS-CoV-2 infection and have been shown to be useful in evaluating vaccine candidates.

Hamsters in group 1 received 20 µg of LNP-formulated full-length S protein mRNA (PTX-B).

Hamsters in Group 2 received 4 µg of LNP-formulated full-length S protein mRNA (PTX-B).

Hamsters in Group 3 received 1 µg of LNP-formulated full-length S protein mRNA (PTX-B).

Group 4 hamsters received formulation buffer (PBS sham/negative vaccine control group).

On Day 0, all hamsters were pre-bleed for baseline analysis. On day 1, all hamsters received the first intramuscular injection (vaccine or control, according to group 1 to group 4). Animals were allowed to acclimate for 7 days before receiving the first vaccine dose. On day 21, a live blood draw was performed on all hamsters. On day 22, all hamsters received a second (booster) vaccination according to groups 1 to 4. On day 29, all hamsters received AAV6-hACE2 intranasally (see, eg, Example 3) to facilitate SARS-CoV-2 infection. On day 38, all hamsters were infected with SARS-CoV-2 via intranasal infection. All animals received a total dose of 7.5×10^5 TCID ₅₀ as determined by back titration. Thereafter, animals were monitored daily for weight loss and signs of disease or distress. Additionally, viral shedding was monitored by collecting oral swabs every other day. Hamsters were euthanized on day 42 for endpoint analysis of: (i) infectivity of lung homogenates; (ii) viral RNA levels in the lungs; (iii) lung histopathology. Monitor animals for any observable clinical signs during the vaccination phase during the study. No obvious adverse reactions were observed. The complete experimental design is described in Table 8. Table 8. Experiment overview Vaccination route Inoculate the hamster intramuscularly into two sites on the hind legs, using a 23-25 gauge, 3/8-1 inch needle, inoculating a total of 200 μL (100 μL per site) Vaccination time 2 doses on day 1 and 22, challenge on day 43 Challenge virus dose route SARS-CoV-2 7.5×10 ^5TCID50 Intranasal

Four animals from each group were randomly selected and euthanized at 4 and 8 days post-infection (dpi). Tissues were collected for determination of viral load by qRT-PCR and infectious virus titer levels and for histopathology. At the end of the experiment (8 dpi), blood was also collected from both groups of animals to assess the titers of neutralizing antibodies.

Animals in the vaccinated group showed no weight loss on average during the course of the experiment. In contrast, hamsters in the sham vaccine group showed moderate average weight loss starting at 3 dpi. Overall, the average weight loss at the end of the trial was 11%. No other significant clinical signs of disease were reported in either group.

After animals were euthanized at 4 and 8 dpi, half of the lungs were placed in formalin for tissue fixation. Tissues were subsequently H&E stained and evaluated by a pathologist blinded to each group. Compared with the vaccinated group, the control group (sham vaccinated) had significantly higher pathology scores at both time points. This suggests that the disease in the unvaccinated group was more severe.

Oral swabs were collected during the experiment for assessment of viral shedding. Interestingly, although viral RNA was detected in both groups throughout the experiment, the actual levels of infectious virus were significantly reduced in vaccinated animals (ranging from 2 to 3 log reductions, as shown in Figure 14) . This suggests that viral shedding was lower in the vaccinated group throughout the experiment and that PTX-B reduced viral shedding, thereby providing a therapeutic benefit.

Examination of viral loads in the turbinates showed a significant reduction in the amount of infectious virus at 4 dpi in the vaccinated group, and undetectable levels of infectious virus at 8 dpi. A similar trend was seen in the lungs, with the notable difference that no infectious virus was detected in the lungs of vaccinated animals at any time point. Viral RNA was detected in the lungs of both vaccinated and unvaccinated groups at both time points.

These data demonstrate that vaccination with PTX-B provides protection against intranasal challenge with SARS-CoV2-2 in a hamster infection model. Example 5. Study on immunogenicity and local tolerance in mice

The goal of this 2-dose immunogenicity and tolerability study is to obtain basic safety data related to the mRNA vaccine in addition to immunological data on different mouse strains for use in other preclinical experiments. On Days 1 and 22, groups of BALB/c mice were administered PTX-B via IM injection at dose levels of 0, 4, or 20 µg, as summarized in Table 9. The clinical signs, body weight changes and skin observations of the main research animals were evaluated by the modified Draize score. Animals in the hematology cohort were sacrificed on day 24 (two days after the second dose), blood was drawn for hematology and organ weights were recorded, gross pathology was assessed, and liver, spleen and injection site tissue were examined microscopically. The interleukin population was sacrificed on day 22 for determination of serum interleukin concentration. Primary study animals were terminated on Day 43 (three weeks after the second dose) and immunogenicity endpoints, hematology, clinical chemistry, liver function testing, gross pathology, and organ weights were assessed. Table 9. Mouse immunogenicity and local tolerance studies - study design Group number test substance Dosage(µg) Dosage volume (µl) Dose concentration(µg/mL) Number of animals main research Hematology interleukin male female male female male female 1 control 0 50 0 10 10 5 5 5 5 2 PTX-B 4 50 0.08 10 10 5 5 5 5 3 PTX-B 20 50 0.4 10 10 5 5 5 5 safety-related endpoints

After the second dose of 20 μg PTX-B, a transient, slight weight loss was observed in both sexes; however, there was no significant difference in mean body weight between the groups at the end of the study (data not shown out).

Test material-related findings at the injection site were noted during clinical observation and gross and microscopic examination; all findings were reversible. Based on the Draize score, redness and/or swelling at the injection site between 4 and 20 µg was occasionally observed, and piloerection was seen in a few female mice for one to two days after the first dose, but these were not considered important. discovery. At termination two days after the second dose, histopathological findings at the injection site included mild to moderate mixed cellular inflammation in both sexes at 4 and 20 µg; this finding was accompanied by edema and in one female at 20 µg. Accompanied by mineralized substances at µg. A dose relationship was noted in the incidence and severity of this finding in females. Inflammation is closely associated with a hard, abnormal consistency and swelling. In addition, at both doses, a few animals experienced slight to mild bleeding, which was closely related to the dark lesions at the injection site or subcutaneous tissue; there was no obvious dose relationship. At the end of the study, no macroscopic findings were observed at the injection site.

Serum interleukin analysis was performed 6 hours after the second dose (day 22) to monitor for interleukin release syndrome, a known potential side effect of LNP-formulated mRNA. Analyze IFN-γ, IL-1β, IL-6, IL-10, MCP-1, and TNF-α using validated immunoassay methods. IL-6 (up to 53-fold and 266-fold higher in males and females, respectively) and MCP-1 (up to 20-fold and 15-fold higher in males and females, respectively) were observed in both sexes at both dose levels ) of PTX-B-related increases in serum concentrations. In general, the magnitude of response is dose-related. For MCP-1, there were no meaningful sex-related differences in responses. For IL-6, the increase was greater in females than in males. Mild increases (up to 2.5-fold) in serum concentrations of IFN-γ were observed in some animals of both sexes. There were no obvious PTX-B-related changes in IL-1β, IL-10 and TNF-α. The observed pattern of interleukin changes is inconsistent with interleukin release syndrome.

Body weight was measured weekly during the study. In the 20 µg group, body weight of both males and females decreased slightly. By the end of the study, all groups had regained body weight (data not shown).

Hematological parameters were measured two days (day 24) and three weeks (day 43) after the second vaccination. At the first time point, the only dose-responsive changes in both sexes were in leukocytes (264% and 420% of control at 4 and 20 µg for males and 420% and 420% for females at 4 and 20 µg, respectively). 329% and 514%) and reticulocytes (males were 69% and 41% of the control at 4 and 20 µg, respectively, and females were 53% and 27% of the control at 4 and 20 µg, respectively); no effects were observed. Effects of red blood cell parameters. All hematological parameters were within normal range on day 43 (white blood cells: 77% and 77% of control at 4 and 20 µg for males, 67% and 75% of control at 4 and 20 µg for females, Reticulocytes (107% and 117% of control at 4 and 20 µg for males, 122% and 129% of control at 4 and 20 µg for females).

Two days after the second dose (day 24), it was noted that the livers of females at 20 μg exhibited minimal hepatocyte cytoplasmic changes, characterized by the accumulation of glycogen-like material. This change was associated with weight gain (absolute and relative to brain weight, 24% to 27%) and light discoloration. However, on day 43, all liver function tests were within the normal range and were not significantly different from the control group.

On day 24, an increase in spleen weight was noted in both sexes at 4 and 20 μg (absolute and relative to brain weight, 32% to 49%). This increase was statistically significant and correlated well with enlargement in females. Micro-correlation cannot be established.

By the end of the study (day 43, three weeks after the second dose), no overall PTX-B-related findings were noted. Although absolute spleen weight still increased at 20 µg (14% to 18%), the magnitude of the increase was significantly less than two days after the second dose, and all hematological parameters were normal.

In conclusion, PTX-B administered to BALB/c mice by IM injection on days 1 and 22 at dose levels of 4 and 20 μg was well tolerated. Limited results were observed after the second dose, mainly increases in serum concentrations of IL-6 and MCP-1, dose-related increases in leukocytes and decreases in reticulocytes, dose-related injection site reactions, and increases in spleen weight, but No micro correlation. Other findings noted only at 20 mcg/dose included slight weight loss and hepatocyte cytoplasmic changes. All findings were fully or partially reversible; as of day 43, test material-related effects were limited to a slight increase in spleen weight at 20 μg/dose. Immunogenicity-Related Endpoints

Splenocytes were collected at the end of the study for analysis by ELISpot (Figure 10, IFN-γ and IL-4 ELISpots of splenocytes from PTX-B immunized mice). Collected from PTX-B mice were stimulated on IFN-γ and IL-4 multiplex ELISpot plates in the presence of SARS-CoV-2 S protein peptide pools S158 and S157 (available, for example, from JPT Peptide Technologies, Berlin, Germany). Splenocytes. Spots were counted after overnight stimulation. Statistics were performed by two-way ANOVA. As shown in Figure 10, a significant increase in IFN-γ expression was observed in both male and female mice self-stimulated with the S158 peptide pool. Similarly, the S157 peptide pool induced a significant increase in IFN-γ expression in male mice. IL-4 performance was not significantly increased by stimulation with either peptide pool. This combination of results indicates a Th1-biased response.

In the SARS-CoV-2 neutralization assay, sera from female and male mice treated with formulation buffer as negative controls did not provide protection against infection with SARS-CoV-2 clinical isolates at any dilution tested. Protection (Figure 11). In contrast, the mean ID50 titers of sera from female and male mice immunized with 4 µg of PTX-B were 1353 and 480, respectively. Sera from mice immunized with 20 μg of PTX-B provided greater protection, with mean ID50 titers of 7645 (females) and 5118 (males), indicating a dose-responsive effect (Figure 11).

To confirm the potent neutralization of sera from mice treated with PTX-B, a second independent pseudovirus neutralization assay was performed. Serum from the negative control group showed no neutralizing ability (Figure 12). Serum from female and male mice treated with 4 µg of PTX-B provided protective activity with ID50 values of 4048 and 1863, respectively. Serum from mice immunized with 20 µg PTX-B showed mean ID50 values of 16390 (female) and 1414 (male).

Immunization with both 4 and 20 μg of PTX-B resulted in strong neutralizing antibody responses in BALB/c mice (Figure 13A to Figure 13C). Serial dilutions of sera from PTX-B-treated mice were performed, and anti-SARS-CoV-2 spike IgG, IgG1, IgG2a, IgG2b, IgM, and IgA were measured using anti-isotype HRP antibodies. The median value is represented by a box plot, which must represent the Tukey analysis of Q1 and Q3 in the interquartile range, and statistical outliers are represented by a single point. The results were dose-responsive and consistent with those demonstrated in previous experiments in C57BL/6 mice. The profile of anti-SARS-CoV-2 anti-spike protein antibodies induced by initial and additional doses of PTX-B showed that this formulated vaccine promoted seroconversion against the SARS-CoV-2 spike protein. SARS-CoV-2 spike protein-specific IgG (Figure 13A, left panel), IgG1 (Figure 13A, right panel), IgG2a (Figure 13B, left panel), IgG2b (Figure 13B , right panel), IgM (Fig. 13C, left panel), and IgA (Fig. 13C, right panel). Example 6. Efficacy of SARS-CoV-2 N, M, N/M protein nucleic acid vaccines

Co-culture assays were performed by mixing fluorescent T cells (labeled with CFSE) with activated dendritic cells (DC) transfected with mRNA for N, M, or N/M mixtures with or without secretion signals. As described in Table 9 below. Specifically, monocytes were isolated on day 1 from peripheral blood mononuclear cells (PBMC) from COVID-19 convalescent patients. Mononuclear dendritic cells (MDDCs) were also differentiated from PBMCs from the same convalescent patients. MDDC were transfected with RNA vaccine on day 4, and the transfected MDDC were co-cultured with PBMC to induce autostimulation of T cells in the co-culture. Table 10. Description of nucleic acid vaccines used in Example 6 and Figure 15 group mRNA vaccine description SEQ ID NO A tdTomato (negative control) 46 B N - sequence encoding N protein 30 C M - sequence encoding M protein 28 D NM - sequence encoding N and M proteins 29 E sNM - sequence with signal peptide encoding N and M proteins 31

Dendritic cells in the co-culture were matured using a cocktail of interleukins containing TNF-a, IL-1b, IL-6 and PGE2 factors. On day 5, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) to monitor lymphocyte proliferation (see, e.g. , Lyons AB, Parish CR (May 1994). "Determination of lymphocyte division by flow Cytometry". Journal of Immunological Methods. 171 (1): 131-7, incorporated herein by reference).

Subsequently, on day 11, cells were stained using fluorescent antibodies to CD4 or CD8 to identify CD4+/CD8+ T cells, and the amount of CFSE labeling in antibody-labeled cells was determined to determine T cell proliferation.

Cells stimulated by DC have low levels of CSFE due to multiple cell divisions. Establish a threshold value to identify cells with low CFSE staining, that is, cells that have undergone 1 or more divisions (e.g., CFSE signal in cells that have divided once is reduced to 1/2; CFSE in cells that have divided more than 1 time) The signal is reduced by more than 1/2). Typically, cells that divide 1 to 7 times are identified as "low CFSE" cells. Therefore, the ratio of [low CFSE staining in mRNA-transfected cells] to [low CFSE staining in control cells] provides the T cell stimulation index (SI). An increased SI value indicates the extent to which the mRNA vaccine stimulates a T cell response.

The stimulation of T cells, that is, the stimulation index (SI), is calculated as follows: SI = [Proliferation of T cells stimulated with DC transfected with mRNA (% CFSE-lo)] divided by [Control with DC (DC containing culture medium) Proliferation of stimulated T cells (% CFSE-lo)]. SI values greater than 2 are considered to indicate biologically significant proliferation. The data are shown in Figure 15. Example 7. Pseudovirus neutralization analysis of VOC using PTX-B

Various patient sera were tested for neutralization capacity using a pseudovirus neutralization assay (see, e.g., Example 2) using pseudotyped to wild-type SARS-CoV-2 variants of concern (VOC) and others as described in Table 11 Pseudovirus with variant S protein. Table 11. Description of S proteins encoded by pseudoviruses in Example 7 and Figures 16 to 18 group Fake virus ID Description of the encoded S protein A WT S protein of the original virus strain from Wuhan, China B D614G WT S protein with D614G mutation C UK variant PANGO lineage B.1.17 S protein D SA variants PANGO lineage B.1.351 S protein E D614G+L452R WT S protein with D614G and L452R F UK+L452R PANGO lineage B.1.17 S protein with L452R mutation G SA+L452R PANGO lineage B.1.351 S protein with L452R mutation H BR variant PANGO lineage P.1 S protein

The sera tested include CBS 13 and CBS 5 (Canadian Blood Services samples from COVID-19 patients), O132 (sera from patients with dual mRNA vaccination), and sera containing NRC VHH72 antibodies (targeting S protein RBD). As shown in Figure 16, various test sera had reduced neutralizing abilities against pseudoviruses encoding the S proteins of different SARS-CoV-2 variants. In general, variants encoding the British and South African S protein variants had reduced neutralizing capacity compared to the WT and D614G variants. The S protein of the South African variant and the S protein of the South African variant including the L452R mutation always showed the lowest neutralization levels.

Next, sera from mice vaccinated with 1 μg or 10 μg of PTX-B or 10 μg of a control mRNA vaccine encoding TdTomato were tested against VOC pseudoviruses, and the ID50 was measured. As shown in Figure 17, sera from mice vaccinated with the control mRNA vaccine (TdTomato) showed no protection from infection. Serum from mice vaccinated with 1 µg of PTX-B showed protection against infection, with a dose-dependent increase in protective efficiency seen at the 10 µg dose. Generally, ID50 values between 2 and 3 in Figure 17 may be protective against infection, and ID values greater than 3 are presumed to be protective. As can be seen, the highest level of protection was demonstrated against the WT (A), UK (C), UK+L452R (F) and D614G+L452R (E) pseudovirus S protein variants, with BR (H), SA (D ) and SA+L452R (G) pseudovirus S protein variants have reduced protective efficacy. In Figure 17, lines connecting dots represent serum from the same mouse. Figure 18 shows a comparison of the protective efficiency of each variant (C, F, E, H, D and G provided in Table 11) relative to the WT SARS-CoV-2 pseudotyped pseudovirus (A in Table 11), And the trend shown in Figure 17 is confirmed. Example 8. Safety, tolerability and immunogenicity in humans Research overview

A Phase Ia/Ib, first-in-human, observer-blinded, randomized, placebo-controlled, ascending-dose study was conducted to evaluate the efficacy of PTX-B vaccine in healthy seronegative adults aged 18 to 64 years and ≥65 years. Safety, tolerability and immunogenicity.

The purpose of this study was to evaluate the safety and tolerability of 2 doses of PTX-B vaccine in healthy seronegative adults aged 18 to 64 years and ≥65 years, and to evaluate the safety and tolerability of 2 doses of PTX-B vaccine in healthy seronegative adults aged 18 to 64 years and ≥ Immunogenicity in healthy seronegative adults aged 65 years.

The safety and tolerability endpoints evaluated are: the incidence of events during the follow-up period after vaccination using the per-protocol (PP) and safety groups. These events include: vital signs during the follow-up period after vaccination and Administration site reactions (e.g., arm examination assessment, including pain, tenderness, erythema/redness, induration/swelling); and adverse events (AEs; e.g., fever, chills, nausea) required to be recorded daily through the third day after each vaccination /vomiting, diarrhea, headache, fatigue, myalgia).

Overall safety was analyzed using both adjusted intention-to-treat (mITT) and safety populations. If the difference between individuals in each treatment group and the PP population is ≤ 5%, the mITT population will not be used for analysis. Adverse events not required to be recorded from day 1 to day 56 were analyzed, as well as medical AEs (days 1 to 56), new chronic diseases (NOCD), serious adverse events [SAE], and adverse events of special concern. Event [AESI] and potential immune-mediated medical condition (PIMMC) from days 1 to 395 (approximately 1 year after last vaccination). Results of targeted physical examinations, vital sign assessments, and clinical safety laboratory tests are also recorded.

Immune response endpoints evaluated include immunogenicity analysis using mITT and safety populations. If the difference between individuals in each treatment group and the PP population is ≤ 5%, the mITT population will not be used for analysis. Antibodies (immunoglobulin (Ig) M, IgG, IgA [enzyme-linked immunosorbent assay]; and neutralizing) were analyzed, and blood/peripheral blood mononuclear cells (PBMC [flow cytometry, enzyme-linked immunosorbent assay] Immunospot analysis]) of cell-mediated immunity.

The study was designed with age and dose escalation and was conducted in seronegative adult individuals with no evidence of recent exposure to severe acute respiratory syndrome (SARS)-CoV-2 or an influenza or respiratory fusion virus identified ( RSV) (febrile or lower respiratory tract infection) viral respiratory disease.

The study is conducted in the following 2 phases:

Phase 1a (approximately 60 individuals) individuals received 2 doses of PTX-B intramuscular (IM) vaccine or placebo 4 weeks apart.

Cohort 1 included 20 healthy individuals aged 18 to 64 years who were administered 16 μg of PTX-B IM vaccine or placebo; 5 sentinel individuals (4 PTX-B: 1 placebo), followed by the remaining 11 PTX-B:4 placebo (cohort expansion) subjects.

Cohort 2 included 20 healthy individuals aged 18 to 64 years who were administered 40 μg of PTX-B IM vaccine or placebo; 5 sentinel individuals (4 PTX-B: 1 placebo), followed by the remaining 11 PTX-B:4 placebo (cohort expansion) subjects.

Cohort 3 included 20 healthy individuals aged 18 to 64 years who were administered 100 μg of PTX-B IM vaccine or placebo; 5 sentinel individuals (4 PTX-B: 1 placebo), followed by the remaining 11 PTX-B:4 placebo (cohort expansion) subjects.

Evaluation of the sentinel cohort includes safety follow-up assessments (including required and unrecorded AEs and safety laboratory assessments) until the third day after vaccination.

Phase 1b (approximately 60 individuals) individuals received 2 doses of PTX-B vaccine 4 weeks apart (15 receiving placebo and 45 on active treatment).

Cohort 4 included 20 healthy individuals ≥65 years of age; 15 individuals were administered PTX-B IM vaccine at 1 dose level based on previous results from Phase 1a, and 5 individuals were administered placebo.

Cohort 5 included 20 healthy individuals ≥65 years of age; 15 individuals were administered PTX-B IM vaccine at 1 dose level based on previous results from Phase 1a, and 5 individuals were administered placebo.

Cohort 6 included 20 healthy individuals aged ≥65 years who were administered PTX-B IM vaccine at 1 dose level based on previous results, and 5 individuals were administered placebo.

Each cohort undergoes approximately 14 days of independent security review before enrolling the next cohort.

Individuals visited clinical sites for screening (days -21 to -1) and on days 1, 8, 28±2, 36, 56±2, 90±3, 180±5, and 395-14. Secure telephone calls were made to individuals on Days 2 and 29. Screening procedures include informed consent, assessment of entry criteria, demographic information, height, weight, body mass index, medical and surgical history, safety assessment (including SAE assessment, documentation of previous medications and surgeries, physical examination, vital sign assessment, clinical hematology and urine samples, nasopharyngeal [NP] swabs, clinics may choose to undergo additional point-of-care testing to confirm eligibility), and blood samples for immunogenicity analysis (antibodies). On Day 1, randomization occurred (overall 15:5 study vaccine:placebo vaccine in Phase 1a and Phase 1b cohorts). On days 1 and 28, eligibility is confirmed and safety assessments are conducted, immunogenicity and cell-mediated immunity blood collection is performed, paper diary training and distribution is performed, and vaccine or placebo administration is performed. Conduct safety assessment, blood and urine samples for safety assessment, and blood samples for immunogenicity analysis and cell-mediated immunity until the end of the study consultation. Assessment of AEs not required to be documented was performed on days 1 to 56. Medical-seeking AE assessments were performed while the individual was at home on Days 1 to 56. NOCD, AESI (including COVID 19 enhanced disease cases), and PIMMC were assessed from Day 1 to the end of the study. SAEs will be assessed throughout the study.

Vaccine and placebo were prepared by unblinded site pharmacists and administered by unblinded CPU personnel at the clinical study site by IM injection into the deltoid muscle of the non-dominant side of the upper arm. Individuals were observed for immediate AEs and/or reactogenicity within approximately 1 hour of vaccine administration. During the follow-up period after each vaccination, individuals were provided with diary cards and trained to record the specific systemic and local symptoms elicited daily, as well as any additional AEs. Individuals are asked to take a photo of the completed diary card and send the photo to the site via text/email to ensure close monitoring of responses.

For each subject, the duration of continuous monitoring of the subject was approximately 14 months. Study inclusion criteria

Individuals must meet all inclusion criteria (numbered 1-8 below) to be eligible to participate in the study. Additionally, seek out racial and ethnic minorities to obtain a diverse research population.

1. The individual has read, understood and signed the informed consent form.

2. Healthy adult men and women aged 18 to 64 years (Phase 1a) or ≥65 years (Phase 1b) (inclusive of endpoints) at screening.

3. SARS-CoV-2 seronegative and reverse transcription polymerase chain reaction (RT-PCR) negative at screening, with no evidence of recent exposure or virality not identified as influenza or RSV (febrile or lower respiratory tract infection) Respiratory diseases.

4. Body mass index at the time of screening ≥18 and ≤30 kg/m2.

5. You must be in good health before participating in the study and have no clinically relevant abnormalities that may interfere with the study evaluation. Older (≥65 years) participants could have stable comorbidities (no medication changes or worsening in the past 3 months).

6. Women of childbearing potential (WOCBP) and men whose sexual partners are WOCBP must be able and willing to use at least one highly effective contraceptive method during the study period (that is, including hysterectomy, bilateral salpingectomy, and bilateral oophorectomy) surgical, hormonal oral [in combination with a spermicide-containing male condom], transdermal, implanted or injected, barrier [i.e., condom, spermicide-containing diaphragm]; intrauterine device; vasectomy Partner [minimum 6 months], clinically infertile partner; or abstinent). Female individuals are considered WOCBP after menarche and until they have been postmenopausal for 12 consecutive months (without other medical reasons) or are otherwise permanently infertile. Individuals of non-reproductive potential are not required to use any other form of contraception during the study. Non-reproductive potential is defined as individual confirmation of: surgical sterilization (e.g., bilateral oophorectomy, bilateral salpingectomy, bilateral cautery occlusion [not acceptable with Essure System®], hysterectomy, or tubal ligation), postmenopausal (defined as Permanent menopause for at least 12 consecutive months before screening); if postmenopausal status is unclear, perform a pregnancy test before vaccination.

7. Women of childbearing age must have a negative pregnancy test before each vaccination. If menopausal status is unclear, a pregnancy test is required.

8. Must be able to attend all consultations during the study (scheduled and unscheduled, as applicable) and comply with all study procedures, including completing daily diary cards after each injection. Study exclusion criteria

Individuals, including individuals ≥65 years of age (for Phase 1b), are not eligible to participate in the study if they meet any of the exclusion criteria (numbered 1 to 35 below), or will be discontinued at the discretion of the investigator if they develop any of the exclusion criteria during the study.

1. A history of acute or chronic medical conditions (including dementia) that, in the opinion of the investigator, would make vaccination unsafe or interfere with assessment of response.

2. History of any medical condition that puts an individual at higher risk for severe illness attributable to SARS-CoV-2 will be excluded, including: chronic kidney disease; COPD (chronic obstructive pulmonary disease); heart disease, such as heart failure , coronary artery disease or cardiomyopathy; any immunocompromised state, including transplantation, historical immunodeficiency, HIV, immunosuppressive drug intake; sickle cell disease; current smoker or >5 pack/year smoking history; Type 2 diabetes.

Individuals with a history of any of the following conditions that may be at increased risk for COVID-19 complications are excluded: Asthma (moderate to severe); Cerebrovascular disease (affecting blood vessels and blood supply to the brain); Cystic fibrosis; High blood pressure ( Hypertension or high blood pressure; neurological conditions such as dementia; liver disease; pulmonary fibrosis (damage or scarring of lung tissue); thalassemia (a blood disorder); type 1 diabetes.

3. Ongoing clinical conditions or history of medications or treatments that may adversely affect the immune system.

4. SARS-CoV-2 seropositive or RT-PCR positive individuals, including before the second dose of PTX-B vaccine.

5. Individuals at increased risk of exposure to SARS-CoV-2 (e.g., health care workers, first responders).

6. Close contact with anyone known to be infected with SARS-CoV-2 within 30 days before vaccine administration.

7. Living in a group setting or group care facility (such as a dormitory, assisted living facility, or nursing home).

8. Individuals with any elevated (Grade 1 or higher) laboratory test assessed by the investigator at screening as clinically significant for age/sex.

9. Individuals with any liver function enzyme elevated (Grade 1 or higher) for age/sex at screening, regardless of assessment of clinical significance (one retest is allowed). The criteria for excluding individuals with elevated liver enzymes are as follows: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase or gamma-glutaminyltransferase >1.5 × upper limit of normal (ULN); total bilirubin >1.5×ULN.

10. History of active neoplastic disease (excluding successfully treated non-melanoma skin cancer) or any hematological malignancy. "Active" is defined as having received treatment within the past 5 years.

11. Long-term (>2 weeks) use of oral or parenteral steroids or high-dose inhaled steroids (>800 μg/day beclomethasone dipropionate or equivalent) within 6 months before screening (nasal and topical steroids are allowed ).

12. History of autoimmune disease, inflammatory disease or PIMMC (Appendix B).

13. Women who are currently pregnant, breastfeeding, or planning to become pregnant between 181 days after registration and randomization.

14. History of Guillain-Barré Syndrome or any degenerative neurological disorder.

15. History of allergic reaction to any injected vaccine.

16. Known or suspected allergy to one or more components of the vaccine.

17. History of alcohol abuse, illicit drug use, physical dependence on any opioid, or any drug abuse or addiction within 12 months of screening.

18. Acute illness or fever (body temperature >37.5C) occurs within 3 days before study enrollment (enrollment may be delayed to allow for complete recovery if acceptable to the researcher).

19. Individuals who are currently participating or planning to participate in research involving experimental agents (vaccines, drugs, biological agents, devices or drugs); or who have undergone experiments within 1 month (3 months for immune globulin) before joining this study agents; or individuals who are expected to receive another experimental agent while participating in this study.

20. Individuals who have received immune globulin or another blood product within 3 months before joining this study, or who are expected to receive immune globulin or another blood product during this study.

21. Individuals who wish to donate blood within 6 months of the first vaccination.

22. Individuals using prescription drugs to prevent SARS-CoV-2.

23. Individuals scheduled to receive another vaccine within 3 months prior to the study should not be given influenza vaccine within 2 weeks of the vaccine, except for those scheduled to receive it within 3 months before the study.

24. Receipt any other SARS-CoV-2 or other experimental coronavirus (MERS, SARS, etc.) vaccine before or at any time during the study.

25. Receive any investigational vaccine or investigational drug within 1 month after enrollment until the end of the study (1 year after the last vaccination).

26. Plan to travel outside Canada from joining to the 56th day.

27. Have a history of surgery or major trauma within 12 weeks after screening, or have surgery planned during the study.

28. Severe blood loss (> 400 mL) or donation of 1 or more units of blood or plasma within 6 weeks before participating in the study.

29. Engaging in strenuous activity or heavy drinking within 72 hours prior to safety laboratory sample collection (assessed by researcher).

30. Positive urine drug abuse screen or alcohol breathalyser test result.

31. Screening for human immunodeficiency virus-1 and -2 antibodies, hepatitis B surface antigen, or hepatitis C virus antibodies is positive.

32. Participate in the planning or implementation of this study.

33. Unwilling or unlikely to comply with research requirements.

34. The individual is an employee, contractor, or friend or relative of any employee of the sponsor, CRO, research site or site affiliate.

35. Individual blood oxygen measurement <90%. research composition

The vaccine product is presented as a 0.2 mg/mL, 2 mL fill in a 3 mL USP/European Pharmacopoeia Type I borosilicate glass vial with a fluororesin-laminated bromobutyl rubber stopper with a red, plastic Pull-top aluminum closure. The PTX-B messenger ribonucleic acid humoral vaccine is a 0.2 mg/mL 0.5 mL injectable solution with multiple dose levels as described.

The placebo was commercially available sodium chloride 0.9% injectable IM solution 0.0 mg/mL 0.5 mL. Overview of research results

This Phase 1, first-in-human, observer-blinded, randomized, placebo-controlled, ascending-dose study evaluated the safety, tolerability, and immunogenicity of PTX-B vaccine in healthy seronegative adults aged 18 to 64 years. sex. The study was designed with dose escalation and was conducted in seronegative adult individuals with no evidence of recent exposure to SARS-CoV-2.

The safety population includes all individuals who provided consent to be randomized and receive any amount of vaccine/placebo. The safety population was used for all safety and immunogenicity analyses, and was analyzed on an actual treatment basis.

The per-protocol population includes all individuals in the safety population who received the specified dose of vaccine/placebo according to the protocol, have serological results, and have no major protocol deviations affecting the primary immunogenicity results, as locked and unblinded in the database by the sponsor previously determined. The PP population is the primary population used for safety endpoint analyses.

The intention-to-treat population was adjusted to include all individuals in the safety population who provided any serological data. The mITT population was used for analysis of immunogenicity endpoints. If the difference between individuals in each treatment group and the PP population was ≤ 5%, the mITT population was used for analysis.

List and summarize immunogenicity data for each cohort and study phase by phase, cohort, and time point using appropriate descriptive statistics.

List and summarize vital signs, clinical laboratory tests, and physical examination results by study phase, cohort, and time point using appropriate descriptive statistics.

The number and percentage of individuals reporting any treatment-emergent adverse event (TEAE) or reactogenicity are summarized by study phase and cohort, and tabulated by system organ class and priority (using MedDRA codes). TEAEs are further classified according to severity and relationship, and for SAEs, they are divided into medical AEs, NOCDs, and AESIs.

In addition, the number of individuals who became infected after vaccination and whether vaccination reduced or worsened the disease are also presented.

Individuals were randomized to receive PTX-B vaccine or placebo in a 3:1 ratio. Dosing occurred within the cohort as follows: starting with the 16 µg dose, followed by 40 µg, and then 100 µg. Each cohort started with a sentinel group of 5 individuals who were dosed first, followed by the rest of the cohort. Adverse events were collected frequently, and an independent safety review committee (iSRC) composed of infectious disease experts and statisticians met frequently to review and approve dosing of the next arm.

A total of 60 individuals were recruited and 58 individuals received two doses of study drug. Two individuals withdrew from the study after receiving one dose of study drug due to personal reasons unrelated to the study drug. Overall, the male to female ratio is exactly 50:50, with 83.3% being white, 13.3% Asian, and 3.3% other.

Adverse events collected were graded as local (at the injection site) or systemic based on industry-standard FDA guidelines for vaccine reactions. Overall, PTX-B was safe and well tolerated at all three dose levels: 16 µg, 40 µg, and 100 µg. There were no serious adverse events. The only local adverse event recorded was pain at the injection site, as expected. There was no redness or swelling at any dose of 40 μg PTX-B. This compares favorably with other mRNA vaccines where redness and swelling have been documented in clinical trials and in general use. Systemic reactions include fatigue, chills and fever, and are generally mild to moderate and well tolerated, with headache being the most common reaction, occurring in up to 60% of patients after the second dose. These results compare favorably with published adverse events for emergency-use approved mRNA vaccines.

PTX-B vaccination induces high anti-S IgG antibodies: Clinical trial participants were vaccinated on days 0 and 28. Plasma samples were collected on day 0 (pre-screening), day 8, day 28 (pre-dose 2) and day 42 for use of a multiplex sandwich-based immunoassay with electrochemistry from the Meso Scale Discovery (MSD) platform Luminescence (ECL) readings determine levels of IgG anti-S protein. All study participants in all three vaccine dose groups (16, 40 and 100 µg) developed strong IgG antibody responses to the spike protein that were detected by day 28 and increased by day 42. No antibodies against the S protein were detected in participants who received placebo. Additionally, the highest antibody levels were found at the medium and high doses. As of day 42, antibody levels in participants vaccinated with PTX-B were more than one log higher than in plasma from convalescent individuals evaluated in the same analysis. Individuals were analyzed for total IgG levels. Just after the first dose (day 28), high levels of anti-spike (shown in Figure 19) and anti-RBD (Figure 19) were induced at all dose levels. Two weeks after the second dose (day 42), levels increased even higher, reaching levels more than 10 times higher than the average of 5 serum samples from convalescent patients (Figure 19). Anti-spike IgG (Au/mL) was also measured in SARS-CoV-2 convalescent patients (Figure 20).

Antibody levels were comparable to those published in a recent Stanford University report evaluating IgG responses in individuals vaccinated with a COVID-19 mRNA vaccine compared with patients with SARS-CoV-2 infection ( Röltgen et al . (2021 April 7). mRNA Vaccination Compared to Infection Elicits an IgG-Predominant Response with Greater SARS-CoV-2 Specificity and Similar Decrease in Variant Spike Recognition. Preprint published on May 11, 2021 from doi.org/10.1101/2021.04 .05.21254952 download).

The high levels of anti-S protein IgG antibodies induced at all three different doses were further confirmed using ELISA. Serum samples from vaccinated individuals were added to spike protein-coated ELISA plates, and bound IgG antibodies were then detected using peroxidase-labeled secondary anti-IgG antibodies. The IgG antibody concentration determined by interpolation on the calibration curve was as high as approximately 1000 ELISA units/ml after a single dose of PTX-B (day 28) (Figure 23). The IgG concentration continued to increase after the second dose, exceeding 10,000 ELISA units/ml (Figure 23).

PTX-B vaccination induces high neutralizing antibody levels: Neutralizing activity in plasma from study participants assessed by S-ACE2 blockade MSD analysis. Here, the antibody blocks the interaction between the S protein and the ACE2 receptor, and the decrease in ECL signal is used to calculate the percent inhibition in plasma at the same dilution. All participants at all three dose levels in the study showed blocking activity by day 28, and all participants achieved 100% blocking activity by day 42 with samples at 1:100 or higher dilutions. These results indicate that PTX-B induces a strong neutralizing antibody response. In addition, quantification of reference standard antibody levels in ng/mL showed that all participants produced neutralizing antibodies by day 28 after the first immunization and by day 42 two weeks after the second immunization Increase 10 times. Neutralizing antibodies in individuals were analyzed by assessing their ability to block the interaction between spike protein and hACE2 in vitro. Using an S:ACE2 receptor blockade assay based on MSD technology, participants vaccinated with PTX-B showed high levels of neutralizing activity in plasma on days 28 and 42 (Figure 21). When percent inhibition was assessed in samples diluted 100-fold or more, all participants had 100% inhibition by day 42. Data published by Röltgen et al. showed that individuals vaccinated with the BioNTech/Pfizer mRNA vaccine showed an average inhibition of 75% at day 42 (1:100 dilution) using the same MSD analysis. These data indicate that PTX-B has enhanced neutralizing ability at all doses.

Neutralizing activity was assessed by S-ACE2 blocking MSD analysis. Quantification of antibody levels in ng/mL is based on the activity of a reference standard. All participants produced neutralizing antibodies by day 28 after the first immunization, with a 10-fold increase by day 42 two weeks after the second immunization (Figure 22). Example 9. Pseudovirus neutralization of anti-COVID19 antibodies induced by PTX-B in humans

Neutralizing activity was further assessed by pseudovirus neutralization assay. Spike pseudotype ΔG-luciferase rVSV virus was generated in ES-293 cells following the protocol reported by Bewley K.R. (Bewley et al., Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays; Nature Protocols, 2021, 16, 3114-3140). Subject pseudovirus stocks to observation procedures to obtain optimal pseudovirus dilutions.

Serum samples from individuals vaccinated with PTX-B were heat treated to inactive components. Non-activated serum samples and reference serum were serially diluted. Incubate pre-diluted test serum and reference serum with pseudovirus diluent at a 1:1 ratio for 1 hour at 37°C. The serum and pseudovirus mixture was then transferred to the plated Vero E6 cells and incubated at 37°C and 5% CO2 for 18 to 22 hours.

Add ONE-Glo EX Luciferase Assay Reagent to the cell plate. The reaction plate was incubated at room temperature for 3 minutes and luciferase levels were read. The midpoint of each curve was determined using 4PL regression using the SoftMax Pro protocol, and the output is reported as the neutralization titer ( _NT50 ) for each sample. The PTX-B vaccine produced neutralizing titers comparable to those obtained in convalescent patients after the first dose (day 28). Two weeks after the second dose of PTX-B (day 42), neutralizing titers increased further and were higher than those obtained in patients since the recovery phase (Figure 24).

After the first dose (day 28; before receiving the second dose), the median neutralization levels reached 0.3 (16 μg dose and 40 μg dose) and 0.8 (100 μg dose) times the levels in convalescent patients, respectively. Two weeks after the second dose (day 42), median neutralization levels increased to 4.0 times (16 μg dose), 8.5 times (40 μg dose), and 23.0 times (100 μg dose) the levels in convalescent patients. The 100 μg dose induced higher binding and neutralizing antibody titers compared to lower doses.

The neutralizing activity of neutralizing antibodies induced by PTX-B vaccine was compared with COVID19 mRNA vaccines BNT162b2 and mRNA-1273. As shown in Tables 12 and 13, the average neutralizing level of neutralizing antibodies induced by the PTX-B vaccine was comparable to that of BNT162b2. Table 12. Average neutralization levels of PTX-B (multiples of recovery period (n=33)) sky 16µg (n=15) 40µg (n=14) 100µg (n=15) 28 0.3 0.3 0.8 42 4.0 8.6 23.0 Table 13. Average neutralization level of BNT162b2* (multiple of recovery period) sky 10µg 20µg 30µg twenty one 0.2 0.2 0.1 28 1.7 3.9 3.8 35 1.0 3.1 1.7 * Data from Walsh, EE et al., Safety and immunogenicity of two RNA based COVID-19 vaccine candidates, N Engl. J. Med ., 2020, 383(25): 2439-2450.

Similarly, compared with the data reported by Anderson EJ et al., Safety and immunogenicity of SARS-CoV-2 mRNA 1273 vaccine in older adults. N Engl. J. Med ., 2020, 383: 2427-2438, PTX-B vaccine The neutralizing activity of the induced neutralizing antibodies was comparable to the neutralizing activity induced by the mRNA-1273 vaccine.

Predict PTX- using the model developed by Khoury , DS et al. The protective efficacy of B vaccine showed that PTX-B was equivalent to the nucleic acid vaccines BNT162b2 and mRNA-1273 (Figure 25). The neutralizing activity of PTX-B vaccine was higher than that of BNT162b2 and mRNA-1273 vaccines (Figure 25). The results showed that PTX-B elicited a stronger response in humans than BNT162b2 and mRNA-1273 vaccines. Example 10. Neutralizing ability of different SAR-CoV-2 variants of concern (VOC)

Identical sera from groups of individuals who received 2 doses (day 0 and day 28) of 16 μg, 40 μg or 100 μg PTX-B vaccine were tested for pseudovirus neutralizing activity against viral variants. Pseudoviruses of the original SAR-CoV-2 virus and its variant α, β and δ VOC were prepared for neutralization analysis. The results shown in Figure 26 indicate that the PTX-B vaccine induced comparable neutralizing activity against the original SAR-CoV-2 strain and alpha, beta and delta VOCs in all three dose cohorts (Figure 26). The neutralizing activity induced by high-dose PTX-B (100 μg) was generally higher than that induced by low-dose (16 μg) and medium-dose (40 μg) PTX-B. Neutralizing antibody responses to SARS-CoV-2 variants in vaccinated Ontario long-term care home residents and workers, 2021 August 8; MedRxiv preprint doi: https://doi.org/10.1101/2021.08.06.2126172) reported that the neutralizing ability obtained by BNT162b2 vaccination was equivalent. Equivalents and categories

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope is not intended to be limited to the above description, but rather as set forth in the appended claims.

In the scope of the claim, unless indicated to the contrary or otherwise obvious from the context, articles such as "a/an" and "the" may mean one or more than one. Unless indicated to the contrary or otherwise apparent from context, if one, more than one, or all group members are present in, used in, or otherwise associated with a given product or method are relevant, a claim or description that includes an "or" between one or more members of the group is deemed to be satisfied. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or method. This disclosure includes embodiments in which more than one or all of the group members are present in, used in, or otherwise associated with a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and permits, but does not require, the inclusion of additional elements or steps. When the term "comprises" is used herein, it also encompasses and discloses the term "consisting of."

Where a range is given, the endpoints are included. Furthermore, it should be understood that, unless otherwise indicated or otherwise apparent from the context and ordinary skill in the understanding, values expressed as ranges may be employed in different embodiments using any specific value or subrange within the stated range, unless The context otherwise expressly provides otherwise, or one-tenth of the unit up to the lower end of the range.

In addition, it should be understood that any specific embodiments belonging to the prior art may be expressly excluded from any one or more technical solutions. Because such embodiments are believed to be known to those of ordinary skill in the art, they may be excluded, even if such exclusion is not expressly set forth herein. Any particular embodiment of a composition (e.g., any therapeutic or active ingredient; any method of production; any method of use; etc.) may be excluded from any one or more solutions for any reason, whether or not related to the existence of prior art.

It is to be understood that the words used are descriptive rather than limiting and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the disclosure in its broader form. .

Although the present disclosure has been described at some length and with some particularity with respect to several embodiments described, it is not intended that the disclosure be limited to any such details or implementations or to any particular implementation. Rather, Reference should be made to the accompanying claims for interpretation in order to provide the broadest possible interpretation of such claims considering the prior art and thus effectively encompass the intended scope of the present disclosure.

[Figure 1] shows the results of SARS-CoV-2 neutralization analysis using virus isolated from a patient in Ontario. Groups 1 to 5 relate to the vaccine formulation administered (see Table 6).

[Fig. 2] shows the results of neutralization analysis using SARS-CoV-2 pseudotyped lentivirus, which encodes a luciferase gene and can infect HEK293T cells. Groups 1 to 5 relate to the vaccine formulation administered (see Table 6).

[Figure 3] shows the ID50 (dilution at which 50% inhibition of infectivity is seen) of neutralization analysis of SARS-CoV-2 clinical isolates and pseudoviruses.

[Fig. 4] shows IFN-γ analysis by ELISpot to determine T cell response to PTX-B immunization.

[Fig. 5] shows the interleukin analysis performed by Luminex on the mice vaccinated with PTX-B on the 1st day and the 22nd day after the first dose and the additional dose.

[Fig. 6A] to [Fig. 6B] show interleukin analysis by flow cytometry of mice vaccinated with PTX-B on days 1 and 22 for the initial and additional injections.

[Fig. 7] shows changes in body weight of mice challenged with SARS-CoV-2.

[Fig. 8] shows the protective efficacy in the AAV6-hACE2 transduction mouse model.

[Fig. 9] shows lung histopathological scores in the AAV6-hACE2 transduced mouse model.

[Fig. 10] shows IFN-γ and IL-4 ELISpot analysis of spleen cells derived from PTX-B immunized mice.

[Fig. 11] Shows protection against infection by SARS-CoV-2 clinical isolates in SARS-CoV-2 neutralization analysis.

[Fig. 12] Shows protection against infection in pseudovirus neutralization analysis.

[Fig. 13A] to [Fig. 13C] show anti-SARS-CoV-2 anti-spike protein antibody profiles.

[Figure 14] shows that in a SARS-CoV-2 challenge study in hamsters, the levels of infectious virus were significantly reduced in vaccinated animals.

[Fig. 15] shows the calculation of T cell stimulation index in a co-culture experiment using SARS-CoV-2 N, M, and N/M protein nucleic acid vaccines.

[Fig. 16] Shows the characterization of serum/antibody neutralization analysis using pseudoviruses encoding S protein variants from SARS-CoV-2 variants of concern (VOC) and other variants.

[Figure 17] shows the protective efficiency (ID50) of PTX-B against infection by SARS-CoV-2 VOC pseudovirus and other pseudovirus variants.

[Figure 18] shows the protective efficiency (ID50) of PTX-B against infection by individual SARS-CoV-2 VOC pseudoviruses and other pseudovirus variants.

[Fig. 19] shows anti-spike protein IgG levels in individuals vaccinated with PTX-B on days 8, 28, and 42 after vaccination with 16, 40, or 100 μg doses.

[Fig. 20] Shows anti-spike protein IgG levels in placebo-treated control individuals compared with levels in the plasma of SARS-CoV-2 convalescent patients.

[Fig. 21] shows the neutralizing activity in samples from individuals vaccinated with PTX-B on days 8, 28, and 42 after vaccination with doses of 16, 40, or 100 μg.

[Figure 22] Shows anti-COVID-19 neutralizing antibody levels in samples from individuals vaccinated with PTX-B on days 8, 28, and 42 after vaccination with 16, 40, or 100 μg doses.

[Fig. 23] Shows anti-COVID-19 neutralizing antibody concentrations from individuals vaccinated with PTX-B on days 28 and 42 after vaccination with 16, 40, or 100 μg doses.

[Figure 24] Shows pseudotyped virus neutralization by anti-COVID-19 neutralizing antibodies from individuals vaccinated with PTX-B on days 28 and 42 after vaccination with 16, 40, or 100 μg doses.

[Fig. 25] shows the prediction of the protective efficacy of PTX-B based on the Khoury model.

[Fig. 26] shows the neutralizing activity induced by PTX-B against the original strain of SAR-CoV-2 and α, β and δ VOCs.

TW202330923A_111136145_SEQL.xml

Claims (59)

A polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein, and wherein the polynucleotide includes a first sequence region, the third A sequence region includes a nucleic acid sequence that is at least 80% identical to a member of the group consisting of SEQ ID NOs: 7, 20, 26, 27, and 32. The polynucleotide of claim 1, wherein the first sequence region is at least 95% identical to SEQ ID NO: 7. The polynucleotide of claim 2, wherein the first sequence region is at least 99% identical to SEQ ID NO: 7. The polynucleotide of claim 3, wherein the first sequence region consists of SEQ ID NO: 7. The polynucleotide of claim 4, wherein the encoded spike protein has the protein sequence of SEQ ID NO: 2. The polynucleotide of any one of claims 1 to 5, wherein at least 50% of the polynucleotide sequence is codon optimized. The polynucleotide of claim 5, wherein the polynucleotide is DNA. The polynucleotide of claim 5, wherein the polynucleotide is RNA. The polynucleotide of claim 8, wherein the polynucleotide is mRNA. The polynucleotide of claim 9, which includes a 5'UTR and a 3'UTR, wherein the 5'UTR includes SEQ ID NO: 47 and the 3'UTR includes SEQ ID NO: 48. The polynucleotide of claim 10, wherein at least one uridine nucleoside is modified to N1-methylpseudouridine. The polynucleotide of claim 11, wherein all uracil nucleosides are modified to N1-methylpseudouridine. A nucleic acid vaccine comprising the polynucleotide of claim 12. As claimed in claim 13, the nucleic acid vaccine is formulated in lipid nanoparticles (LNP). For example, the nucleic acid vaccine of claim 14, wherein the LNP contains (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 %. Ear% conjugated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (a) 50 to 65 mol% of cationic lipids; (b) non-cationic lipids, which contains 4 to 10 mol% of phospholipids and 30 to 40 mol% of Cholesterol; and (c) 0.5 to 2 mol% of conjugated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0 to 10 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0 to 10 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0 to 10 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterol; and (d) about 0.5 to 10 mol% PEGylated lipid. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0.5 to 3 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterol; and (d) about 0.01 to 10 mol% PEGylated lipid. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterol; and (d) about 0.1 to 20 mol% PEGylated lipid. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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 a pegylated lipid. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0.5 to 2 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0.5 to 20 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 5 to 10 mol% PEGylated lipids. Such as the nucleic acid vaccine of claim 14, wherein the LNP contains (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% % sterols; and (d) about 0.5 to 15 mol% PEGylated lipids. For example, the nucleic acid vaccine of claim 14, wherein the LNP contains (a) 45 to 55 mol% of 3D-P-DMA; (b) 8 to 10 mol% of DSPC; (c) 36 to 42 mol% of DSPC Cholesterol; and (d) 1.4 to 1.8 mol% PEG-DMA. For example, the nucleic acid vaccine of claim 29, wherein the 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% PEG-DMA. A pharmaceutical composition comprising the nucleic acid vaccine of claim 30 and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 31, which contains about 0.2 mg/mL of the mRNA described in claim 9. The pharmaceutical composition of claim 32 is suitable for intramuscular (IM) injection. For example, the pharmaceutical composition of claim 32 is packaged in a 3 mL glass vial with a 2 mL filler. A method of inducing an immune response in an individual, comprising administering a nucleic acid vaccine according to any one of claims 13 to 30 or a pharmaceutical composition according to any one of claims 31 to 34. The method of claim 35, wherein the immune response includes a T cell response. The method of claim 35, wherein the immune response includes a B cell response. The method of claim 35, further comprising administering a booster after the first administration. The method of claim 35, wherein the immune response of the subject is dose-dependent. The method of claim 39, wherein the dose-dependent immune response comprises inducing one or more of SARS-CoV-2 spike protein-specific IgG, IgG1, IgG2a, IgG2b, IgM and IgA antibodies in the individual. The method of claim 39, wherein the dose-dependent immune response includes inducing one or more of IL-2+ T cells, IL-4+ T cells, and IFN-γ+ T cells. A method of treating COVID-19 in an individual, comprising administering to the individual a nucleic acid vaccine according to any one of claims 13 to 30 or a pharmaceutical composition according to any one of claims 31 to 34. A method of preventing COVID-19 in an individual, comprising administering to the individual a nucleic acid vaccine according to any one of claims 13 to 30 or a pharmaceutical composition according to any one of claims 31 to 34. A method of alleviating or improving the physiological effects or symptoms of COVID-19 in an individual, comprising administering to the individual a nucleic acid vaccine according to any one of claims 13 to 30 or a medicine according to any one of claims 31 to 34 composition. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is from about 5 µg to about 100 µg. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 16 μg. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 40 μg. The method of any one of claims 42 to 44, wherein the dose of the mRNA administered to the individual is about 100 μg. The method of any one of claims 42 to 44, wherein the volume of the pharmaceutical composition administered to the individual is from about 0.025 mL to about 0.5 mL. 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. The method of any one of claims 42 to 50, comprising administering a second dose of the nucleic acid vaccine or pharmaceutical composition between about 1 week and about 5 weeks after the first dose. The method of claim 51, wherein the second dose is administered about 4 weeks after the first dose. The method of any one of claims 42 to 44, wherein anti-spike protein IgG antibodies are detected in the individual as of day 28 after receiving the first dose of the nucleic acid vaccine or pharmaceutical composition. The method of claim 53, wherein the amount of the anti-spike protein IgG antibody present in the individual increases by day 42 after receiving the second dose of the nucleic acid vaccine or pharmaceutical composition. 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. A method for inducing the production of SARS-CoV-2 neutralizing antibodies in an individual, which includes administering to the individual a nucleic acid vaccine according to any one of claims 13 to 30 or a pharmaceutical composition according to any one of claims 31 to 34 things. The method of claim 56, wherein the SARS-CoV-2 neutralizing antibody can be detected in the individual as of day 28 after administration. The method of claim 57, wherein the production of the SARS-CoV-2 neutralizing antibody in the individual increases on day 42 after receiving the second dose of the nucleic acid vaccine or pharmaceutical composition. The method of claim 57, wherein the level of SARS-CoV-2 neutralizing antibodies increases 10-fold by day 42.

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