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

Abstract

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

Description

[Technical Field]

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

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel coronavirus strain that began infecting mammals in China in 2019 and has spread to a pandemic. SARS-CoV-2 infection causes coronavirus disease 2019 (known as "COVID-19"), which affects mammals in various ways, ranging from asymptomatic individuals to those with a wide range of symptoms from mild to severe illness or death.

[0003] Vaccines are an effective way to provide preventative protection against infectious diseases. Currently, there are limited vaccines available for the prevention, mitigation, and / or treatment of COVID-19. Treatment for COVID-19 is limited to managing the symptoms and / or side effects of the disease. Therefore, there remains a strong need for COVID-19 vaccines, including formulations that deliver vaccines to a range of different target T cells. [Summary of the Invention]

[0004] This invention provides a nucleic acid vaccine, compositions and formulations comprising the nucleic acid vaccine, and methods of using the same to prevent coronavirus infection for the prevention, mitigation, and treatment of COVID-19. The nucleic acid vaccine may include a polynucleotide encoding at least one antigenic protein of SARS-CoV-2, a fragment thereof, or a variant thereof. The SARS-CoV-2 antigenic protein is a structural protein of SARS-CoV-2. The structural protein may be a spike protein, a membrane protein, a nucleocapsid phosphoprotein, or an envelope protein. Non-limiting examples of the amino acid sequences of such structural proteins are shown in Table 1 (SEQ ID Nos: 1-6 and 15-19).

[0005] This document provides a COVID-19 nucleic acid vaccine for a method of vaccinating an individual against COVID-19, wherein the nucleic acid vaccine may include at least one polynucleotide encoding at least one structural protein of SARS-CoV-2 or a fragment thereof.

[0006] This document provides a method for inducing an immune response in an individual by administering an effective amount of the nucleic acid vaccine described herein. The immune response may be, but is not limited to, a T-cell response or a B-cell response. As a non-limiting example, the immune response may be generated by a single administration of the nucleic acid vaccine described herein. As another non-limiting example, the immune response may be generated by a booster administration of the nucleic acid vaccine described herein. Administration of the pharmaceutical composition may generate a dose-responsive immune response in an individual. As a non-limiting example, a dose-responsive immune response may include the induction of one or more of SARS-CoV-2 spike protein-specific IgG, IgG1, IgG2a, IgG2b, IgM, and IgA antibodies in the individual. As another non-limiting example, a dose-responsive immune response may include the 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 reactions in the individual.

[0007] This article provides a method for treating and / or preventing COVID-19 in individuals by administering the nucleic acid vaccine described herein.

[0008] This article provides pharmaceutical compositions and formulations for the treatment and prevention of nucleic acid vaccines for COVID-19.

[0009] The nucleic acid vaccines described herein can be formulated in one or more lipid nanoparticles (LNPs).

[0010] This document also provides a nucleic acid vaccine for COVID-19, comprising approximately 0.2 mg / mL mRNA, wherein the mRNA contains a coding region having a nucleic acid sequence that is at least 95% identical to that of SEQ ID NO: 7. In some embodiments, the mRNA of the nucleic acid vaccine disclosed herein contains a coding region having a nucleic acid sequence as described in SEQ ID NO: 7. The nucleic acid vaccine can be prepared as a 2 mL filler in a 3 mL glass vial.

[0011] In some embodiments, administering a nucleic acid vaccine to an individual comprises administering approximately 5 µg to approximately 100 µg of mRNA to the individual. For example, the method may comprise administering approximately 16 µg of mRNA to the individual. Alternatively, the method may comprise administering approximately 40 µg of mRNA to the individual. Alternatively, the method may comprise administering approximately 100 µg of mRNA to the individual.

[0012] In some embodiments, administering a nucleic acid vaccine to an individual comprises administering approximately 0.025 mL to approximately 0.5 mL of the nucleic acid vaccine to the individual. For example, the method may include administering approximately 0.025 mL of the nucleic acid vaccine to an individual, administering approximately 0.05 mL of the nucleic acid vaccine to an individual, administering approximately 0.08 mL of the nucleic acid vaccine to an individual, administering approximately 0.2 mL of the nucleic acid vaccine to an individual, or administering approximately 0.5 mL of the nucleic acid vaccine to an individual.

[0013] In some embodiments of the provided method, delivery includes intramuscular (IM) injection of a nucleic acid vaccine into an individual.

[0014] Nucleic acid vaccines may be administered to individuals in the form of a first dose of nucleic acid vaccine, followed by a second dose of nucleic acid vaccine approximately 1 to 5 weeks later. In some embodiments, the second dose of nucleic acid vaccine is administered approximately 4 weeks after the first dose.

[0015] In some embodiments, anti-spike protein IgG antibodies were detected in individuals up to day 28 after receiving the first dose of the nucleic acid vaccine.

[0016] In some embodiments, anti-spike protein IgG antibodies were detected in individuals up to day 28 after receiving the first dose of the nucleic acid vaccine, and were enhanced in individuals up to day 42 after receiving the second dose of the nucleic acid vaccine.

[0017] In some embodiments, the number of anti-spike protein IgG antibodies in an individual is increased by up to 10 times compared to the average number of anti-spike protein IgG antibodies in serum samples from recovered SARS-CoV-2 patients.

[0018] In some embodiments, SARS-CoV-2 neutralizing antibodies were detected in individuals up to day 28 after the first dose of the nucleic acid vaccine.

[0019] In some embodiments, SARS-CoV-2 neutralizing antibodies are enhanced in individuals up to day 42 after the second dose of the nucleic acid vaccine.

[0020] Therefore, the present invention provides a COVID-19 nucleic acid vaccine for a method of administering a COVID-19 vaccine to an individual, wherein the nucleic acid vaccine comprises about 0.2 mg / mL mRNA, wherein the mRNA comprises a nucleic acid sequence having at least 95% identity with SEQ ID NO: 7, and wherein the nucleic acid vaccine is formulated for intramuscular (IM) injection and formulated in lipid nanoparticles (LNP).

[0021] Details of various embodiments are set forth in the following embodiments. Other features, objectives, and advantages will become apparent from the embodiments and the scope of the claims.

Implementation Method

[0048] Cross-reference to related applications

[0049] This application claims Canadian Application No. 3,096,009, filed on October 9, 2020; Application No. 3,107,232, filed on January 26, 2021; Application No. 3,113,094, filed on March 23, 2021; Application No. 3,116,284, filed on April 23, 2021; and Application No. 3,096,009, filed on April 30, 2021. Priority claims to No. 116,932; No. 3,118,329, filed May 12, 2021; No. 3,128,078, filed August 9, 2021; No. 3,128,660, filed August 19, 2021; and No. 3,132,188, filed September 28, 2021, the contents of which are each incorporated herein by reference in their entirety. Sequence List

[0050] This application is filed together with an electronic sequence list. The sequence list file, named 2092_1004PCT_SL.txt, was created on October 1, 2021, and has a size of 198,523 bytes. Information from the electronic sequence list is incorporated herein by reference in its entirety. I. Introduction

[0051] The following description sets forth exemplary compositions, methods, parameters, and the like. However, it should be understood that this description is not intended to limit the scope of the invention, but rather is provided instead as an illustrative example.

[0052] This document describes nucleic acid vaccines, specifically polynucleotides (e.g., mRNA), compositions, formulations, methods, and / or uses of nucleic acid vaccines comprising polynucleotides encoding one or more antigenic proteins of SARS-CoV-2, fragments thereof, or variants thereof, for the prevention, mitigation, and / or treatment of COVID-19. The antigenic protein may be a structural protein of SARS-CoV-2. The structural protein may be a spike (S) protein, a membrane (M) protein, a nucleocapsid (N) phosphoprotein, or an envelope (E) protein.

[0053] In some embodiments, at least one component of the nucleic acid vaccine is a polynucleotide encoding at least one of an antigenic protein of SARS-CoV-2 or a fragment or variant of the antigenic protein. The antigenic protein may be a structural protein of SARS-CoV-2. The polynucleotide may be an RNA polynucleotide, such as an mRNA polynucleotide.

[0054] In some embodiments, the nucleic acid vaccine includes at least one mRNA polynucleotide encoding at least one of the structural proteins of SARS-CoV-2 or fragments or variants of the structural protein.

[0055] In some embodiments, the polynucleotide may be designed to encode one or more polypeptides of interest from SARS-CoV-2, or fragments or variants thereof. Such polypeptides of interest from SARS-CoV-2 may include, but are not limited to, complete polypeptides, multiple polypeptides, polypeptide fragments, or polypeptide variants, which may be independently encoded by one or more regions, or partially or entirely, of a polynucleotide from SARS-CoV-2. As used herein, the term "polynucleotide of interest" means any polypeptide selected to encode within the polynucleotide described herein or whose function is influenced by the polynucleotide described herein. Any of the peptides or polypeptides described herein may be antigenic (also known as immunogenic).

[0056] As used herein, "peptide" means a polymer of amino acid residues (natural or non-natural) most commonly linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function or origin. In some embodiments, the polypeptide of interest is an antigen encoded by a polynucleotide as described herein.

[0057] In some embodiments, the encoded polypeptide is less than about 50 amino acids, and the polypeptide is hereinafter referred to as a peptide. If the polypeptide is a peptide, its length will be at least about 2, 3, 4, or at least 5 amino acid residues. Therefore, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be single molecules or multi-molecule complexes, such as dimers, trimers, or tetramers. They may also comprise single-chain 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 amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids.

[0058] The term "peptide variant" refers to a molecule whose amino acid sequence differs from the native or reference sequence. Compared to the native or reference sequence, the amino acid sequence variant may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence. Typically, the variant will have at least about 50% homology with the native or reference sequence, and more preferably, it will have at least about 80%, or at least about 85%, more preferably at least about 90%, or even more preferably at least about 95% homology with the native or reference sequence.

[0059] In some embodiments, a "variant mimic" is provided. As used herein, the term "variant mimic" is a mimic containing one or more amino acids that will mimic the activation sequence. For example, glutamate can serve as a mimic of azophospho-threonine and / or azophospho-serine. In some embodiments, an "amino acid sequence variant" is provided.

[0060] When applied to amino acid sequences, "homology" is defined as the percentage of similarity between residues in a candidate amino acid sequence and residues in a second amino acid sequence after sequence alignment and, if necessary, the introduction of vacancies to achieve the maximum percentage of homology. The methods and computer programs used for alignment are well known in the art. It should be understood that homology depends on the calculation of the percentage of similarity, but its value may vary due to vacancies and penalties introduced in the calculation.

[0061] When applied to polypeptide sequences, “homology” refers to the corresponding sequence of another species that has a substantially similarity to the second sequence of the second species.

[0062] As used herein, “analyte” is intended to include peptide variants that differ in that one or more amino acid alterations, such as substitution, addition or deletion of amino acid residues that still maintain one or more characteristics of the parent or starting peptide.

[0063] In some embodiments, the present invention covers several types of peptide-based compositions, including variants and derivatives. These include substitutional, insertional, deletion, and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant," but generally refers to a molecule that is modified and / or altered in any way relative to a reference molecule or a starting molecule.

[0064] For example, a sequence tag or amino acid, such as one or more lysines, may be added to the peptide sequence described herein (e.g., at the N-terminus or C-terminus). The sequence tag can be used for peptide purification or localization. Lysines can be used to improve peptide solubility or allow biotin labeling. Alternatively, amino acid residues in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein may be omitted as appropriate, thereby providing a truncated sequence. Alternatively, depending on the intended use of the sequence, such as when the sequence is part of a larger, soluble sequence linked to a solid carrier, certain amino acids (e.g., C-terminal or N-terminal residues) may be omitted.

[0065] When referring to a polypeptide, a “substitution variant” is a variant in which at least one amino acid residue in the native or starting sequence is removed and replaced by a different amino acid inserted at the same position. The substitution can be monosubstituted, in which only one amino acid in the molecule is substituted, or the substitution can be polysubstituted, in which two or more amino acids in the same molecule are substituted.

[0066] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include the substitution of another nonpolar residue with a nonpolar (hydrophobic) residue such as isoleucine, valine, or leucine. Similarly, examples of conservative substitution include the substitution of another polar residue with a polar (hydrophilic) residue, such as between arginine and lysine, between glutamic acid and aspartic acid, and between glycine and serine. Additionally, the substitution of another basic residue with a basic residue such as lysine, arginine, or histidine, or the substitution of another acidic residue with an acidic residue such as aspartic acid or glutamic acid, are further examples of conservative substitution. Examples of nonconservative substitutions include replacing nonpolar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, and methionine with polar (hydrophilic) residues such as cysteine, glutamic acid, glutamic acid, or lysine and / or replacing polar residues with nonpolar residues.

[0067] When referring to a polypeptide, an "insertion variant" is a variant in which one or more amino acids are inserted at a specific position adjacent to the native or starting sequence. "Immediately adjacent" amino acid means that it is connected to the α-carboxyl or α-amino functional group of the amino acid.

[0068] When referring to peptides, "deletional variants" are variants in which one or more amino acids are removed from the native or starting amino acid sequence. Typically, deletion variants will have one or more amino acid deletions in a specific region of the molecule.

[0069] When referring to polypeptides, "covalent derivatives" include modifications and / or post-translational modifications of native or starting proteins using organic or non-protein derivatizing agents. Covalent modifications are conventionally introduced by reacting the target amino acid residues of the protein with an organic derivatizing agent capable of reacting with selected side-chain or terminal residues, or by utilizing post-translational modification mechanisms that function in selected recombinant host cells. The resulting covalent derivatives are suitable for procedures targeting residues important for identifying biological activity, immunoassays, or for the immunoaffinity purification of recombinant glycoproteins to prepare anti-protein antibodies. Such modifications are performed within the capabilities of a person generally skilled in the art and without improper experimentation.

[0070] When referring to a polypeptide, "characteristic" is defined as a unique component of the molecule based on its amino acid sequence. Characteristic features of polypeptides encoded by polynucleotides as described herein include surface appearance, local conformational shape, folds, loops, hemi-loops, domains, semi-domains, sites, terminals, or any combination thereof.

[0071] As used herein, when referring to a polypeptide, the term “surface appearance” refers to the polypeptide-based component of a protein that appears on the outermost surface.

[0072] As used herein, when referring to a polypeptide, the term “local conformational shape” means the polypeptide-based protein structural representation located within a definable protein space.

[0073] As used herein, when referring to polypeptides, the term "fold" refers to the configuration of the amino acid sequence obtained at the lowest possible energy. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary folding include β-sheets and α-helices. Examples of tertiary folding include domains and regions formed by the aggregation or separation of forces with higher energy. Regions formed in this manner include hydrophobic and hydrophilic pockets, and analogues thereof.

[0074] As used herein, when relating to peptide conformation, the term “turn” means a change in the orientation of the backbone of a peptide or polypeptide and may involve bending of one, two, three or more amino acid residues.

[0075] As used herein, when referring to a polypeptide, the term "loop" refers to a structural feature of the polypeptide that can be used to reverse the orientation of the polypeptide backbone. When a loop is found in a polypeptide and only the orientation of the backbone is changed, it may contain four or more amino acid residues. Oliva et al. have identified at least five classes of protein loops (J. Mol Bio., l266 (4): 814-830; 1997). The loop may be open or closed. A closed loop or "cyclic" loop may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between bridging portions. Such bridging portions may be contained in the cysteine-cysteine ​​bridge (Cys-Cys) typical of polypeptides with disulfide bonds, or the bridging portions may be non-protein-based, such as the dibromoacetic acid agent used herein.

[0076] As used herein, when referring to a polypeptide, the term "semi-cyclic" means a portion of the identified ring having at least half of the amino acid residues of the ring from which that portion is derived. It should be understood that a ring may not always contain an even number of amino acid residues. Thus, in cases where a ring contains or is identified as containing an odd number of amino acids, the semi-cyclic part of an odd-numbered ring will contain an integer portion of that ring or the next integer portion (number of amino acids in the ring / 2 + / - 0.5 amino acids).

[0077] As used herein, when referring to a polypeptide, the term "domain" means a polypeptide motif having one or more identifiable structural or functional features or properties (e.g., binding force, acting as a protein-protein interaction site).

[0078] As used herein, when referring to a polypeptide, the term "half-domain" means a portion of an identified domain that has at least half of the amino acid residues of the domain from which that portion is derived. It should be understood that a domain may not always contain an even number of amino acid residues. Therefore, in cases where a domain contains or is identified as containing an odd number of amino acids, the half-domain of an odd-numbered domain will contain an integer portion of that domain or the next integer portion (the number of amino acids in the domain / 2 + / - 0.5 amino acids). For example, a domain identified as containing 7 amino acids may produce a half-domain containing 3 or 4 amino acids (7 / 2 = 3.5 + 1 - 0.5 = 3 or 4). It should also be understood that subdomains may be identified within a domain or half-domain, and these subdomains may not possess all the structural or functional characteristics identified in the domain or half-domain from which they are derived. It should also be understood that amino acids containing any of the domain types mentioned herein do not necessarily have to be linked along the polypeptide backbone (i.e., non-adjacent amino acids can fold structurally to create domains, half-domains, or subdomains).

[0079] 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. A site indicates a location within a peptide or polypeptide that may be modified, manipulated, altered, derived, or varied within the molecule of the polypeptide described herein.

[0080] As used herein, when referring to a polypeptide, the term "termini" or "terminus" refers to the end of a peptide or polypeptide. Such a terminus is not limited to the first or last site of the peptide or polypeptide, but may include other amino acids in the terminal region. The polypeptide-based molecules described herein may be characterized by having an N-terminus (terminated by an amino acid having a free amino group (NH2)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). In some cases, the proteins described herein consist of multiple polypeptide chains (polymers, oligomers) linked together by disulfide bonds or by non-covalent forces. Such proteins will have multiple N-termini and C-termini. Alternatively, the terminus of a polypeptide may be modified such that, depending on the specific circumstances, it begins or ends with a non-polypeptide-based portion (such as an organic conjugate).

[0081] Once any feature is identified or defined as a desired component of the polypeptide encoded by the polynucleotide described herein, any of several manipulations and / or modifications of such feature may be performed by moving, exchanging, reversing, deleting, randomizing, or copying. Furthermore, it should be understood that feature manipulation can produce the same results as the molecular modifications described herein. For example, manipulation involving domain deletion will result in a change in molecular length, just as modifying nucleic acids to encode molecules smaller than full length.

[0082] In peptides, the term "modification" refers to a modification relative to a standard set of 20 amino acids. Modifications can be various. In some embodiments, these regions may contain one, two, or more (as the case may) modifications.

[0083] Modification and manipulation can be achieved by methods known in this art, such as, but not limited to, site-directed mutagenesis or a priori incorporation during chemical synthesis. The activity of the modified molecule can then be tested using in vitro or in vivo analyses, such as those described herein or any other suitable screening analyses known in this art.

[0084] In some embodiments, the polypeptide may comprise a common sequence discovered through several rounds of experiments. As used herein, a "common" sequence is a single sequence, which represents a set of sequences that allow for variation at one or more sites.

[0085] As those skilled in this art will recognize, protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of the relevant polypeptide. For example, this document provides any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical to the reference protein). The length of the protein fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids. In another example, any protein comprising an elongated segment of about 20, about 30, about 40, about 50, or about 100 or more amino acids may be used according to the nucleic acid vaccine described herein, such amino acids being approximately 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any of the sequences described herein. In some embodiments, the polypeptide used according to the nucleic acid vaccine described herein includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.

[0086] Therefore, the polynucleotides of the present invention encode peptides or polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications relative to the reference sequence, specifically the peptide or polypeptide sequence disclosed herein. The polynucleotides may also contain substitutions, insertions and / or additions, deletions and covalent modifications relative to the polynucleotide reference sequence.

[0087] A reference molecule (peptide or polynucleotide) may share a certain degree of consistency with the designed molecule (peptide or polynucleotide). As is known in this art, the term "consistency" refers to the relationship between the sequences of two or more peptides, polypeptides, or polynucleotides, as determined by comparing such sequences. In this art, consistency also means the degree of sequence correlation between them, as determined by the number of matches between strings of two or more amino acid residues or nucleosides. Consistency measures the percentage of consistent matches between the smaller of two or more sequences using gap alignments (if present), which are processed by a specific mathematical model or computer program (e.g., an "algorithm"). The consistency of related peptides can be readily calculated using known methods. Such methods include, but are not limited to, those described in: *Computational Molecular Biology*, Lesk, AM, ed., Oxford University Press, NY, 1988; *Biocomputing: Informatics and Genome Projects*, Smith, DW, ed., Academic Press, NY, 1993; *Computer Analysis of Sequence Data*, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, NJ, 1994; *Sequence Analysis in Molecular Biology*, von Heinje, G., Academic Press, 1987; *Sequence Analysis Primer*, Gribskov, M. and Devereux, J., ed., M. Stockton Press, NY. 1991; and Carillo et al., SIAM J. Applied Math. 48: 1073; 1988).

[0088] In some embodiments, the encoded polypeptide variant may have the same or similar activity as the reference polypeptide. Alternatively, the variants can have altered activity (e.g., increased or decreased) relative to the reference polypeptide. In general, a variant of a particular polynucleotide or polypeptide described herein will have at least about 40%, 45%, 50 with a particular reference polynucleotide or polypeptide, as determined by resorting to sequence alignment programs and parameters known herein and by those familiar with the technology %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence consistency. Such alignment tools include the other tools of the BLAST program group (Stephen F. Altschul et al., Gapped BLAST and PSLBLAST: a new generation of protein database search programs, Nucleic Acids Res. 1997, 25:3389–3402). Other tools are described in this paper, specifically in the definition of “consistency”. II.The composition of the present invention SARS-CoV-2

[0089] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus strain that can cause the coronavirus disease known as "COVID-19" COVID-19 affects mammals in different ways, ranging from asymptomatic individuals to individuals with a wide range of symptoms in the range from mild symptoms to severe disease or death. To date, about 80% of COVID-19 patients have mild to moderate symptoms, and about 20% can develop complications such as severe pneumonia, acute respiratory distress syndrome, sepsis, and even death. The list of symptoms associated with COVID-19 is constantly changing as doctors and scientists learn more about COVID-19 and how it affects the body, but some of the symptoms recognized to date include fever or chills, cough, shortness of breath or shortness of breath, fatigue, body aches, muscle soreness, headache, sore throat, congestion or runny nose, nausea and / or vomiting, diarrhea and a new loss of taste or smell.

[0090] The SARS-CoV-2 genome encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as non-structural proteins (named nsp1 to nsp16) and accessory proteins. The viral spike protein is typically the primary mediator for viral entry into cells. The SARS-CoV-2 spike protein binds to its receptor, human ACE2 (hACE2), via its receptor-binding domain (RBD) and is activated by human proteases via proteolytic degradation. Another characteristic of the SARS-CoV-2 spike protein is the presence of a functional furin cleavage site at the S1-S2 boundary (S1 being the receptor-binding unit and S2 the membrane fusion unit). This site can pre-activate many viruses, including SARS-CoV-2, for entry. The SARS-CoV-2 spike protein has been used as a protective antigen to elicit neutralizing antibodies in various vaccine development strategies. Membrane and envelope proteins are used for viral assembly. The envelope protein (E) forms a homopentamer cation channel that is important for viral pathogenicity (Mandala et al., Nature Structural and Molecular Bio. 2020, 27: 1202-1208). The nucleocapsid protein packages the viral genome into a helical ribonucleocapsid (RNP) and plays a role in viral self-assembly (Chang et al.; The SARS coronavirus nucleocapsid protein - Forms and functions; Antiviral Res. 2014; 103:39-50; the contents of which are incorporated herein by reference in full). Furthermore, nucleocapsid proteins in SARS-CoV-2 can regulate host cell mechanisms and participate in the regulation of the viral life cycle.

[0091] While not wishing to be bound by theory, it appears that SARS-CoV-2 binds to the human receptor ACE2 (hACE2). The receptor-binding domain (RBD) in the spike protein appears to be the most variable part of the coronavirus genome. Six RBD amino acids have been shown to be essential for binding to the ACE2 receptor, and the SARS-CoV-2 genome appears to possess an RBD that binds with high affinity to ACE2 in humans, ferrets, cats, and other species with high receptor homology (Anderson et al.; The Proximal Origin of SARS-CoV-2; Nature Medicine, 2020; 26(4): 450-452; the contents of which are incorporated herein by reference in their entirety).

[0092] In some embodiments, the polynucleotide encoding a full-length polypeptide of a structural protein of the nucleic acid vaccine described herein, or a fragment or variant of a structural protein of SARS-CoV-2, such as a spike protein, nucleocapsid protein, envelope protein, or membrane protein.

[0093] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode more than one fragment or variant of the structural proteins of SARS-CoV-2, such as spike proteins, nucleocapsid proteins, envelope proteins and / or membrane proteins.

[0094] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a mutant variant of one of the structural proteins, or a fragment of a mutant variant of a structural protein of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change in one of the structural proteins of SARS-CoV-2, where aspartic acid is changed to glycine.

[0095] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes 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 in the spike protein of SARS-CoV-2, where aspartic acid is changed to glycine. As a non-limiting example, the variant may be a single amino acid change in the spike protein of SARS-CoV-2, where aspartic acid at position 614 is changed to glycine (D614G) (Korber et al.; Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus; Cell; 2020, 182(4): 812-827; the contents of which are incorporated herein by reference in their entirety).

[0096] In some embodiments, the nucleic acid vaccine described herein may encode one or more proteins, peptides, fragments thereof, or variants of the structural proteins of SARS-CoV-2. Non-limiting examples of proteins, peptides, fragments thereof, or variants of the structural proteins of SARS-CoV-2 are provided in Table 1. NCBI reference numbers are also provided in the table where known. Table 1. Structural Protein Sequences of SARS-CoV-2 Serial number identifier (SEQ ID NO) describe sequence type 1 Spike protein (NCBI reference number: YP_009724390.1) (“S protein”) protein 2 spike protein with D614G mutation protein 3 Envelope proteins (NCBI reference number: YP_009724392.1) protein 4 Membrane proteins (NCBI reference number: YP_009724393.1) protein 5 Nucleocapsid phosphoprotein (NCBI reference number: YP_009724397.2) protein 6 B.1.351 (South Africa) variant spike protein protein 15 B.1.17 (UK) variant spike protein protein 16 Spike protein with D614G and L452R mutations protein 17 The B.1.17 (UK) variant spike protein with the L452R mutation protein 18 The B.1.351 (South Africa) variant spike protein with the L452R mutation protein 19 P.1 (Brazil) variant spike protein protein

[0097] 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.

[0098] In some embodiments, the nucleic acid vaccine may be an mRNA vaccine that produces one or more structural proteins, peptides, fragments or variants of SARS-CoV-2 upon translation. Therefore, the polynucleotide of the mRNA vaccine is an mRNA polynucleotide encoding one or more structural proteins, peptides, fragments or variants of SARS-CoV-2.

[0099] In one embodiment, the coding sequence of the mRNA vaccine described herein may be based on the coding sequence of the spike (S) protein from the genome of the SARS-CoV-2 Wuhan-Hu-1 isolate (Genbank: NM908947.3, complete genome sequence). In some embodiments, a code variation of a single amino acid change from D614 to G614 is introduced to match the amino acids of currently prevalent viral strains.

[0100] Non-limiting examples of RNA sequences encoding structural proteins, peptides, fragments, or variants of SARS-CoV-2 are provided in Table 2. Table 2. Sequences of the spike protein of SARS-CoV-2 Serial number identifier (SEQ ID NO) describe sequence type 7 The coding region of the spike protein with the D614G mutation RNA 20 The coding region of the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) RNA twenty one coding region of M protein RNA twenty two The coding regions of N and M proteins RNA twenty three coding region of N protein RNA twenty four Signal peptide and the coding regions of N and M proteins RNA 25 Sequence encoding the receptor-binding domain (RBD) of the S protein RNA 26 Sequence encoding the S protein of mRNA with a mutated furin protease site RNA 27 Sequence encoding the spike protein with the D614G mutation RNA 28 Sequence encoding the M protein RNA 29 Sequences encoding N and M proteins RNA 30 Sequence encoding N protein RNA 31 Sequence containing a signal peptide and encoding N and M proteins RNA 32 Sequence encoding the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) RNA

[0101] In some embodiments, the mRNA sequence encoding the spike protein of SARS-CoV-2 with the D614G mutation includes the coding region of SEQ ID NO: 7 or a fragment or variant thereof.

[0102] In some embodiments, the mRNA sequence encoding the spike protein of SARS-CoV-2 with the D614G mutation comprises SEQ ID NO: 27, or a fragment or variant thereof.

[0103] In some embodiments, the nucleic acid vaccine may comprise a region encoding any of the sequences listed in Table 1, or a fragment or variant thereof. The nucleic acid vaccine may comprise a hybrid or chimeric region, or a mimic or variant. In some embodiments, the nucleic acid vaccine may comprise any of the polynucleotide sequences listed in Table 3. Table 3. Exemplary sequences in nucleic acid vaccines for the treatment or prevention of COVID-19 Serial number identifier (SEQ ID NO) describe sequence type 8 Sequence encoding the M protein DNA 9 Sequences encoding N and M proteins DNA 10 Sequence encoding N protein DNA 11 Sequence containing a signal peptide and encoding N and M proteins DNA 12 Sequence encoding the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) DNA 33 The coding region of the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) DNA 34 coding region of M protein DNA 35 The coding regions of N and M proteins DNA 36 coding region of N protein DNA 37 Signal peptide and the coding regions of N and M proteins DNA 38 Sequence encoding the M protein DNA 39 Sequences encoding N and M proteins DNA 40 Sequence encoding N protein DNA 41 Sequence containing a signal peptide and encoding N and M proteins DNA 42 Sequence encoding the spike protein with the D614G mutation DNA 43 Sequence encoding the spike protein of SARS-CoV-2 variant B.1.351 (South African variant) DNA 50 Sequence encoding the receptor-binding domain (RBD) of the S protein DNA 51 Sequence encoding the S protein with a mutated furin protease site DNA

[0104] Any sequence or variant thereof mentioned in Tables 1-3 may also be used in the memory-enhancing vaccine described herein.

[0105] In some embodiments, the nucleic acid vaccine described herein encodes a protein, fragment thereof, or variant thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the protein provided by the amino acid sequences in Table 1. In the case of two or more polypeptide sequences, the term "identical" or percentage "identical" refers to the same two or more sequences. Percentage identity between polypeptide sequences can be determined using algorithms known in this art, such as BLAST and CLUSTAL.

[0106] The sequence of the SARS-CoV-2 protein or a fragment thereof or a variant thereof may be obtained from any source. In some embodiments, the sequence of the SARS-CoV-2 protein or a fragment thereof or a variant thereof is derived from a strain capable of infecting or at risk of infecting human individuals.

[0107] In some embodiments, the sequence of the SARS-CoV-2 protein or its fragments or variants may be modified or optimized (such as codon optimization) to be expressed in a particular cell or host organism.

[0108] In some embodiments, the nucleic acid vaccine described herein may be a multivalent vaccine. A multivalent vaccine may include a polynucleotide encoding at least two different proteins, peptides, fragments thereof, or variants of SARS-CoV-2. As a non-limiting example, the polynucleotide may encode the same or different structural proteins. As a non-limiting example, the polynucleotide may encode the same structural protein, but different variants of the structural protein.

[0109] In some embodiments, the nucleic acid vaccine encodes the full-length S protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes an S protein fragment of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a receptor binding domain (RBD) fragment of the spike protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the spike protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes an S protein sequence (e.g., full length, fragment, or variant) of SARS-CoV-2, wherein the S protein has a mutated furin cleavage site. S protein furin cleavage site mutants will remove or disable the furin cleavage site in the S protein (e.g. between the S1 and S2 borders). In some viral envelope proteins, disruption of furin cleavage sites was found to enhance performance and stability. In some embodiments, the nucleic acid vaccine encodes an S protein sequence (e.g., full length, fragment, or variant) of SARS-CoV-2, wherein the S protein includes a D614G mutation. Nucleic acid vaccines encoding the S protein of SARS-CoV-2, fragments or variants thereof may also include signal peptide and / or at least one linker (e.g., GSG linker) sequence and one or more of the sequences in the nucleic acid vaccine may be optimized by a codon.

[0110] In some embodiments, the nucleic acid vaccine encodes the full-length M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes an M protein fragment of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the topological domain of the M protein of SARS-CoV-2 (e.g., a virion surface or intraviral region). In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of an M protein (e.g., a full-length protein or fragment) of SARS-CoV-2. Nucleic acid vaccines encoding the M protein of SARS-CoV-2, fragments or variants thereof may also include signal peptide and / or at least one linker (e.g., GSG linker) sequence and one or more of the sequences in the nucleic acid vaccine may be optimized by a codon.

[0111] In some embodiments, the nucleic acid vaccine encodes the full-length N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a fragment of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the RNA-binding domain of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a dimer domain of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the N protein of SARS-CoV-2 (e.g., the full-length protein or a fragment). Nucleic acid vaccines encoding the N protein of SARS-CoV-2, its fragments, or variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences in the nucleic acid vaccine may be codon-optimized.

[0112] In some embodiments, the nucleic acid vaccine encodes the full-length E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a fragment of the SARS-CoV-2 E protein. In some embodiments, the nucleic acid vaccine encodes a topological domain (e.g., a region on or within a viral particle) of the SARS-CoV-2 E protein. In some embodiments, the nucleic acid vaccine encodes a transmembrane domain of the SARS-CoV-2 E protein. In some embodiments, the nucleic acid vaccine encodes a variant of the SARS-CoV-2 E protein (e.g., the full-length protein or a fragment). Nucleic acid vaccines encoding the SARS-CoV-2 E protein, its fragments, or variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences in the nucleic acid vaccine may be codon-optimized.

[0113] 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, their fragments, or variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences in the nucleic acid vaccine may be codon-optimized. In some embodiments, the sequences encoding two different structural proteins of SARS-CoV-2, their fragments, or variants, of the nucleic acid vaccine are constructed as a single polynucleotide.

[0114] In some embodiments, the nucleic acid vaccine encodes at least three different sequences of structural protein fragments of SARS-CoV-2 or their variants. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment, or variant sequences of SARS-CoV-2 and the M protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment, or variant sequences of SARS-CoV-2 and the N protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S protein, fragment, or variant sequences of SARS-CoV-2 and the E protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M protein, fragment, or variant sequences of SARS-CoV-2 and the S protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N protein, fragment, or variant sequences of SARS-CoV-2 and the S protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E protein, fragment, or variant sequences of SARS-CoV-2 and the S protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M protein, fragment, or variant sequences of SARS-CoV-2 and the N protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M protein, fragment, or variant sequences of SARS-CoV-2 and the E protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N protein, fragment, or variant sequences of SARS-CoV-2 and the M protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N protein, fragment, or variant sequences of SARS-CoV-2 and the E protein, fragment, or variant sequence of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E protein, fragment, or variant sequences of SARS-CoV-2 and the N protein, fragment, or variant sequence of SARS-CoV-2. In some 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, 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 E protein, fragment, or variant sequence of SARS-CoV-2.In some embodiments, the nucleic acid vaccine encodes the S protein, fragment, or variant sequence of SARS-CoV-2, the N protein, fragment, or variant sequence of SARS-CoV-2, and the E protein, fragment, or variant sequence of SARS-CoV-2. In 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, fragment, or variant sequence of SARS-CoV-2. Nucleic acid vaccines encoding at least three different sequences of structural protein fragments of SARS-CoV-2 or their variants may also include a signal peptide and / or at least one linker (e.g., the GSG linker) sequence, and one or more sequences in the nucleic acid vaccine may be codon-optimized. SARS-CoV-2 variants.

[0115] SARS-CoV-2 is a member of the Coronaviridae family. Multiple variants of SARS-CoV-2 (sometimes referred to as "virus strains" or "lineages") have been identified globally. The nomenclature of SARS-CoV-2 variants used in this specification is consistent with the PANGO nomenclature for new viral lineages (Rambaut, Andrew et al., A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nature microbiology, 2020, 5:: 1403-1407, the contents of which are incorporated herein by reference in their entirety). Near-real-time data related to PANGO SARS-CoV-2 lineages or variants can be accessed online using the Nextstrain SARS-CoV-2 analysis user interface (nextstrain.org / ncov / global).

[0116] Up to the present invention, multiple PANGO lineage variants of SARS-CoV-2 have been identified, including the following (the numbers in parentheses represent the number of cases in each submitted PANGO lineage: A (37); A.1 (8); A.11 (2); A.12 (1); A.19 (5); A.2 (6); A.2.2 (9); A.2.4 (5); A.2.5 (12); A.21 (8); A.22 (1); A.23 (2); A.23.1 (40); A.24 (2); A.25 (1); A.28 (4); A.3 (3); A.5 (5); A.6 (1); AD.2 (1); AE.1 (1); AE.2 (2); AE.4 (1); AE.5 (1); AE.7 (1); AE.8 (1); AG.1 (1); AY.1; AY.2; AY.3; B (47); B.1 (374); B.1.1 (237); B.1.1.1 (40); B.1.1.10 (2); B.1.1.111 (2); B.1.1.121 (1); B.1.1.133 (2); B.1.1.141 (5); B.1.1.142 (6); B.1.1.153 (6); B.1.1.157 (1); B.1.1.159 (3); B.1.1.160 (1); B.1.1.161 (1); B.1.1.163 (8); B.1.1.170 (1);B.1.1.174 (1); B.1.1.176 (2); B.1.1.180 (1); B.1.1.186 (2); B.1.1.189 (4); B.1.1.198 (2); B.1.1.200 (1); B.1.1.204 (2); B.1.1.207 (6); (22); B.1.1.216 (9); B.1.1.219 (1); B.1.1.222 (32); B.1.1.226 (1); B.1.1.230 (1); B.1.1.231 (4); B.1.1.232 (1); B.1.1.241 (1);B.1.1.243 (1); B.1.1.25 (26); B.1.1.263 (2); B.1.1.265 (1); B.1.1.27 (6); B.1.1.273 (1); B.1.1.274 (7); B.1.1.28 (34); B.1.1.280 (3); B.1.1.284 (5); B.1.1.294 (7); B.1.1.297 (1); B.1.1.300 (1); B.1.1.301 (1);B.1.1.304 (1);B.1.1.306 (5);B.1.1.312 (3);B.1.1.315 (2);B.1.1.316 (4);B.1.1.317 (8);B.1.1.318 (1);B.1.1.326 (1);B.1.1.328 (3);B.1.1.33 (14);B.1.1.330 (6);B.1.1.331 (1);B.1.1.333 (4);B.1.1.337 (2);B.1.1.344 (2);B.1.1.345 (1);B.1.1.348 (29);B.1.1.350 (1);B.1.1.351 (2);B.1.1.354 (7);B.1.1.355 (2);B.1.1.359 (2);B.1.1.365 (1);B.1.1.366 (1);B.1.1.368 (1);B.1.1.37 (1);B.1.1.372 (2);B.1.1.374 (5);B.1.1.375 (9);B.1.1.381 (1);B.1.1.383 (1);B.1.1.388 (1);B.1.1.389 (17);B.1.1.39 (3);B.1.1.394 (3);B.1.1.397 (4);B.1.1.398 (2);B.1.1.40 (2);B.1.1.404 (1);B.1.1.410 (3);B.1.1.411 (3);B.1.1.413 (3);B.1.1.416 (6);B.1.1.419 (2);B.1.1.420 (4);B.1.1.428 (2);B.1.1.429 (2);B.1.1.430 (1);B.1.1.432 (8);B.1.1.434 (1);B.1.1.447 (1);B.1.1.448 (2);B.1.1.451 (1);B.1.1.464 (2);B.1.1.485 (1);B.1.1.487 (5);B.1.1.50 (16);B.1.1.514 (2);B.1.1.516 (2);B.1.1.517 (1);B.1.1.519 (106);B.1.1.521 (1);B.1.1.54 (2);B.1.1.56 (1);B.1.1.57 (1);B.1.1.63 (7);B.1.1.7 (534);B.1.1.70 (10);B.1.1.71 (1);B.1.1.99 (1);B.1.108 (1);B.1.110.3 (1);B.1.111 (29);B.1.116 (1);B.1.126 (2);B.1.128 (3);B.1.13 (1);B.1.139 (2);B.1.146 (1);B.1.149 (1);B.1.153 (2);B.1.160 (65);B.1.160.14 (1);B.1.160.15 (1);B.1.160.25 (1);B.1.160.28 (1);B.1.160.8 (1);B.1.160.9 (1);B.1.164 (2);B.1.170 (2);B.1.177 (71);B.1.177.11 (1);B.1.177.12 (1);B.1.177.15 (1);B.1.177.18 (1);B.1.177.21 (7);B.1.177.32 (4);B.1.177.35 (1);B.1.177.4 (1);B.1.177.40 (2);B.1.177.42 (1);B.1.177.43 (1);B.1.177.44 (2);B.1.177.46 (3);B.1.177.49 (1);B.1.177.51 (1);B.1.177.52 (3);B.1.177.53 (1);B.1.177.54 (2);B.1.177.59 (1);B.1.177.6 (1);B.1.177.60 (23);B.1.177.68 (1);B.1.177.73 (6);B.1.177.76 (1);B.1.177.77 (1);B.1.177.78 (1);B.1.177.79 (1);B.1.177.81 (5);B.1.177.82 (1);B.1.177.83 (1);B.1.177.86 (3);B.1.189 (2);B.1.192 (7);B.1.195 (4);B.1.2 (222);B.1.210 (2);B.1.214 (6);B.1.214.2 (1);B.1.219 (5);B.1.22 (3);B.1.22.1 (16);B.1.220 (1);B.1.221 (27);B.1.221.1 (1);B.1.223 (1);B.1.229 (1);B.1.23 (2);B.1.232 (1);B.1.234 (20);B.1.236 (3);B.1.237 (3);B.1.240 (7);B.1.240.1 (14);B.1.241 (1);B.1.243 (34);B.1.256 (1);B.1.258 (61);B.1.258.11 (1);B.1.258.17 (17);B.1.258.2 (1);B.1.258.22 (1);B.1.258.23 (1);B.1.260 (2);B.1.273 (1);B.1.277 (1);B.1.279 (1);B.1.281 (4);B.1.289 (1);B.1.291 (2);B.1.3 (1);B.1.306 (1);B.1.308 (1);B.1.311 (5);B.1.324 (1);B.1.329 (1);B.1.334 (1);B.1.338 (1);B.1.346 (1);B.1.349 (2);B.1.351 (199);B.1.356 (3);B.1.357 (1);B.1.36 (56);B.1.36.1 (5);B.1.36.10 (2);B.1.36.16 (33);B.1.36.17 (1);B.1.36.18 (12);B.1.36.19 (1);B.1.36.21 (1);B.1.36.22 (6);B.1.36.29 (6);B.1.36.31 (5);B.1.36.34 (3);B.1.36.38 (1);B.1.36.8 (3);B.1.360 (1);B.1.361 (3);B.1.362 (8);B.1.367 (3);B.1.369 (12);B.1.369.1 (1);B.1.370 (1);B.1.371 (1);B.1.375 (1);B.1.379 (1);B.1.380 (9);B.1.393 (1);B.1.396 (2);B.1.398 (11);B.1.399 (1);B.1.400 (4);B.1.404 (2);B.1.409 (5);B.1.411 (19);B.1.416 (16);B.1.420 (6);B.1.426 (1);B.1.427 (25);B.1.428 (4);B.1.429 (58);B.1.438 (4);B.1.441 (4);B.1.451 (1);B.1.456 (4);B.1.459 (15);B.1.462 (1);B.1.465 (1);B.1.466 (4);B.1.466.1 (1);B.1.466.2 (34);B.1.468 (7);B.1.469 (1);B.1.470 (11);B.1.471 (5);B.1.476 (1);B.1.478 (1);B.1.479 (1);B.1.480 (2);B.1.492 (1);B.1.497 (28);B.1.499 (24);B.1.504 (1);B.1.505 (1);B.1.509 (3);B.1.517 (6);B.1.517.1 (16);B.1.523 (3);B.1.524 (21);B.1.525 (16);B.1.526 (8);B.1.526.1 (6);B.1.526.2 (2);B.1.527 (2);B.1.530 (9);B.1.535 (2);B.1.540 (1);B.1.541 (1);B.1.544 (8);B.1.547 (1);B.1.551 (1);B.1.558 (3);B.1.560 (1);B.1.561 (6);B.1.564 (2);B.1.565 (6);B.1.568 (3);B.1.575 (4);B.1.576 (1);B.1.577 (5);B.1.581 (1);B.1.582 (7);B.1.587 (1);B.1.588 (4);B.1.595.4 (1);B.1.596 (17);B.1.596.1 (1);B.1.600 (8);B.1.603 (2);B.1.605 (1);B.1.609 (6);B. 1.617.1;B.1.617.2;B.1.617.3;B.1.619 (1);B.1.620 (12);B. 1. 621 (65);B.1.67 (1);B.1.84 (1);B.1.91 (4);B.1.94 (1);B.12 (1);B.27 (3);B.28 (1);B.3 (6);B.31 (2);B.35 (4);B.4 (13);B.4.1 (1);B.4.2 (1);B.4.6 (2);B.4.7 (2);B.40 (3);B.42 (2);B.43 (1);B.45 (1);B.53 (2);B.55 (2);B.56 (1);B.6 (23);B.6.3 (1);B.6.6 (5);B.6.7 (1);B.6.8 (29);C.1 (2);C.1.1 (1);C.11 (5);C.12 (3);C.13 (1);C.14 (4);C.16 (18); C.17 (2); C.18 (1); C.2 (5); C.2.1 (11); C.23 (2); C.26 (5); C.29 (1); C.30 (1); C.32 (1); C.35 (10); C.36 (14); C.36.1 (1); (10); C.4 (3); C.8 (2); C. 37 (54); D.2 (33); L.3 (8); N.2 (1); N.3 (4); N.4 (14); N.5 (8); N.6 (3); N.7 (4); N.9 (4); P.1 (57); P.2 (47); P.6 (2);P.7 (3);Q.1 (6), Q.3 (7); Q.4 (2); Q.5 (1); Q.6 (1). Q.8 (7), R.1 (9); S.1 (1); U.2 (1); U.3 (1); W.1 (1); Y.1 (2); and Z.1 (1). .

[0117] From an epidemiological perspective, variants are generally classified as variants of concern (VOCs), variants of concern (VOCs), and variants with serious consequences (VOCs). 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.

[0118] VOI may have certain genetic markers associated with changes in receptor binding, reduced neutralization of antibodies against previous infections or vaccinations, reduced therapeutic efficacy, and increased predictive value for potential diagnostic impact, transmissibility, or disease severity. In some cases, VOI has specific genetic markers that predict changes in transmission, diagnosis, treatment, or immune evasion, or that may lead to an increased proportion of cases or unique outbreaks. SARS-CoV-2 VOIs include, for example, PANGO lineages B.1.1.7 (α), B.1.351 (β); B.1.427 / 429 (ε); B.1.526 (ι); B.1.525 (η); B.1.617.1 (κ); B.1.617.2 (δ); B.1.621 (μ); C37 (λ); P.1 (γ) and P.2.

[0119] VOCs may include variants that provide evidence of increased transmissibility, more severe illness (increased hospitalizations or deaths), significantly reduced neutralizing activity of antibodies produced during prior infection or vaccination, reduced effectiveness of treatment or vaccines, or failure of diagnostic tests. In some cases, VOCs may provide evidence of impacting diagnosis, treatment, and vaccines; widespread interference with diagnostic testing targets; evidence of significantly increased resistance to one or more classes of therapies; evidence of significantly reduced neutralizing activity of antibodies produced during prior infection or vaccination; evidence of reduced protection against severe illness induced by vaccines; evidence of increased transmissibility; or evidence of increased disease severity. SARS-CoV-2 VOCs may include, for example, PANGO lineages B.1.1.7 (α), P.1 (γ), B.1.351 (β), B.1.427 and B.1.429 (ε), B.1.526 (ι), B.1.525 (η), B.1.617.1 (κ), B.1.617.2 (δ), B.1.621 (μ), and C37 (λ).

[0120] VOHC may provide clear evidence that preventative measures or medical mechanisms (MCMs) have significantly reduced effectiveness relative to previously prevalent variants. In some cases, VOHC impacts medical mechanisms (MCMs) by demonstrating diagnostic failure, evidence of significantly reduced vaccine effectiveness, disproportionately high numbers of vaccine breakthrough cases, extremely low protection against vaccine-induced severe disease, significantly reduced sensitivity to multiple Emergency Use Authorization (EUA) or approved therapeutics, more severe clinical disease, and increased hospitalization rates.

[0121] 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 thereof, 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 thereof, or variants thereof. In some embodiments, the nucleic acid vaccine encodes a polypeptide containing a specific mutation called D614G.

[0122] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.526 SARS-CoV-2 variant (i.e., the ι variant), such as one or more of the following: spike protein substitutions for L5F, T95I, D253G, S477N, E484K, D614G and / or A701V; ORF1a substitutions for L3201P, T265I and / or Δ3675 / 3677; ORF1b substitutions for P314L and / or Q1011H; ORF3a substitutions for P42L and Q57H; ORF8 substitution for T11I; and / or 5'UTR substitution for R81C.

[0123] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.525 SARS-CoV-2 variant (i.e., the n variant), such as one or more of the following: spike protein substitutions for A67V, Δ69 / 70, Δ144, E484K, D614G, Q677H and / or F888L; ORF1b substitution for P314F; ORF1a substitution for T2007I; M protein substitution for I82T; N protein substitutions for A12G and / or T205I; and / or 5'UTR substitution for R81C.

[0124] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the P.2 SARS-CoV-2 variant, such as one or more of the following: spike protein substitution for E484K, D614G and / or V1176F; ORF1a substitution for L3468V and / or L3930F; ORF1b substitution for P314L; N protein substitution for A119S, R203K, G204R and / or M234I; 5'UTR substitution for R81C.

[0125] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.1.7 SARS-CoV-2 variant (i.e., the α variant), such as one or more of the following: spike protein substitutions Δ69 / 70, Δ144Y, E484K, S494P, N501Y, A570D, D614G and / or P681H.

[0126] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the P.1 SARS-CoV-2 variant (i.e., the γ variant), such as one or more of the following: spike protein substitutions K417N / T, E484K, N501Y and / or D614G.

[0127] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.351 SARS-CoV-2 variant (i.e., the β variant), such as one or more of the following: spike protein substitutions K417N, E484K, N501Y, and / or D614G. The B.1.351 variant is also known as the South African variant because it originated in South Africa.

[0128] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.427 SARS-CoV-2 variant, such as one or more of the following: spike protein substitution L452R and / or D614G.

[0129] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.429 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions S13I, W152C, L452R and / or D614G.

[0130] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.617.1 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions G142D, E154K, L452R, E484Q, D614G, P681R and / or Q1071H.

[0131] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides comprising one or more mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant (i.e., the δ 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 comprising one or more mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions V70F, A222V, W258L, and / or K417N.

[0132] In some embodiments, the nucleic acid vaccine encodes one or more polypeptides containing one or more mutations or substitutions present in the B.1.617.3 SARS-CoV-2 variant, such as one or more of the following: spike protein substitutions T19R, G142D, L452R, E484Q, D614G, P681R and / or D950N.

[0133] In some embodiments, the nucleic acid vaccine encodes a SARS-CoV-2 spike protein selected from one or more of the following substitutions and / or deletions, such as proteins, peptides, fragments, or variants: A570D, A67V, A701V, D253G, D614G, E484K, F888L, K417N / T, L452R, L5F, N501Y, P681H, Q677H, S13I, S477N, S494P, T95I, V1176F, W152C, Δ144, Δ144Y, and Δ69 / 70.

[0134] In some embodiments, the nucleic acid vaccine code includes SARS-CoV-2 ORF1a selected from one or more of the following substitutions and / or deletions: L3201P, T265I, T2007I, L3468V, Δ3675-3677 and L3930F.

[0135] In some embodiments, the nucleic acid vaccine encoding includes one or more of the following substituted SARS-CoV-2 ORF1b: P314F, P314L and Q1011H.

[0136] In some embodiments, the nucleic acid vaccine encoding includes one or more of the following substituted SARS-CoV-2 ORF3a: P42L and Q57H.

[0137] In some embodiments, the nucleic acid vaccine encoding includes SARS-CoV-2 ORF8 replaced by T11I.

[0138] In some embodiments, the nucleic acid vaccine encoding includes the SARS-CoV-2 5'UTR replaced by R81C.

[0139] In some embodiments, the nucleic acid vaccine encodes the SARS-CoV-2 M protein, such as a protein, peptide, fragment, or variant, which is replaced by the I82T.

[0140] In some embodiments, the nucleic acid vaccine encodes a SARS-CoV-2 N protein substituted with one or more of the following, such as a protein, peptide, fragment, or variant: A12G, A119S, R203K, G204R, T205I, and M234I. Components of the nucleic acid vaccine

[0141] In some embodiments, the polynucleotides described herein encode at least one polypeptide of interest, such as one or more proteins, peptides, fragments thereof, or variants of SARS-CoV-2. The SARS-CoV-2 proteins, peptides, fragments thereof, or variants of the present invention may be wild-type, wherein they are derived from an infectious agent or modified (e.g., structural proteins or fragments thereof and variants thereof are engineered, designed, or artificial). They may have any combination of the features described herein.

[0142] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode one or more peptides or polypeptides of interest. Such peptides or polypeptides are structural proteins of SARS-CoV-2 or fragments or variants thereof, used for the prevention, mitigation, and / or treatment of COVID-19. As a non-limiting example, such peptides or polypeptides may act as antigens or antigen molecules (preferably as immunogenic molecules). The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a nucleotide polymer. Such polymers are commonly referred to as polynucleotides.

[0143] Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), diol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with aL-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-a-LNA with 2'-amino functionalization), vinyl nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) or mixtures or combinations thereof.

[0144] In some embodiments, the in vitro transcription (IVT) enzyme synthesis method can be used to prepare linear polynucleotides (referred to as "IVT polynucleotides") encoding one or more proteins, peptides, fragments or variants of SARS-CoV-2 of the present invention.

[0145] In some embodiments, the nucleic acid vaccine may include "chimeric polynucleotides" having portions or regions of different sizes and / or encoding proteins (e.g., structural proteins of SARS-CoV-2). A "chimera" is an entity having two or more incongruous or heterogeneous portions or regions. As used herein, a "portion" or "region" of a polynucleotide is defined as any portion of the polynucleotide that is less than the entire length of the polynucleotide.

[0146] In some embodiments, the nucleic acid vaccine comprises 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). 0, 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).

[0147] In some embodiments, the nucleic acid vaccine includes at least one polynucleotide encoding at least one peptide or polypeptide of interest. In another embodiment, the polynucleotide may be non-coding.

[0148] In some embodiments, the length of the region encoding at least one peptide or polypeptide of interest of the polynucleotide of the nucleic acid vaccine is greater than about 30 nucleotides (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,3...). 00, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). As used herein, such regions may be referred to as “coding regions” or “regional coding”.

[0149] In some embodiments, the polynucleotide of the nucleic acid vaccine is or acts as messenger RNA (mRNA). As used herein, the term "messenger RNA (mRNA)" means any polynucleotide that encodes at least one peptide or polypeptide of interest and is capable of being translated to produce the encoded peptide or polypeptide of interest in vitro, in vivo, in situ, or ex vivo.

[0150] The minimum length of the polynucleotide region of a nucleic acid vaccine may be a length sufficient to encode a nucleic acid sequence that is sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, this length may be sufficient to encode 2-30 amino acids, such as peptides of 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. This length may be sufficient to encode peptides of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or not longer than 40 amino acids, such as peptides not longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids. Examples of dipeptides that the polynucleotide sequence may encode may include, but are not limited to, carnosine and methylcarnosine.

[0151] The length of the polynucleotide region of a nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants of SARS-CoV-2 used for the prevention, mitigation and / or treatment of COVID-19 may be greater than about 30 nucleotides. The length may be, but is not limited to, at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,6 ...2 700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides.In some embodiments, 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 (up 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.

[0152] The nucleic acid vaccine described herein may be an mRNA vaccine. An mRNA vaccine comprises at least one mRNA molecule that, upon translation, produces at least one peptide or polypeptide of interest for the prevention, mitigation, and / or treatment of COVID-19. Generally, an mRNA molecule generally includes at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. mRNA components: start codon and stop codon.

[0153] In some embodiments, the mRNA includes a region for initiating translation. This region may include any translation initiation sequence or signal, including a start codon. As a non-limiting example, the region includes a start codon. In some embodiments, the start codon may be "ATG", "ACG", "AGG", "ATA", "ATT", "CTG", "GTG", "TTG", "AUG", "AUA", "AUU", "CUG", "GUG", or "UUG".

[0154] In some embodiments, the mRNA includes a region that terminates translation. This region may include any translation termination sequence or signal, including a stop codon. As a non-limiting example, the region includes a stop codon. In some embodiments, the stop codon may be "TGA", "TAA", "TGA", "TAG", "UGA", "UAA", "UGA", or "UAG".

[0155] In some embodiments, the length of the start or stop translation region may be independently in the range of 3 to 40, for example 5-30, 10-20, 15 or at least 4, or 30 or fewer nucleotides. In addition, in addition to start and / or stop codons, such regions may also contain one or more signal and / or restriction sequences.

[0156] In some embodiments, a masking agent may be used to mask a first start codon or an alternative start codon to increase the probability that translation will begin on a start codon or alternative start codon downstream of the masked start codon or alternative start codon.

[0157] In some embodiments, a start codon may be removed from the polynucleotide sequence so that translation of the polynucleotide begins at a codon that is not a start codon. Translation of the polynucleotide may begin at a codon following the removed start codon or at a downstream start codon or an alternative start codon. The polynucleotide sequence with the start codon removed may further include at least one masking agent for the downstream start codon and / or alternative start codon to control or attempt to control the initiation of translation, the length of the polynucleotide, and / or the structure of the polynucleotide. mRNA component: coding region

[0158] 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 the peptide or polypeptide of interest include one or more proteins, peptides, fragments thereof, or variants of SARS-CoV-2 for the prevention, mitigation, and / or treatment of COVID-19. mRNA component: non-translated region

[0159] The polynucleotides of the nucleic acid vaccines described herein may contain one or more regions or portions that act as or are used as untranslated regions (UTRs). The wild-type UTR of a gene is transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start point and continues to the start codon, but does not include the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. Although not wishing to be bound by theory, UTRs may play a role in the stability of nucleic acid molecules and in translation. Variants of the UTR may be utilized in which one or more nucleotides are added to or removed from the end, including A, T, C, or G.

[0160] In some embodiments, the length of the UTR of the polynucleotide of the nucleic acid vaccine may independently be in the range of 15-1,000 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).

[0161] Wild-type 5' UTRs include features that play a role in translation initiation because such 5' UTRs include sequences such as the Kozak sequence, which are known to be involved in how ribosomes initiate the translation of many genes. 5' UTR formation is also known to involve secondary structures for elongation factor binding. Other non-UTR sequences (e.g., introns or portions of intron sequences) may also be used as regions or subregions that can increase protein yield and polynucleotide content.

[0162] It is known that natural or wild-type 3' UTRs contain embedded adenosine and uridine elongations. These AU-rich tags are particularly prevalent in genes with high turnover rates. The introduction, removal, or modification of 3' UTR-rich AU elements (AREs) can be used to regulate the stability of polynucleotides in nucleic acid vaccines.

[0163] UTRs from any gene may be incorporated into regions of polynucleotides of nucleic acid vaccines. Alternatively, artificial UTRs of variants that are not wild-type regions may also be used in polynucleotides of nucleic acid vaccines. The placement orientation of such UTRs or portions thereof may be the same as in the transcripts from which they are derived, or their orientation or position may be varied. As used herein, the term “alteration” in relation to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. As a non-limiting example, 5' or 3' UTRs may be inverted, shortened, or elongated from one or more other 5' or 3' UTRs from different parental sequences.

[0164] In some embodiments, the lateral regions are selected from transcript families of proteins that share common functions, structures, and characteristics. For example, the polypeptide of interest may belong to a protein family that is expressed in a particular cell, tissue, or at a certain time during development. UTRs from any such genes may be exchanged with any other UTR from the same or different protein families to produce 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, location, origin, or mode of expression.

[0165] 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 at the 5' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. In some embodiments, the 5' UTR is separated from the 5' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine by 1, 2, 3, 4, 5, 6 or more nucleotides; for example, a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 5' UTR from the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 91% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR having at least 94% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 96% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5' UTR having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR having a sequence that is at least 99% sequence identical to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 5'UTR having a sequence that is at least 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).

[0166] 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 at the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. In some embodiments, the 3'UTR is separated from the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine by 1, 2, 3, 4, 5, 6 or more nucleotides; for example, a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 3'UTR from the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 91% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 94% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 96% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having a sequence that is at least 99% sequence identical to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having a sequence that is at least 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).

[0167] 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 at the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. In some embodiments, the 3'UTR is separated from the 3' of the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine by 1, 2, 3, 4, 5, 6 or more nucleotides; for example, a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 3'UTR from the start codon encoding the SARS-CoV-2 polypeptide sequence of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 91% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 92% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 94% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 96% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 99% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein may contain a 3'UTR, which has a sequence that is at least 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). mRNA components: Cap and IRES sequences.

[0168] In some embodiments, the polynucleotides of the nucleic acid vaccines disclosed herein may include a 5' cap structure. The 5' cap structure of natural mRNA participates in nuclear export, increases mRNA stability, and binds to mRNA cap-binding protein (CBP). This protein associates with poly(A)-binding protein via CBP to form a mature circular mRNA species responsible for mRNA stability and translation capacity in cells. The cap further facilitates the removal of 5' proximal introns during mRNA splicing.

[0169] In some embodiments, the 5' end cap region of the polynucleotide of the nucleic acid vaccine may contain a single end cap or a series of nucleotides forming the end cap. The length of the end cap region may be 1 to 10, such as 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides. In some instances, the end cap region may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the end cap is absent.

[0170] In some embodiments, end-cap analogs, also referred to herein as synthetic end-cap analogs, chemical end-caps, chemical end-cap analogs, or structural or functional end-cap analogs, can be used in nucleic acid vaccines. End-cap analogs may be chemically (e.g., non-enzymatic) or enzymatically synthesized, and their chemical structure may differ from that of natural (e.g., endogenous, wild-type, or physiological) 5'-caps, but they retain end-cap function.

[0171] In some embodiments, the 5' end cap of the polynucleotide of the nucleic acid vaccine may include an endogenous end cap or an end cap analog. As a non-limiting example, the 5' end cap may contain a guanine analog. Suitable guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine (m1G), 2'-fluoro-guanosine, 7-dezo-guanosine, 8-side-oxy-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0172] Those familiar with this technique will understand that the 5' cap can be generated via enzymes or other synthetic methods. Endogenous mRNA molecules are 5'-capped, with a 5'-ppp-5'-triphosphate bond formed between the terminal guanylic acid cap residue and the sense nucleotide transcribed from the 5' end of the mRNA molecule. This 5'-guanylic acid cap can then be methylated to produce an N7-methyl-guanylic acid residue. The ribose of the nucleotide transcribed from the 5' end and / or proximal end of the mRNA may also be 2'-O-methylated, depending on the case. 5'-uncapping via hydrolysis and cleavage of the guanylic acid cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0173] The polynucleotides of the nucleic acid vaccines described herein, such as mRNA, can be modified to include a non-hydrolyzable end-cap structure, thereby preventing end-cap removal and thus increasing the mRNA half-life. Since end-cap hydrolysis requires cleavage of the 5'-ppp-5' phosphodiester bond, modified nucleotides can be used during the end-capping reaction. For example, a vaccinia virus end-capping enzyme, available from companies such as New England Biolabs (Ipswich, MA), can be used with α-thioguanosine nucleotides according to the manufacturer's instructions to generate a thiophosphate bond in the 5'-ppp-5' end cap. Additional modified guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, can be used.

[0174] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminus and / or the 5'-preterminal nucleotide of the mRNA at the 2'-hydroxyl group of the sugar ring (as described above). Multiple unique 5'-cap structures can be used to generate nucleic acid molecules, such as the 5'-cap of mRNA molecules.

[0175] 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 in chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5'-caps while retaining end-cap function. End-cap analogs can be chemically (e.g., non-enzymatically) or enzymatically synthesized and linked to nucleic acid molecules, such as mRNA molecules.

[0176] For example, the anti-reverse cap analog (ARCA) cap contains two guanines linked by 5'-5'-triphosphate groups, one of which contains an N7 methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which can be equivalently designated as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA). The N7- and 3'-O-methylated guanine provides the terminal portion of the capped nucleic acid molecule (e.g., mRNA).

[0177] Another example is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0178] Although cap analogs allow for the addition of caps to nucleic acid molecules during in vitro transcription reactions, up to 20% of transcripts remain capless. This, along with the structural differences between cap analogs and endogenous 5'-cap structures, can lead to reduced translational capacity and decreased cell stability.

[0179] In exemplary embodiments of the present invention, polynucleotides (e.g., mRNA) may be capped post-transcriptionally using an enzyme. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can create a typical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of mRNA and the guanine capping nucleotide, wherein the capping guanine contains N7 methylation and the 5'-terminal nucleotide of mRNA contains 2'-O-methyl. Such structures are referred to as Cap 1 structures. In some embodiments, compared to other 5' capping analog structures known in the art, such as those described herein, Cap 1 structures provide higher translational efficiency and cellular stability and reduced activation of pro-inflammatory cytokines. Cap structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')N1mpNp (Cap 1), and 7mG(5')-ppp(5')N1mpN2mp (Cap 2).

[0180] In one embodiment, the polynucleotide of the nucleic acid vaccine described herein comprises a Cap 1 structure.

[0181] Because polynucleotides, such as mRNA, can be capped post-transcriptionally, and because this process is more efficient, up to 100% of polynucleotides, such as mRNA, can be capped. This contrasts with approximately 80% of capped analogues ligated to mRNA during in vitro transcription reactions.

[0183] 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, the IRES plays an important role in initiating protein synthesis in the absence of a 5' cap structure. The IRES may serve as a single ribosome binding site or as one of multiple ribosome binding sites for mRNA. mRNA component: tailed region

[0184] 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 quadruplets.

[0185] In some embodiments, the mRNA includes a chain-terminating nucleoside. Non-limiting examples of chain-terminating nucleosides include 2'-O methyl, F, and locked nucleosides (LNA).

[0186] In some embodiments, the length of the tailing region of the polynucleotide in the nucleic acid vaccine may range from absent 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.

[0187] In some embodiments, the poly-A tail can also be added after the construct is output from the kernel.

[0188] In some embodiments, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide (such as an mRNA molecule) during RNA processing to increase stability. Immediately following transcription, the 3' end of the transcript may be cleaved to release a 3' hydroxyl group. Then, a poly-A polymerase adds the adenine nucleotide chain to the RNA. This process, known as polyadenylation, adds the poly-A tail, which may 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.

[0189] In some embodiments, the length of the poly-A tail (when present) is greater than 30 nucleotides (e.g., at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000 nucleotides). In some embodiments, the poly-A tail region comprises 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).

[0190] In some embodiments, the length of the poly-A tail is about 99 nucleotides (SEQ ID NO: 44).

[0191] In some embodiments, the poly-A tail is designed relative to the length of the entire polynucleotide or the length of a specific region of the polynucleotide. This design may be based on the length of the coding region, the length of a specific feature or region, or the length of the final product expressed by the self-polynucleotide.

[0192] In this case, the length of the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the polynucleotide or its characteristics. 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 the total length of the construct, the construct region, or the total length of the construct minus 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater of the poly-A tail. Furthermore, engineered binding sites of poly-A binding proteins and polynucleotide binding can enhance performance. Signal sequence

[0193] In some embodiments, the polynucleotides of the nucleic acid vaccine may also encode additional features that facilitate the transport of the polypeptide to therapeutically relevant sites. One such feature that facilitates protein transport is a signal sequence. As used herein, a "signal sequence" or "signal peptide" is a polynucleotide or polypeptide of about 9 to 200 nucleotides (3 to 60 amino acids) in length, incorporated into the 5' end of the coding region or the encoded N-terminal polypeptide. In some embodiments, the addition of such sequences enables the encoded polypeptide to be transported to the endoplasmic reticulum via one or more secretory pathways. After protein transport, some signal peptides are cleaved from the protein by signal peptidase.

[0194] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein includes a signal sequence comprising SEQ ID NO: 45 (DNA) or SEQ ID NO: 49 (RNA). Codon optimization

[0195] Polynucleotides of nucleic acid vaccines, regions, portions, or subregions thereof, may be codon-optimized. Codon optimization methods are known in this art and can be applied to achieve one or more of a number of objectives. These objectives include, but are not limited to, matching codon frequencies in the target organism and the host organism to ensure correct folding; altering GC content to increase mRNA stability or reduce secondary structures; minimizing the execution of tandem repeat codons or bases that may impair gene construction or expression; customizing transcription and translation control regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins; adding, removing, or reorganizing protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; regulating translation rates to allow proper folding of protein domains; or reducing or eliminating problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in this art, and non-limiting examples include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), and / or proprietary methods. In some embodiments, the ORF sequence is optimized using an optimization algorithm. The codon selections for each amino acid are given in Table 4. Table 4. Codon Selection Single-letter naming convention Amino acid name Codon selection A alanine GCT, GCC, GCA, GCG C Cysteine TGT, TGC D Aspartic acid GAT, GAC E glutamic acid GAA, GAG F Phenylan TTT, TTC G Glycine GGT, GGC, GGA, GGG H histidine CAT, CAC I Isoleucine ATT, ATC, ATA K lysine AAA, AAG L Leucine CTT, CTC, CTA, CTG, TTA, TTG M Methionine ATG N Asparagine AAT, AAC P proline CCT, CCC, CCA, CCG Q glutamic acid CAA, CAG R Arginine CGT, CGC, CGA, CGG, AGA, AGG s serine TCT, TCC, TCA, TCG, AGT, AGC Sec selenocysteine UGA in mRNA in the presence of selenocysteine ​​insertion elements (SECTS) termination stop codon TAA, TAG, TGA T threonine ACT, ACC, AC A, ACG V Valine GTT, GTC, GTA, GTG w tryptophan TGG γ Tyrosine TAT, TAC

[0196] In some embodiments, the nucleic acid vaccine is vectorized after codon optimization. Non-limiting examples of vectors include, but are not limited to, plasmids, viruses, myxosomes, and artificial chromosomes. Modification

[0197] The nucleic acid vaccines (including mRNA vaccines) of the present invention may include one or more modifications. The term "modification" or, where appropriate, "modified" refers to a modification of A, G, U, or C ribonucleotides. Generally, these terms are not intended to refer to ribonucleotide modifications in the 5'-terminal cap portion of naturally occurring mRNA. In polypeptides, the term "modification" refers to a modification relative to the standard set of 20 amino acids.

[0198] As described herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof ("nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside that includes a phosphate group or other backbone bond (nucleoside inter-bond).

[0199] Modifications can be various. In some embodiments, the coding region, non-translated region, flanking region, and / or terminal or tailed region may contain one, two, or more (as the case may vary) nucleoside or nucleotide modifications. In some embodiments, the nucleic acid vaccine of the present invention comprises one or more modifications that, compared to unmodified polynucleotides, make the nucleic acid molecule more resistant to degradation in the cell and / or more stable in the cell when introduced into the cell.

[0200] The polynucleotides of the nucleic acid vaccines described herein may include any useful modifications, such as modifications to sugars, nucleobases, or nucleoside internucleotides (e.g., modifications to the phosphate ester / phosphodiester bond / phosphodiester backbone). One or more pyrimidine nucleobase atoms may be substituted or replaced, for example, by substitution of an amino group, a thiol group, an alkyl group (e.g., methyl or ethyl), a halogen group (e.g., chlorine or fluorine) atom or group, depending on the situation. In some embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and nucleoside internucleotides. Modifications according to the invention may be modifications of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or mixtures thereof. Additional modifications are described herein.

[0201] In some embodiments, the modification includes nucleotides modified with 2'-O-methyl or 2'-O-methoxyethyl (2'-OMe and 2'-MOE, respectively).

[0202] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein may contain at least one of the modifications described herein.

[0203] The polynucleotides of the nucleic acid vaccines described herein may include combinations of modifications to sugars, nucleobases and / or nucleoside bonds.

[0204] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that can be used in the vaccines of the present invention include, but are not limited to, any modifications as described in PCT Publication WO2017070626, the contents of which are incorporated herein by reference in their entirety, including, for example, modifications or deletions of nucleotides (or codons) encoding one or more N-linked glycosylation sites in a translated polypeptide. Modifications of vaccines that can be used in the present invention 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 invention may further include features or modifications as described in PCT patent applications published in WO2020255063, WO2020182869, WO2016011222, WO2016011226, WO2016005004, WO2016000792, WO2015176737, WO2015085318, WO2015048744 and WO2015034925, and U.S. patent applications published in US20200254086, US20200206362, US20180311336 and US20180303929; the contents of each of these publications are incorporated herein by reference in their entirety.

[0205] For example, polynucleotides comprising the mRNA molecules of the nucleic acid vaccines described herein may include the following modifications: The nucleoside internucleotide bonds of the polynucleotide may be partially or completely modified. The polynucleotide may contain modifications to one or more nucleobases. The polynucleotide may contain 5-methylcytosine replacing all cytosine nucleobases / cytidine nucleotides. Furthermore, the polynucleotide may have one or more modifications to one or more sugar subunits of the nucleoside. The sugar modification may be one or more locked nucleic acids (LNAs) or 2'-O-methoxyethyl modifications ("2'-MOE"). The polynucleotide may be designed as a patterned array with sugar, nucleobase, or bond modifications. In some embodiments, the polynucleotide may contain modifications that maximize stability. In some embodiments, the polynucleotide may be completely modified with 2'-MOE-sugar. Modified nucleobases

[0206] 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.

[0207] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one nucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), and 5-methoxy-uridine (mo5U). Uridine 5-oxyacetic acid (cmo5U), methyl uridine 5-oxyacetic acid (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxymethylhydroxymethyl-uridine (chm5U), 5-carboxymethylhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5- Methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-aminomethylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauronic acid methyl-uridine (τm5U), 1-tauronic acid methyl-pseudouridine, 5-tauronic acid methyl-2-thio-uridine (τm5s2U) 1-Taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., deoxythymidine with nucleobase), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyl-Dihydrouridine (m5D), 2-Thio-Dihydrouridine, 2-Thio-Dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-Thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-Thio-pseudouridine, N1-Methyl-pseudouridine (also known as 1-methylpseudouridine (m1ψ)), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-Thio-uridine (inm5s2U), α-Thio-uridine, 2'-O-Methyl-uridine (Um), 5,2'-O-Dimethyl-uridine ( m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-aminomethoxymethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-arasu-uridine, 2'-F-uridine, 2'-OH-arasu-uridine, 5-(2-methoxycarbonylvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0208] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudo-isocytidine, 3-methyl-cytidine (m3C), N4-acetylated-cytidine (ac4C), 5-methylated-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (…). hm5C), 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deazon-pseudocytidine, 1-methyl-1-deazon-pseudocytidine, zebularine, 5- Aza-Zebralin, 5-methyl-Zebralin, 5-aza-2-thio-Zebralin, 2-thio-Zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine ( m5Cm), N4-acetylated-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-methylated-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arasacchar-cytidine, 2'-F-cytidine and 2'-OH-arasacchar-cytidine.

[0209] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deazo-adenine, 7-deazo-8-aza-adenine, 7-deazo-2-amino-purine, 7-deazo-8-aza-2-amino-purine, 7-deazo-2,6-diamino-purine, and 7-deazo-8-aza-2,6-diamino-purine. N-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylaminomethylmethyl-adenosine (g6A), N6-threonylaminomethylmethyl N6-methyl-N6-threonylaminomethyladenosine (m6t6A), 2-methylthio-N6-threonylaminomethyladenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxyn-valine-adenosine (hn6A), 2-methylthio-N6-hydroxyn-valine-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, α- Thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyl-adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinose-adenosine, 2'-F-adenosine, 2'-OH-arabinose-adenosine, and N6-(19-amino-pentaenodecyl ester)-adenosine.

[0210] In some embodiments, the modified nucleobase is modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 4-demethyl-wyoside (imG-14), isowyoside (imG2), wyoside (yW), peroxywyoside (o2yW), hydroxywyoside (OHyW), undermodified hydroxywyoside (OHyW*), 7-deazono-guanosine, Q nucleoside (Q), epoxy-Q nucleoside (oQ), and hemi-guanosine. Lactosyl-Q nucleoside (galQ), Mannosyl-Q nucleoside (manQ), 7-cyano-7-deazo-guanosine (preQ0), 7-aminomethyl-7-deazo-guanosine (preQ1), Archipelone (G+), 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy -Guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-sideoxy-guanine, 7-methyl-8-sideoxy-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, α-thio-guanine, 2 '-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im) and 2'-O-riboguanosine (phosphate) (Gr(p)).

[0211] The nucleobases of a nucleotide may be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. For example, each nucleobase may be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase may also include, for example, naturally occurring and synthetic derivatives of the base, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen (e.g., 8-bromo), 8-amino, 8-thiol, 8-thiouracil, etc. Alkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen (specifically 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deadenine, 7-deadenine, 3-deadenine, deadenine, 7-deadenine, 3-deadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazine, 9-deadenine, imidazo[4,5-d]pyrazol, thiazo[4,5-d]pyrimidine, pyrazol-2-one, 1,2,4-triazine, tazo; and 1,3,5-triazine.

[0212] Various sugar modifications, nucleotide modifications, and / or nucleotide bonds (e.g., backbone structure) can be introduced at various positions in the polynucleotides described herein. Those skilled in the art will understand that nucleotide analogs or other modifications can be located at any position in the polynucleotide without substantially reducing its function. The polynucleotides of the present invention may contain about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any or more of A, G, T / U, or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 95%). % 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%).

[0213] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein may be modified into a cyclic nucleic acid. The ends of the polynucleotide may be linked 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. The cyclic polynucleotide may further include other structural and / or chemical modifications relating to A, G, T / U, or C ribonucleotides / deoxyribonucleotides.

[0214] 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 should be understood that since nucleotides (sugar, base, and phosphate portions, such as bonds) can each be modified, any modification to any portion of a nucleotide or nucleoside will constitute a modification.

[0215] In some embodiments, the polynucleotide is modified in at least 10% of only one component of the nucleotide, wherein such component is a nucleobase, sugar, or bond between nucleosides. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleobase, sugar, or bond of the polynucleotide described herein may be modified.

[0216] As a non-limiting example, all uracil nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all guanine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all guanine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all cytosine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different. In some embodiments, all adenine nucleosides of the polynucleotides in the nucleic acid vaccine are modified. These modifications may be the same or different.

[0217] In one embodiment of the present invention, the polynucleotide of the nucleic acid vaccine is modified to include N1-methyl-pseudouridine nucleotide. Sugar modification

[0218] Modified nucleosides and nucleotides that can be incorporated into polynucleotides (e.g., RNA or mRNA, as described herein) can be modified on the sugars of ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or substituted with several different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, a halogen, a substituted C1-6 alkyl group, a substituted C1-6 alkoxy group, a substituted C6-10 aryloxy group, a substituted C3-8 cycloalkyl group, a substituted C3-8 cycloalkoxy group, a substituted C6-10 aryloxy group, a substituted C6-10 aryl-C1-6 alkoxy group, a substituted C1-12 (heterocyclic)oxy group, a sugar (e.g., ribose, pentose, or any sugar described herein), polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR, wherein R is H or a substituted alkyl group, and n is 0 to 20. (e.g., integers of 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16 and 4 to 20); "locked" nucleic acid (LNA), wherein the 2'-hydroxyl group is connected to the 4'-carbon of the same ribose by a C1-6 alkyl or C1-6 heteroalkyl bridge, wherein exemplary bridges include methylene, propyl, ether or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0219] In some embodiments, the polynucleotide, such as the mRNA of the nucleic acid vaccine described herein, contains at least one sugar modification. Generally, the RNA comprises a glycosylribose as a 5-membered ring with oxygen. Exemplary, non-limiting modified nucleotides include the substitution of oxygen in the ribose (e.g., with S, Se, or an alkyl group, such as methylene or ethyl); the addition of a double bond (e.g., to substitute the ribose with a cyclopentenyl or cyclohexenyl group); cyclic condensation of the ribose (e.g., to form a 4-membered ring of cyclobutane or oxygen); cyclic expansion of the ribose (e.g., to form a 6- or 7-membered ring with an additional carbon or heteroatom, such as anhydrous hexitol, altritol, mannitol, cyclohexyl, cyclohexenyl, and N-hydroxyl (also having an aminophosphate backbone)); and polycyclic forms (e.g., tricyclic and "unlocked" forms, such as glycol nucleic acid (GNA)). (For example, R-GNA or S-GNA, wherein the ribose is replaced by an ethylene glycol unit linked to a phosphodiester bond), threononucleotides (TNA, wherein the ribose is replaced by an α-L-threofuranosyl-(3'→2') group), and peptide nucleotides (PNA, wherein the ribose and phosphodiester backbone are replaced by 2-aminoethyl-glycine bonds). The glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in the ribose. Therefore, polynucleotide molecules (including mRNA) as described herein may include nucleotides containing, for example, arabinose as a sugar.

[0220] Non-limiting examples of sugar modifications may include the modifications provided in Table 5. The polynucleotides of the present invention may have one or more nucleotides carrying the modifications provided in Table 5. In some embodiments, each of the polynucleotides described herein carries any of the modifications provided in Table 5, or does not carry any of the modifications provided in Table 5. Table 5. Nucleotide Sugar Modifications Nucleotides Structural description DNA 2'-O-methyl(2'-OMe) 2'F-RNA 2'F-ANA 4'S-RNA UNA LNA 4'S-FANA 2'-O-methoxyethyl (2'-MOE) 2'-O-allyl 2'-O-ethylamine 2'-O-cyanoethyl 2'-O-acetal 4'-C-aminomethyl-2'-O-methylRNA 2'-azido group Methylene-cLNA N-MeO-aminoBNA N-Me-aminooxy BNA 2',4'-BNA NC [NMe] MC ONA tc-DNA CeNA ANA HNA

[0221] In some embodiments, at least one 2' position (OH in RNA or H in DNA) of the nucleotide sugar of the polynucleotide is substituted with -OMe, referred to as 2'-OMe. In some embodiments, at least one 2' position (OH in RNA or H in DNA) of the nucleotide sugar of the polynucleotide is substituted with -F, referred to as 2'-F. Nucleoside internucleotide bond

[0222] The polynucleotides of the present invention may include any modification to the nucleoside internucleotides (e.g., to the phosphate ester / phosphodiester bond / phosphate diester backbone). In the context of the polynucleotide backbone, the terms "phosphate ester" and "phosphodiester" are used interchangeably. The backbone phosphate ester group may be modified by replacing one or more oxygen atoms with different substituents. Furthermore, the modified nucleosides and nucleotides may include the complete replacement of the unmodified phosphate ester portion with another nucleoside internucleotide bond as described herein. Examples of modified phosphate ester groups include, but are not limited to, thiophosphates, methylphosphates, selenophosphates, boranophosphates, boranophosphate esters, hydrophosphonates, aminophosphates, diaminophosphates, alkyl or arylphosphonates, and triphosphates. In dithiophosphates, both non-linked oxygen atoms are replaced with sulfur. Phosphate linkers can also be modified by replacing oxygen with nitrogen (bridging amino phosphates), sulfur (bridging thiophosphates), and carbon (bridging methylene-phosphonates).

[0223] An α-thio-substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides via non-natural thiophosphate backbone bonding. Thiophosphate DNA and RNA exhibit increased nuclease resistance, followed by a longer half-life in the cellular environment. It is anticipated that thiophosphate-linked polynucleotide molecules will also reduce innate immune responses through weaker binding / activation to cellular innate immune molecules.

[0224] In a specific embodiment, the modified nucleoside includes α-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine or 5'-O-(1-thiophosphate)-pseuuridine).

[0225] In some embodiments, the polynucleotide contains at least one thiophosphate bond or methylphosphonate bond between the nucleotides.

[0226] In some embodiments, the polynucleotide comprises at least one 5'-(E)-phosphonovinyl ester (5'-E-VP) and a phosphate ester analog as a modification.

[0227] In one embodiment of the present invention, the polynucleotide (e.g., mRNA) of the COVID-19 nucleic acid vaccine may be modified. (Variance)

[0228] The valence of the nucleic acid vaccine of the present invention can vary. "Valence" refers to the number of antigenic components in the nucleic acid vaccine or the polynucleotide of the nucleic acid vaccine. The antigenic components of the nucleic acid vaccine may be on the same polynucleotide or on different polynucleotides. In some embodiments, the nucleic acid vaccine may be monovalent. In some embodiments, the nucleic acid vaccine may be bivalent. In some embodiments, the nucleic acid vaccine may be trivalent. In some embodiments, the nucleic acid vaccine may be multivalent, and may contain 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 antigens or antigenic portions, such as, but not limited to, antigenic peptides. As a non-limiting example, the antigenic peptide may be one or more fragments or variants of the structural protein of SARS-CoV-2. Synthetic enzyme method: in vitro transcriptase synthesis.

[0229] The cDNA encoding the polynucleotide of the nucleic acid vaccine described herein can be transcribed using an in vitro transcription (IVT) system. This system typically includes a transcription buffer, nucleotide triphosphates (NTPs), a ribonuclease inhibitor, and a polymerase. The NTPs may be manufactured in-house, optionally from a supplier, or synthesized as described herein. The NTPs may be selected from, but are not limited to, the NTPs described herein, including natural and non-natural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, and polymerase variants.

[0230] In some embodiments, the DNA template is removed from the IVT reaction using DNase I enzyme. The digested DNA and nucleotides are then removed during oligo dT purification of the mRNA. This purification method is based on the affinity of the poly-A tail of the mRNA for the poly-dT column bed. Centrifugation can be used, but may not be necessary to remove the digested DNA and nucleotides. After purification using a reverse-phase column (e.g., SDVB) to remove double-stranded RNA from the mRNA, ultrafiltration followed by one or more filtration steps can be used. After purification, residual DNA can be measured using PCR to confirm that DNA in plastid regions outside the region transcribed into mRNA has been removed. In some embodiments, where product concentration is required, perfiltration followed by one or more filtration steps can be used to remove any biological load (e.g., biomolecules or other biological material).

[0231] Any number of RNA polymerases or variants can be used for the synthesis of polynucleotides in the nucleic acid vaccines described herein. RNA polymerases can be modified by inserting or deleting amino acids from the RNA polymerase sequence.

[0232] Polynucleotide or nucleic acid synthesis reactions can be performed using enzymatic methods employing polymerases. Polymerases catalyze the formation of phosphodiester bonds between nucleotides in polynucleotide or nucleic acid chains. Currently known DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis. The DNA polymerase I (pol I) or A polymerase family, including the Klenow fragment of *E. coli*, Bacillus DNA polymerase I, *Taq* DNA polymerase, and T7 RNA and DNA polymerase, is one of the most thoroughly studied families. Another large family is the DNA polymerase a (pol a) or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from bacteriophages T4 and RB69. Although these polymerase families employ similar catalytic mechanisms, they differ in terms of acceptor specificity, acceptor analog binding efficiency, primer elongation extent and rate, DNA synthesis mode, exonuclease activity, and sensitivity to inhibitors. Solid-phase chemical synthesis

[0233] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be manufactured entirely or partially using solid-phase technology. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a reactant solution. Impurities and excess reagents are washed away, and purification is not required after each step. This process can be automated on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows for the rapid production of polynucleotides or nucleic acids on a relatively large scale, making some polynucleotides or nucleic acids commercially available.

[0234] In some embodiments, automated solid-phase synthesis is used, wherein the chain is synthesized along the 3' to 5' direction. The hydroxyl group at the 3' end of the nucleoside is plugged into the solid support via a chemically cleavable or photoly cleavable linker. Activated nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG, and dT), ribonucleotides (A, C, G, and U), or chemically modified nucleosides, are sequentially added to the nucleosides bound to the support. At the end of the synthesis, a cleavage agent such as ammonia or ammonium hydroxide is added to remove all protecting groups and release the polynucleotide chain from the solid support. Light can also be applied to cleave the polynucleotide chain. The product can then be further purified by high-performance liquid chromatography (HPLC) or electrophoresis. Liquid-phase chemical synthesis

[0235] The synthesis of polynucleotides for the nucleic acid vaccines described herein by sequentially adding monomer building blocks can be carried out in the liquid phase. Covalent bonds are formed between monomers or between the terminal functional groups of the growing chain and the incoming monomers. Functional groups that do not participate in the reaction must be temporarily protected. After each monomer building block is added, the reaction mixture must be purified before the next monomer building block is added. Functional groups at one end of the chain must be deprotected before reacting with the next monomer building block. Liquid-phase synthesis is time-consuming, labor-intensive, and cannot be automated. Despite its limitations, liquid-phase synthesis can still be used for the large-scale preparation of short polynucleotides. Because the system is homogeneous, it does not require large amounts of excess reagents and is cost-effective in this respect. Quantification and purification

[0236] 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 fluids" include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or preejaculate fluid, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual blood, pus, sebum, vomitus, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood. Alternatively, exosomes can be taken from organs selected from the following groups: lungs, heart, pancreas, stomach, intestines, bladder, kidneys, ovaries, testes, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0237] In exosome quantification methods, no more than 2 mL of sample is obtained from an individual, and exosomes are separated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanofiltration, immunoadsorption capture, affinity purification, microfluidic separation, or combinations thereof. In the analysis, the level or concentration of polynucleotides can be the expression level, presence, absence, truncation, or alteration of the administered construct. This level has favorable correlation with one or more clinical phenotypes or with the analysis of biomarkers for human diseases. The analysis can be performed using construct-specific probes, cell counting, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof, while exosomes can be separated using immunohistochemical methods, such as enzyme-linked immunosorbent assay (ELISA). Exosomes can also be separated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanofiltration, immunoadsorption capture, affinity purification, microfluidic separation, or combinations thereof.

[0238] These methods enable researchers to monitor the levels of remaining or delivered polynucleotides in real time. This is possible because the polynucleotides described herein differ from their endogenous forms due to structural modifications.

[0239] In some embodiments, methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis) can be used to quantify polynucleotides. A non-limiting example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). Quantitatively quantified polynucleotides can be analyzed to determine whether the polynucleotides are of an appropriate size and to check that the polynucleotides have 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, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography-mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE).

[0240] The purification of polynucleotides for nucleic acid vaccines described herein may include, but is not limited to, polynucleotide cleaning, quality assurance, and quality control. Cleaning may be performed by methods known in this art, such as, but not limited to, AAGEN-COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes (EX-IQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). When the term "purified" is used in connection with polynucleotides, such as "purified polynucleotides" means polynucleotides isolated from at least one contaminant. As used herein, "contaminant" is any substance that makes another unsuitable, impure, or inferior. Thus, purified polynucleotides (e.g., DNA and RNA) exist in a form or setting different from their natural occurrence or setting, or in a form or setting different from their state or setting prior to treatment or purification methods.

[0241] 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

[0242] The nucleic acid vaccines described herein can be used as therapeutic or preventative agents. In some embodiments, the present invention provides a pharmaceutical composition comprising at least one medically acceptable carrier and a nucleic acid vaccine, i.e., a nucleic acid vaccine for COVID-19. Therefore, pharmaceutical compositions comprising the nucleic acid vaccine described herein can be used to prevent, alleviate, and / or treat COVID-19.

[0243] This document provides nucleic acid vaccines and pharmaceutical compositions thereof that can be used in combination with one or more pharmaceutically acceptable excipients. The pharmaceutical compositions may, where appropriate, contain one or more additional active substances, such as therapeutic and / or prophylactic active substances. The pharmaceutical compositions of the nucleic acid vaccines described herein may be sterile and / or pyrogen-free.

[0244] In some embodiments, the composition is administered to humans, human patients, or individuals. For the purposes of this invention, the phrase "active ingredient" generally refers to a nucleic acid vaccine delivered as described herein, or a polynucleotide contained therein, such as a polynucleotide encoding one or more proteins, peptides, fragments or variants of SARS-CoV-2, for the prevention, mitigation and / or treatment of COVID-19.

[0245] Although the description of pharmaceutical compositions provided herein is primarily directed toward pharmaceutical compositions intended 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 non-human animals, including non-human mammals. It should be fully understood that modifications to pharmaceutical compositions intended for administration to humans are necessary to make the compositions suitable for administration to a variety of animals, and that a generally skilled veterinary pharmacologist may design and / or make such modifications using only standard experimental design (if applicable). Individuals covered by the administration of pharmaceutical compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or poultry, including commercially relevant poultry such as poultry, chickens, ducks, geese, and / or turkeys. Formulations

[0246] Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, as used herein, including, but not limited to, any and all solvents, dispersion media, diluents or other liquid media, dispersants or suspending agents, surfactants, isotonants, thickeners or emulsifiers, preservatives and the like, suitable for the desired particular dosage form. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient media is covered within the scope of this invention unless any known carrier medium is incompatible with the substance or its derivatives, such as producing any adverse biological effects or otherwise interacting harmfully with any other component of the pharmaceutical composition.

[0247] Formulations of the pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacology. Generally, such preparation methods include the steps of combining an active ingredient with an excipient and / or one or more other adjuncts, and subsequently, as needed and / or desired, dividing, shaping and / or encapsulating the product into desired single-dose or multi-dose units.

[0248] The pharmaceutical compositions according to the present invention can be prepared, packaged, and / or sold in batches, in single unit doses, and / or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of the active ingredient to be administered to an individual and / or a suitable fraction of that dose, such as half or one-third of such a dose.

[0249] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition according to the present invention will vary depending on the individual's identity, body type, and / or condition and further depending on the route of administration of the composition. For example, the composition may contain between 0.1% and 100%, such as between 0.5% and 50%, between 1% and 30%, between 5% and 80%, or at least 80% (w / w) of the active ingredient.

[0250] In some embodiments, the formulation described herein may contain at least one nucleic acid vaccine composition, such as a nucleic acid vaccine for COVID-19, or an mRNA vaccine for COVID-19. As a non-limiting example, the formulation may contain 1, 2, 3, 4, or 5 nucleic acid vaccine 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 (e.g., mRNA vaccine) compositions with different sequences. In some embodiments, the formulation contains at least three nucleic acid vaccine (e.g., mRNA vaccine) compositions with different sequences. In some embodiments, the formulation contains at least four nucleic acid vaccine (e.g., mRNA vaccine) compositions with different sequences. In some embodiments, the formulation contains at least five nucleic acid vaccine (e.g., mRNA vaccine) compositions with different sequences.

[0251] The nucleic acid vaccine composition of the present invention may be formulated with one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g., from a reservoir of nucleic acid vaccine compositions); (4) alter biodistribution (e.g., target the nucleic acid vaccine composition to a specific tissue or cell type); (5) increase the translation of protein-encoded proteins in vivo; and / or (6) alter the release characteristics of protein-encoded proteins in vivo.

[0252] In addition to conventional excipients (such as any and all solvents, dispersion media, diluents, or other liquid media), the dispersants or suspending agents, surfactants, isotonics, thickeners or emulsifiers, preservatives, and excipients of the present invention may include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with the nucleic acid vaccine composition (e.g., for transplantation into an individual), hyaluronidase, nanoparticle mimics, and combinations thereof. Therefore, formulations of the present invention may include one or more excipients, each present together to increase the stability of the nucleic acid vaccine composition and / or increase the amount of cell transfection of the nucleic acid vaccine composition. Furthermore, the nucleic acid vaccine compositions of the present invention may be formulated using self-assembled nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients, and delivery agents for use in formulating nucleic acids of the nucleic acid vaccine compositions of the present invention are disclosed in PCT Patent Application Publication WO 2013 / 090648, the contents of which are incorporated herein by reference in their entirety. Lipids

[0253] The nucleic acid vaccine composition of the present invention can be formulated with one or more lipids.

[0254] The synthesis of lipids has been extensively described, and formulations containing such compounds are particularly suitable for delivering oligonucleotides or nucleic acids (see Mahon et al., Bioconjug Chem. 2010, 21:1448-1454; Schroeder et al., JIntern Med. 2010, 267:9-21; Akinc et al., Nat Biotechnol. 200, 8 26:561-569; Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869; Siegwart et al., Proc Natl Acad Sci US A. 2011, 108:12996-3001; all of which are incorporated herein by reference in their entirety).

[0255] Although such lipids have been used to efficiently deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci US A. 2008, 105:11915-11920; Akinc et al., Mol Ther. 2009, 17:872-879; Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; all of which are incorporated herein by reference in their entirety), the present invention covers formulations thereof and their use in delivering at least one pharmaceutically acceptable carrier, including in nucleic acid vaccines. Complexes, micelles, liposomes, or particles containing such lipids can be prepared, and thus can be effectively delivered of nucleic acid vaccine compositions after injection of lipid formulations via local and / or systemic routes. Lipid complexes containing nucleic acid vaccine compositions can be administered by various methods, including but not limited to intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), or intraventricular (ICV) administration.

[0256] In vivo delivery of nucleic acids can be affected by many parameters, including but not limited to formulation composition, the nature of particle PEGylation, loading level, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are incorporated herein by reference in full). For example, small variations in the anchor chain length of poly(ethylene glycol) (PEG) lipids can have a significant impact on in vivo efficacy. In vivo activity can be tested for formulations with different lipid types, including but not limited to penta[3-(1-lauroaminopropyl)]-triethyltetramine 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 full), C12-200 (including derivatives and variants), and MD1.

[0257] The lipid class referred to in this paper as "98N12-5" was revealed by Akinc et al., Mol Ther. 2009, 17:872-879, and the contents of that literature are incorporated herein by reference in their entirety.

[0258] The lipid referred to herein as "C12-200" is disclosed in Love et al., Proc Natl Acad Sci US A. 2010, 107:1864-1869 and Liu and Huang, Molecular Therapy. 2010, 669-670; the contents of these references are incorporated herein by reference in their entirety. Lipid formulations may include particles containing three or four or more components in addition to nucleic acid vaccine compositions. For example, formulations having certain lipids include, but are not limited to, 98N12-5 and may contain 42% lipids, 48% cholesterol, and 10% PEG (C14 alkyl chain length). As another example, formulations having certain lipids include, but are not limited to, C12-200 and may contain 50% lipids, 10% distearate phosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

[0259] In some embodiments, the nucleic acid vaccine composition formulated with lipids is used for systemic intravenous administration. For example, the final optimized intravenous formulation, which is optimized to allow greater than 90% distribution to the liver, uses a nucleic acid vaccine composition and comprises a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipids, having a final weight ratio of approximately 7.5:1 total lipids to nucleic acid vaccine composition and a C14 alkyl chain length on the PEG lipids with an average particle size of approximately 50-60 nm. (See Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are incorporated herein by reference in their entirety). In another example, intravenous formulations using C12-200 lipids (see PCT patent application publication WO2010129709, the contents of which are incorporated herein by reference in their entirety) can have a C12-200 / distearate phosphatidylcholine / cholesterol / PEG-DMG molar ratio of 50 / 10 / 38.5 / 1.5, wherein a total lipid:nucleic acid weight ratio of 7:1 and an average particle size of 80 nm can effectively deliver nucleic acid vaccine 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).

[0260] In some embodiments, formulations containing MD1 lipids can be used to efficiently deliver nucleic acid vaccine compositions to hepatocytes in vivo. The properties of optimized lipid formulations for intramuscular or subcutaneous routes can vary significantly depending on the target cell type and the ability of the formulation to diffuse into the bloodstream via the extracellular matrix. While particle sizes smaller than 150 nm may be required for efficient hepatocyte delivery due to the size of endothelial pores (see Akinc et al., Mol Ther. 2009, 17:872-879, the contents of which are incorporated herein by reference in their entirety), delivery of formulations to other cell types (including, but not limited to, endothelial cells, bone marrow cells, and muscle cells) using lipid-formulated nucleic acid vaccine compositions may not be subject to similar size limitations.

[0261] The in vivo delivery of siRNA to other non-hepatic cells, such as bone marrow cells and endothelial cells, using lipid modulators has been reported (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009, 19:3112-3118; 8th International Judah Folkman Conference, Cambridge, MA, October 8-9, 2010; the contents of these references are incorporated herein by reference in their entirety). For effective delivery to bone marrow cells, such as monocytes, lipid modulators may have a similar component molar ratio. Different ratios of lipids to other components (including but not limited to distearate phosphatidylcholine, cholesterol, and PEG-DMG) can be used to optimize the formulation of nucleic acid vaccine compositions for delivery to different cell types, including but not limited to hepatocytes, bone marrow cells, and muscle cells. For example, component molar ratios may include, but are not limited to, 50% C12-200, 10% distearate phosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG (see Leuschner et al., Nat Biotechnol 2011, 29:1005-1010; the contents of which are incorporated herein by reference in their entirety). Local delivery of nucleic acids to cells via subcutaneous or intramuscular delivery using lipid formulations may not require all the formulation components needed for systemic delivery, and therefore may consist only of lipids and the nucleic acid vaccine composition. Liposomes

[0262] The nucleic acid vaccine composition of the present invention can be formulated using one or more liposomes.

[0263] In some embodiments, the pharmaceutical composition of the nucleic acid vaccine composition includes liposomes. Liposomes are artificially prepared vesicles that may consist primarily of a lipid bilayer and can be used as delivery media for nutrients and pharmaceutical formulations. Liposomes can have different sizes, such as, but not limited to, multilayer vesicles (MLVs) with diameters of hundreds of nanometers and containing a series of concentric bilayers separated by narrow aqueous compartments, small single-cell vesicles (SUVs) with diameters less than 50 nm, and large monolayer vesicles (LUVs) with diameters between 50 nm and 500 nm. Liposome design may include, but is not limited to, opsonins or ligands, to improve liposome attachment or activation events with unhealthy tissues, such as, but not limited to, endocytosis. Liposomes may contain low or high pH to improve the delivery of pharmaceutical formulations.

[0264] The formation of liposomes may depend on physicochemical characteristics, such as, but not limited to, the encapsulated pharmaceutical formulation and liposome components; the nature of the medium in which the liposomes are dispersed; the effective concentration of the encapsulated substance and its potential toxicity; any other processes involved in the application and / or delivery of the vesicles; the optimal size, polydispersity and shelf life of the vesicles for the intended application; and batch-to-batch reproducibility and the possibility of large-scale production of safe and efficient liposome products.

[0265] In some embodiments, pharmaceutical compositions comprising the nucleic acid vaccines described herein may include, but are not limited to, liposomes, such as liposomes formed from 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), SMARTICLES® / NOV340 (Marina Biotech, Bothell), 1,2-dilinolenoyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleno-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (DLin-KC2-DMA), and MC3 (US Patent Application Publication US20100324120; the contents of which are incorporated herein by reference in their entirety), neutral DOPC (1,2-diolenoyl-sn-glycero-3-phosphocholine) liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer)). Biology & Therapy 2006, 5(12): 1708-1713); the contents of which are incorporated herein by reference in full), hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel) and liposomes capable of delivering small molecule drugs, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[0266] In some embodiments, pharmaceutical compositions comprising the nucleic acid vaccines described herein may include, but are not limited to, liposomes, such as liposomes formed from synthetic stable plasmid-lipid particles (SPLP) or stable nucleic acid lipid particles (SNALP), which have been previously described and demonstrated to be suitable for in vitro and in vivo oligonucleotide delivery (see Wheeler et al. Gene Therapy. 1999, 6:271-281; ​​Zhang et al. Gene Therapy. 1999, 6:1438-1447; Jeffs et al. Pharm Res. 2005, 22:362-372; Morrissey et al., Nat Biotechnol. 2005, 2:1002-1007; Zimmermann et al., Nature. 2006, 441:111-114; Heyes et al. J Contr Rel. 2005, 107:276-287; Semple et al. Nature). Biotech. 2010, 28:172-176; Judge et al. J Clin Invest. 2009, 119:661-673; de FougerollesHum Gene Ther. 2008, 19:125-132; each of these contents is incorporated herein by reference in its entirety. Wheeler et al.'s original manufacturing method was a detergent dialysis method, which was subsequently modified by Jeffs et al. and referred to as the spontaneous vesicle formation method. In addition to nucleic acid vaccine compositions, liposome formulations may consist of 3 to 4 lipid components. As a non-limiting example, liposomes may contain, but are not limited to, 55% cholesterol, 20% distearate phosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. In another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipids, wherein the cationic lipids may be 1,2-distearate-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 about 50 mol% to about 85 mol% of the total lipids present in the particle, cationic lipids; about 13 mol% to about 49.5 mol% of the total lipids present in the particle, non-cationic lipids; and about 0.5 mol% to about 2 mol% of the total lipids present in the particle, binding lipids that inhibit particle aggregation, as described in WO2009127060 issued to Maclachlan et al., the contents of which are incorporated herein by reference in their entirety. In another example, the nucleic acid-lipid particle may be any nucleic acid-lipid particle disclosed in US2006008910 issued to Maclachlan et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the nucleic acid-lipid particle may comprise cationic lipids of Formula I, non-cationic lipids, and binding lipids that inhibit particle aggregation.

[0267] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in lipid vesicles, which may have cross-linking between functionalized lipid bilayers.

[0268] In some embodiments, the liposomes may contain the sugar-modified lipids disclosed in U.S. Patent No. US5,595,756 to Bally et al., the contents of which are incorporated herein by reference in their entirety. The lipids may be gangliosides and cerebrosides in an amount of about 10 mol%.

[0269] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in liposomes comprising cationic lipids. The liposomes may have a molar ratio (N:P ratio) of nitrogen atoms in the cationic lipids to phosphates in the nucleic acid vaccine composition between 1:1 and 20:1, as described in PCT Patent Application Publication No. WO2013006825, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the liposomes may have an N:P ratio greater than 20:1 or less than 1:1.

[0270] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in a lipid-polycationic complex. The formation of the lipid-polycationic complex may be achieved by methods known in the art and / or as described in U.S. Patent Application Publication No. 20120178702, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the polycation may include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyguanine, and / or polyarginine, and cationic peptides described in PCT Patent Application Publication No. WO2012013326, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the nucleic acid vaccine composition may be formulated in a lipid-polycationic complex, which may further include neutral lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0271] Liposome formulations can be influenced by factors including, but not limited to, the selection of cationic lipid components, cationic lipid saturation, the nature of PEGylation, the ratio of all components, and biophysical parameters such as size. In one example by Semple et al. (Semple et al., Nature Biotech. 2010, 28:172-176; the contents of which are incorporated herein by reference in their entirety), the liposome formulation consisted of 57.1% cationic lipids, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.

[0272] In some embodiments, the pharmaceutical composition may be formulated with any of the amphoteric liposomes disclosed in: PCT Patent Application Publication No. WO 2008043575 issued to Panzner and U.S. Patent No. US 8,580,297 issued to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety. Amphoteric liposomes may comprise a mixture of lipids, including cationic amphiphilic molecules, anionic amphiphilic molecules, and, where appropriate, one or more neutral amphiphilic molecules. Amphoteric liposomes may comprise an amphoteric compound based on an amphiphilic molecule, wherein the head group of the amphiphilic molecule is substituted with one or more amphoteric groups. In some embodiments, the pharmaceutical composition may be formulated with an amphoteric lipid comprising one or more amphoteric groups having an isoelectric point of 4 to 9, as disclosed in U.S. Patent Application Publication No. 20140227345 to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated with liposomes comprising a sterol derivative as disclosed in U.S. Patent No. 7312206 to Panzner et al. (Novosom), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated with amphoteric liposomes comprising at least one amphiphilic cationic lipid, at least one amphiphilic anionic lipid, and at least one neutral lipid, or with liposomes comprising at least one amphiphilic lipid having both a positive and a negative charge and at least one neutral lipid, wherein the liposomes are stable at pH 4.2 and pH 7.5, as disclosed in U.S. Patent No. 7,780,983 to Panzner et al., the contents of which are incorporated herein by reference in their entirety. In some embodiments, the pharmaceutical composition may be formulated with liposomes capable of encapsulating the nucleic acid vaccine composition of the present invention, such liposomes comprising a serum-stable mixture of lipids taught in U.S. Patent Application Publication No. 20110076322 to Panzner et al., the contents of which are incorporated herein by reference in their entirety. The lipid mixture comprises phosphatidylcholine and phosphatidylethanolamine in a ratio ranging from about 0.5 to about 8. The lipid mixture may also include pH-sensitive anionic and cationic amphiphilic molecules, making the mixture amphoteric, negatively or neutral at pH 7.4 and positively charged at pH 4. The drug / lipid ratio can be adjusted to target the liposomes to specific organs or other sites in the body. In some embodiments, the lipid system loaded with the nucleic acid vaccine composition of the present invention is prepared by the method disclosed in U.S. Patent Application Publication No. 20120021042 to Panzner et al., the contents of which are incorporated herein by reference in their entirety.The method includes mixing an aqueous solution of a polyanionic surfactant with an alcoholic solution of one or more amphiphilic molecules and buffering the mixture to an acidic pH, wherein the one or more amphiphilic molecules readily form amphoteric liposomes at the acidic pH, thereby forming amphoteric liposome complexes in a suspension of the surfactant.

[0273] The nucleic acid vaccine composition of the present invention can be formulated using one or more lipid complexes.

[0274] In some embodiments, the nucleic acid vaccine composition may be formulated as a lipid complex, such as, but not limited to, the ATUPLEXTM system, the DACC system, the DBTC system, and other siRNA-lipid complex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, MA), and targeted and non-targeted nucleic acid delivery based on polyethyleneimine (PEI) or protamine (Aleku et al. Cancer Res. 2008, 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther, 2012, 50:76-78; Santel et al., Gene Ther, 2006, 13:1222-1234; Santel et al., Gene Ther., 2006, 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010, 23:334-344; Kaufmann et al. Microvasc Res., 2010, 80:286-293; Weide et al. J Immunother., 2009, 32:498-507; Weide et al. J Immunother., 2008, 31:180-188; Pascolo., Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., J. Immunother., 2011, 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci US A. 2007, 6;104:4095-4100; deFougerollesHum Gene Ther.2008, 19:125-132; each of these contents is incorporated herein by reference in its entirety. Lipid nanoparticles (LNPs)

[0275] In some embodiments, the nucleic acid vaccine composition of the present invention can be formulated in lipid nanoparticles (LNPs). Generally, LNPs are characterized as small solid or semi-solid particles having an outer lipid layer having a hydrophilic outer surface exposed to a non-LNP environment; an internal space that can be aqueous (vesicle-like) or non-aqueous (micelle-like); and at least one hydrophobic intermembrane space. The LNP membrane can be layered or non-layered and can consist of 1, 2, 3, 4, 5 or more layers. In some embodiments, the LNP may contain a load or payload entering its internal space, entering its intermembrane space, entering its outer surface, or any combination thereof.

[0276] The LNPs used herein are known in this art and generally contain cholesterol (which contributes to stability and promotes membrane fusion), phospholipids (which provide structure for the LNP bilayer and also help endosome escape), polyethylene glycol (PEG) derivatives (which reduce LNP aggregation and "protect" LNPs from nonspecific endocytosis by immune cells) and ionizable lipids (which complex with negatively charged RNA and enhance endosome escape), forming an LNP-forming composition.

[0277] The components of the LNP can be selected based on the desired target, orientation, load, size or other desired characteristics or properties.

[0278] LNP may be the lipid nanoparticles described in PCT Patent Application Publication No. WO2012170930, the contents of which are incorporated herein by reference in their entirety.

[0279] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in an LNP containing at least one cationic lipid.

[0280] In some embodiments, the cationic lipids that can be used in the formulations of the present invention may be selected from, but are not limited to, the cationic lipids described in the following: PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, etc. The contents of U.S. Patent Nos. 2012054365, 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 are hereby incorporated herein by reference in their entirety. Cationic lipids may also be selected from, but are not limited to, Formula A as described in, the following: PCT Patent Application Publications Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365 and WO2012044638, the contents of which are incorporated herein by reference in their entirety. Alternatively, the cationic lipid may be selected from, but is not limited to, the formula CLI-CLXXIX of PCT Patent Application No. WO2008103276, the formula CLI-CLXXIX of U.S. Patent No. 7,893,302, the formula CLI-CLXXXXII of U.S. Patent No. 7,404,969, and the formulas I-VI of U.S. Patent Publication No. US20100036115; the contents of which are each incorporated herein by reference in their entirety. The cationic lipid may be a multivalent cationic lipid, such as the cationic lipid disclosed in U.S. Patent No. 7,223,887 to Gaucheron et al., the contents of which are incorporated herein by reference in their entirety. The cationic lipid may have a positively charged head group comprising two quaternary amino groups and a hydrophobic portion comprising four hydrocarbon chains, as described in U.S. Patent No. 7,223,887 to Gaucheron et al. Cationic lipids may be biodegradable, such as those disclosed in U.S. Patent Application Publication No. 20130195920 to Maier et al., the contents of which are incorporated herein by reference in their entirety. Cationic lipids may have one or more biodegradable groups located in the lipid portion of the cationic lipid, as described in Formulas I-IV of US20130195920 to Maier et al.In some embodiments, the cationic lipids may also be those disclosed in US20130156845 and US 20130129785, WO 2012047656, WO 2010144740, WO 2013086322, or WO 2012016184, all of which are incorporated herein by reference in their entirety.

[0281] As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonadecano-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethylhexadecano-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentadecano-16,19-diene-8-amine, (13Z,16Z)-N,N-dimethyltetradecano-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethyltetradecano-12,15-diene-4-amine, (14Z,17Z)-N,N-dimethyltetradecano-14,17-diene-6-amine, (15Z,18Z)-N,N-di Methyltetracos-15,18-diene-7-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltridec-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethyloctadec-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexadec-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethyldi Pentadecyl-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethyltridecyl-22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyltridecyl-21,24-diene-9-amine, (18Z)-N,N-dimethylheptadecyl-18-en-10-amine, (17Z)-N,N-dimethylhexadecyl-17-en-9-amine, (19Z,22Z)-N,N-dimethylhexadecadecyl-19,22-diene-7-amine, N,N-dimethylheptadecane-10-amine, (20Z,23Z)-N-ethyl-N-methylhexadecadecyl-20,23-diene-10-amine, 1-[(11Z,14Z)-l-nonyl] [Eicosene-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptadec-20-en-10-amine, (15Z)-N,N-dimethylheptadec-15-en-10-amine, (14Z)-N,N-dimethylheptadec-14-en-10-amine, (17Z)-N,N-dimethylheptadec-17-en-10-amine, (24Z)-N,N-dimethyltridec-24-en-10-amine, (20Z)-N,N-dimethylheptadec-20-en-10-amine, (22Z)-N,N-dimethyltridec-22-en-10-amine, (16Z)-N,N-dimethylpentadecan-16-en-8-amine, (12Z,(15Z)-N,N-dimethyl-2-nonyldocosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadec-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecano-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecano-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]docosa-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R, 2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecano-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadec-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradec-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodec-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadec-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecano-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecano-8-amine, RN,N -Dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)prop-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-octyl-5-en-1-yloxy]prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-octyl-5-en-1-yloxy]prop-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)methyl} (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-(nonoxy)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-dien-1-yloxy]-3-(octyloxy)prop-2-amine, (2S)-N,N-Dimethyl-1-[(6Z,9Z,12Z)-octadec-6,9,12-trien-1-yloxy]-3-(octoxy)prop-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentoxy)prop-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylprop-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]- N,N-dimethyl-3-(octyloxy)prop-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylprop-2-amine, (2S)-1-[( [13Z)-(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylprop-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, 1-[(9Z)-hexadecane-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, (2R)-N,N-dimethyl [(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]prop-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S, 2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)prop-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)prop-2-amine and (1lE,20Z,23Z)-N,N-dimethyl-2,Nicocarb-11,20,2-trien-10-amine, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the lipid may be a cleavable lipid, such as that described in PCT Patent Application Publication No. WO2012170889, the contents of which are incorporated herein by reference in their entirety.

[0282] In some embodiments, the nanoparticles described herein may comprise at least one cationic polymer described herein and / or known in the art.

[0283] In some embodiments, the cationic lipids may be synthesized by methods known in the art and / or as described in the following: PCT patent applications No. WO2012040184, No. WO2011153120, No. WO2011149733, No. WO2011090965, No. WO2011043913, No. WO2011022460, No. WO2012061259, No. WO2012054365, No. WO2012044638, No. WO2010080724 and No. WO201021865, the contents of which are each incorporated herein by reference in their entirety.

[0284] In some embodiments, the pharmaceutical composition of the nucleic acid vaccine composition may include at least one of the polyethylene glycol-modified lipids described in PCT Patent Application Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety.

[0285] In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulation may be increased or decreased and / or the carbon chain length of the PEG lipids may be modified within the C14 to C18 range to alter the pharmacokinetics and / or biodistribution of the LNP formulation. As a non-limiting example, the LNP formulation may contain a lipid molar ratio of 1-5% PEG-c-DOMG compared to cationic lipids, DSPCs, and cholesterol. In some embodiments, the LNP formulation of the nucleic acid vaccine composition may contain a lipid molar ratio of 3% PEG-c-DOMG. In some embodiments, the LNP formulation of the nucleic acid vaccine composition may contain a lipid molar ratio of 1.5% PEG-c-DOMG.

[0286] In some embodiments, PEG-c-DOMG may be replaced with PEG lipids, such as, but not limited to, PEG-DSG (1,2-distearyl-sn-glycerol, methoxy polyethylene glycol) or PEG-DPG (1,2-dispalmolyl-sn-glycerol, methoxy polyethylene glycol). 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.

[0287] In some embodiments, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000), a cationic lipid known in the art. In some embodiments, the LNP formulation may contain PEG-DMG 2000 and at least one other component. In some embodiments, the LNP formulation may contain PEG-DMG 2000, DSPC, and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, LNP formulations may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (see, for example, Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS, 2012, 109(36): 14604-14609; incorporated herein by reference in its entirety).

[0288] As another non-limiting example, the nucleic acid vaccine composition described herein can be formulated as nanoparticles delivered via a non-enteral route, as described in U.S. Patent Application Publication No. US20120207845, the contents of which are incorporated herein by reference in their entirety.

[0289] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated with a plurality of cationic lipids, such as the first and second cationic lipids described in U.S. Patent Application Publication No. US20130017223 to Hope et al., the contents of which are incorporated herein by reference in their entirety. The first cationic lipid may be selected based on a first characteristic and the second cationic lipid may be selected based on a second characteristic, wherein the characteristics may be determined as outlined in US20130017223. In some embodiments, the first and second characteristics are complementary.

[0290] The 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 a solid core as described in U.S. Patent Publication No. US20120276209 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.

[0291] In some embodiments, the nucleic acid vaccine composition of the present invention may be complexed with a cationic amphiphilic molecule in an oil-in-water (o / w) emulsion, such as that described in European Publication No. EP2298358 to Satishchandran et al., the contents of which are incorporated herein by reference in their entirety. The cationic amphiphilic molecule may be a cationic lipid, modified or unmodified spermine, bupivacaine, or benzalkonium chloride, and the oil may 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 the oil-in-water emulsion (see, for example, the complex described in EP2298358 to Satishchandran et al., the contents of which are incorporated herein by reference in their entirety).

[0292] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated using a composition comprising a mixture of cationic compounds and neutral lipids. As a non-limiting example, the cationic compound may be of formula (I) disclosed in PCT Patent Application Publication No. WO 1999010390 to Ansell et al., the contents of which are described herein by reference in their entirety, and the neutral lipid may be selected from the group consisting of diacetylphosphatidylcholine, diacetylphosphatidylethanolamine, ceramide, and sphingomyelin. In another non-limiting example, the lipid formulation may comprise cationic lipids of formula A disclosed in U.S. Patent Publication No. US 20120101148 to Akinc et al., neutral lipids, sterols, and PEG or PEG-modified lipids, the contents of which are incorporated herein by reference in their entirety.

[0293] In some embodiments, the LNP formulation can be formulated by the methods described in International Publication Nos. WO2011127255 or WO2008103276. As a non-limiting example, the nucleic acid vaccine composition of the present invention can be encapsulated in any of the lipid nanoparticle (LNP) formulations described in WO2011127255 and / or WO2008103276; the contents of each of these documents are incorporated herein by reference in their entirety.

[0294] In some embodiments, the LNP formulations described herein may comprise a polycationic composition. 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 comprising polycationic compositions may be used for in vivo and / or in vitro delivery of the nucleic acid vaccine compositions described herein.

[0295] In some embodiments, the LNP formulations described herein may additionally include a permeability enhancer molecule. A non-limiting permeability enhancer molecule is described in U.S. Patent Publication No. US20050222064, the contents of which are incorporated herein by reference in their entirety.

[0296] The nanoparticle formulation may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid vaccine composition (e.g., a nucleic acid vaccine for COVID-19). As a non-limiting example, the carbohydrate carrier may include, but is not limited to, anhydride-modified plant glycogen or glycogenic material, plant glycogen octenyl succinate, plant glycogen β-dextrin, and anhydride-modified plant glycogen β-dextrin. (See, for example, PCT Patent Application Publication No. WO2012109121; the contents of which are incorporated herein by reference in their entirety).

[0297] Lipid nanoparticle formulations can be modified by replacing 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 may be potential sources of toxicity. The rapid metabolism of rapidly eliminating lipids can improve the tolerability and therapeutic index of lipid nanoparticles by doses ranging from 1 mg / kg to 10 mg / kg in rats. Ester bonds, including those involved in enzymatic degradation, can improve the degradation and metabolic profiles of the cationic component while maintaining the activity of the reLNP formulation. The ester bond may be internally located within the lipid chain or terminally located at the end of the lipid chain. Internal ester bonds can replace any carbon in the lipid chain.

[0298] In some embodiments, the nucleic acid vaccine composition is formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLNs) may be spherical with an average diameter between 10 and 1000 nm. SLNs have a solid lipid core matrix that is soluble in lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. The lipid nanoparticles may be self-assembled 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).

[0300] In some embodiments, formulations comprising the nucleic acid vaccine compositions described herein may 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 of these contents is incorporated herein by reference in its entirety. An example of passive targeting of hepatocytes by formulations 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 the in vivo binding and uptake of these formulations into hepatocytes (Akinc et al., Mol Ther. 2010, 18:1357-1364; the contents of which are incorporated herein by reference in their entirety).Compounds can also selectively target substances by expressing different ligands on their surface, as exemplified by, but not limited to, folic acid, transferrin, N-acetylglucosamine (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., 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., 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; PeerJ 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; their contents are incorporated herein by reference in their entirety.

[0302] In some embodiments, the nucleic acid vaccine composition of the present invention may 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, the nucleic acid vaccine composition may be encapsulated within a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulation" means to close, surround, or cover. Since encapsulation involves formulations of the compositions of the present invention, encapsulation may be substantial, complete, or partial. The term "substantially encapsulated" means that at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9%, or more than 99.999% of the pharmaceutical composition of the present invention may be closed, surrounded, or covered within a delivery agent. "Partial encapsulation" means that less than 10, 10, 20, 30, 40, 50, or fewer of the pharmaceutical composition or compound of the present invention may be encapsulated, surrounded, or covered within a delivery agent. Advantageously, encapsulation can be determined by measuring the escape or activity of the pharmaceutical composition of the present invention 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 pharmaceutical composition of the present invention is encapsulated in a delivery agent.

[0303] Nucleic acid vaccine compositions may be encapsulated into lipid nanoparticles or rapidly eliminating lipid nanoparticles, and the lipid nanoparticles or rapidly eliminating lipid nanoparticles may then be encapsulated into polymers, hydrogels, and / or surgical sealants described herein and / or known in this art. As non-limiting examples, polymers, hydrogels, or surgical sealants may be PLGA, 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).

[0304] In some embodiments, the lipid nanoparticles may be encapsulated in any polymer known in the art that can form a gel when injected into an individual. As another non-limiting example, the lipid nanoparticles may be encapsulated in a biodegradable polymer matrix.

[0305] 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, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS®, and cellulose derivatives such as aqueous dispersions of ethyl cellulose (AQUACOAT® and SURELEASE®).

[0306] In some embodiments, the controlled release and / or targeted delivery formulation may comprise at least one biodegradable polyester, which may contain polycationic side chains. Biodegradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the biodegradable polyester may include PEG-bonded components to form a polyethylene glycol-modified polymer.

[0307] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated with targeted lipids having a targeting portion, such as that disclosed in U.S. Patent Application Publication No. 20130202652 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the targeting portion of Formula I of US 20130202652 to Manoharan et al. may be selected to facilitate the localization of lipids to desired organs, tissues, cells, cell types or subtypes or organelles. Non-limiting targeting portions covered in this invention include transferrin, paramethoxybenzamide, RGD peptide, prostate-specific membrane antigen (PSMA), fucose, antibodies or aptamers.

[0308] In some embodiments, the nucleic acid vaccine composition of the present invention may be encapsulated in therapeutic nanoparticles. The therapeutic nanoparticles may be formulated by methods described herein and known in the art, such as, but not limited to, PCT Patent Application Publications Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723 and WO2012054923, and U.S. Patent Application Publications Nos. US20110262491 and US2010010464. US Patent Nos. 5, 20100087337, 20100068285, 20110274759, 20100068286, and 20120288541, and US Patent Nos. 8,206,747, 8,293,276, 8,318,208, and 8,318,211, the contents of which are each incorporated herein by reference in their entirety. Therapeutic polymer nanoparticles can be identified by the method described in US Publication No. US20120140790, the contents of which are incorporated herein by reference in their entirety.

[0309] In some embodiments, the therapeutic nanoparticles can be formulated for sustained release. As used herein, "sustained release" means a pharmaceutical composition or compound that meets a release rate over a specific period of time. This period of time may include, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained-release nanoparticles may comprise polymers and therapeutic agents, such as, but not limited to, the nucleic acid vaccine compositions of the present invention (see PCT Patent Application Publication No. WO2010075072 and U.S. Publications Nos. US20100216804, US20110217377, and US20120201859, the contents of which are each incorporated herein by reference in their entirety).

[0310] In some embodiments, the therapeutic nanoparticles may be formulated to be target-specific. As a non-limiting example, the 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 embodiments, the therapeutic nanoparticles may be formulated to be cancer-specific. As a non-limiting example, the therapeutic nanoparticles may be formulated as nanoparticles described in the following: PCT Patent Application Publications Nos. WO2008121949, WO2010005726, WO2010005725 and WO2011084521, and U.S. Patent Application Publications Nos. US20100069426, US20120004293 and US20100104655, the contents of which are each incorporated herein by reference in their entirety.

[0311] In some embodiments, the nanoparticles of the present invention may comprise a polymer matrix. As a non-limiting example, the nanoparticles may comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl transbutenedioate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphoradenene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), or combinations thereof.

[0312] In some embodiments, the therapeutic nanoparticles comprise a diblock copolymer. In some embodiments, the diblock copolymer may comprise a combination of PEG and a polymer such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), or combinations thereof.

[0313] As a non-limiting example, the therapeutic nanoparticles comprise PLGA-PEG block copolymers (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, each of which is incorporated herein by reference in its entirety). In another non-limiting example, the therapeutic nanoparticles are stealthy 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, the contents of which are incorporated herein by reference in their entirety).

[0314] In some embodiments, the therapeutic nanoparticles may comprise multiblock copolymers, such as, but not limited to, the multiblock copolymers described in U.S. Patent Nos. 8,263,665 and 8,287,910, the contents of which are each incorporated herein by reference in their entirety.

[0315] In some embodiments, the block copolymers described herein may be included in a polyionic composite comprising nonpolymeric micelles and block copolymers. (See, for example, U.S. Publication No. US20120076836, the contents of which are incorporated herein by reference in their entirety).

[0316] In some embodiments, the nanoparticles used to deliver the nucleic acid vaccine described herein comprise block copolymers. Non-limiting examples of block copolymers include formulas I, II, III, IV, V, VI, and VII of PCT Patent Application Publication No. WO2015017519, the contents of which are incorporated herein by reference in their entirety.

[0317] In some embodiments, the therapeutic nanoparticles may comprise 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 copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof.

[0318] In some embodiments, the therapeutic nanoparticles may comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(aminoamine) dendrimer, poly(β-amino ester) (see, for example, U.S. Patent No. 8,287,849, the contents of which are incorporated herein by reference in their entirety) and combinations thereof.

[0319] In some embodiments, the therapeutic nanoparticles may comprise at least one biodegradable polyester, which may contain polycationic side chains. Biodegradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. Biodegradable polyesters may include PEG-bonded polymers to form polyethylene glycol-modified polymers.

[0320] In some embodiments, the therapeutic nanoparticles may include binding to at least one targeting ligand. The targeting ligand may 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).

[0321] In some embodiments, the therapeutic nanoparticles may be formulated in an aqueous solution that can be used to target cancer (see PCT Patent Application Publication No. WO2011084513 and U.S. Patent Application Publication No. US20110294717, the contents of which are incorporated herein by reference in their entirety).

[0322] In some embodiments, the nucleic acid vaccine composition may be encapsulated in, linked to, and / or bound to a synthetic nanocarrier. The synthetic nanocarrier includes, but is not limited to, those described in the following: PCT Patent Application Publication Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, and WO20121... The contents of patent applications No. 49301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and U.S. Patent Application Publications Nos. US20110262491, US20100104645, US20100087337 and US20120244222 are each incorporated herein by reference in their entirety. Nanoparticle carriers can be formulated using methods known in this art and / or described herein. As a non-limiting example, the synthetic nanocarrier can be formulated by the methods described in PCT Patent Application Publications Nos. WO2010005740, WO2010030763, and WO201213501, and U.S. Patent Publications Nos. US20110262491, US20100104645, US20100087337, and US2012024422, the contents of which are each incorporated herein by reference in their entirety. The synthetic nanocarrier formulation can be lyophilized by the methods described in PCT Patent Application Publication No. WO2011072218 and U.S. Patent No. 8,211,473; the contents of which are each incorporated herein by reference in their entirety.

[0323] In some embodiments, the synthetic nanocarrier may contain reactive groups to release the nucleic acid vaccine composition described herein (see PCT Patent Application Publication No. WO20120952552 and U.S. Patent Application Publication No. US20120171229, the contents of which are incorporated herein by reference in their entirety).

[0324] In some embodiments, the synthetic nanocarrier can be formulated for targeted release. In some embodiments, the synthetic nanocarrier can be formulated to release the nucleic acid vaccine composition at a specified pH and / or after a desired time interval. As a non-limiting example, the synthetic nanoparticles can be formulated to release the nucleic acid vaccine composition after 24 hours and / or at a pH of 4.5 (see PCT Patent Application Publications Nos. WO2010138193 and WO2010138194, and U.S. Patent Application Publications Nos. US20110020388 and US20110027217, the contents of which are each incorporated herein by reference in their entirety).

[0325] In some embodiments, the synthetic nanocarrier can be formulated for controlled release and / or sustained release of the nucleic acid vaccine composition described herein. As a non-limiting example, the synthetic nanocarrier for sustained release can be formulated by methods known in the art, described herein, and / or as described in PCT Patent Application Publication No. WO2010138192 and U.S. Patent Application Publication No. US20100303850, the contents of which are each incorporated herein by reference in their entirety.

[0326] In some embodiments, the nanoparticles may be optimized for oral administration. The nanoparticles may comprise at least one cationic biopolymer, such as, but not limited to, polyglucosamine or derivatives thereof. As a non-limiting example, the nanoparticles may be formulated by the method described in U.S. Publication No. US20120282343, the contents of which are incorporated herein by reference in their entirety.

[0327] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in a modular composition such as that described in U.S. Patent No. 8,575,123 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the modular composition may comprise a nucleic acid (e.g., the nucleic acid vaccine composition of the present invention), at least one endosome cleavage component, and at least one targeting ligand. The modular composition may have a formula such as any of those described in US 8,575,123 to Manoharan et al.

[0328] In some embodiments, the nucleic acid vaccine composition of the present invention may be encapsulated in a lipid formulation to form stable nucleic acid-lipid particles (SNALP), as described in, for example, U.S. Patent No. 8,546,554 to de Fougerolles et al., the contents of which are incorporated herein by reference in their entirety. The lipids may 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 range of: 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 comprises 40% 2,2-dilinole-4-dimethylaminoethyl-[1,3]-dioxane (lipid A), 10% dioleoylphosphatidylcholine (DSPC), 40% cholesterol, and 10% polyethylene glycol (PEG)-C-DOMG (moles percentage), with a particle size of 63.0 ± 20 nm and a nucleic acid / lipid ratio of 0.027.

[0329] The nucleic acid vaccine composition of the present invention can be formulated with nucleic acid-lipid particles containing an endosomal destabilizer, as disclosed in U.S. Patent No. 7,189,705 to Lam et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the endosomal destabilizer may be Ca2+ ions.

[0330] In some embodiments, the nucleic acid vaccine composition of the present invention may 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 herein by reference in their entirety. Akinc et al. teach that FLiPs may comprise at least one of a single-stranded or double-stranded oligonucleotide, wherein the oligonucleotide has been bound to at least one of an emulsion or liposome that has been aggregated, blended, or bound together with the bound oligonucleotide. As disclosed in US 8,148,344 to Akinc et al., such particles have unexpectedly shown to effectively deliver oligonucleotides to the heart, lungs, and muscles.

[0331] In some embodiments, the nucleic acid vaccine composition of the present invention can be delivered to cells using a composition comprising an expression vector in a lipid formulation, as described in U.S. Patent No. 6,086,913 to Tam et al., the contents of which are incorporated herein by reference in their entirety. The composition disclosed by Tam is serum stable and comprises an expression vector containing first and second inverted repeat sequences from adeno-associated virus (AAV), a rep gene from AAV, and a nucleic acid fragment. The expression vector in Tam is compounded with lipids.

[0332] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated with lipid formulations disclosed in U.S. Publication No. 20120270921, issued to de Fougerolles et al., the contents of which are incorporated herein by reference in their entirety. In one non-limiting example, the lipid formulation may comprise a cationic lipid having Formula A as described in US 20120270921. In another non-limiting example, a composition of exemplary nucleic acid-lipid particles disclosed in Table A of US 20120270921 may be used with the nucleic acid vaccine composition of the present invention.

[0333] In some embodiments, the nucleic acid vaccine composition of the present invention may be completely encapsulated in the lipid particles disclosed in U.S. Publication No. 20120276207, issued to Maurer et al., the contents of which are incorporated herein by reference in their entirety. The particles may comprise a lipid composition comprising pre-formed lipid vesicles, an charged therapeutic agent, and a destabilizer to form a mixture of the pre-formed vesicles and the therapeutic agent in a destabilizing solvent, wherein the destabilizing solvent effectively destabilizes the membrane of the pre-formed lipid vesicles without destroying the vesicles.

[0334] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated with conjugated lipids. In a non-limiting example, the conjugated lipids may have a formula such as that described in U.S. Publication No. 20120264810 to Lin et al., the contents of which are incorporated herein by reference in their entirety. The conjugated lipids may form lipid particles, which further comprise cationic lipids, neutral lipids, and lipids capable of reducing aggregation.

[0335] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in a neutral liposome formulation, such as that disclosed in U.S. Publication No. 20120244207, issued to Fitzgerald et al., the contents of which are incorporated herein by reference in their entirety. The phrase "neutral liposome formulation" means a liposome formulation having a near-neutral or neutral surface charge at physiological pH. Physiological pH may be, for example, from 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 liposome formulations are ionizable lipid nanoparticles (iLNPs). Neutral liposome formulations may include ionizable cationic lipids, such as DLin-KC2-DMA.

[0336] In some embodiments, the nucleic acid vaccine compositions of the present invention may be formulated with charged lipids or amino lipids. As used herein, the term "charged lipid" is intended to include lipids having one or two aliphatic or aliphatic alkyl chains and a quaternary amino head group. The quaternary amine carries a permanent positive charge. The head group may include, where appropriate, 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 alter the pKa of an ionizable group relative to the pKa of a structurally similar compound that does not have a quaternary amine (e.g., a quaternary amine replaced by a tertiary amine). In some embodiments, the charged lipid is referred to as an "amino lipid". In a non-limiting example, an amino lipid may be any amino lipid described in U.S. Publication No. US20110256175 to Hope et al., the contents of which are incorporated herein by reference in their entirety. For example, the amino lipids may have the structures disclosed in Hope's Tables 3-7, such as structure (II), DLin-K-C2-DMA, DLin-K2-DMA, DLin-K6-DMA, etc. The resulting pharmaceutical formulations may be lyophilized according to Hope. In another non-limiting example, the amino lipids may be any amino lipids described in US 20110117125, granted to Hope et al., the contents of which are incorporated herein by reference in their entirety, such as lipids with structure (I), DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA, etc. In another non-limiting example, the amino lipid may have structures (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) or 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 may be any charged lipid described in EP2509636 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.

[0337] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated using an associative complex. In a non-limiting example, the associative complex comprises one or more compounds having a structure defined by formula (I), PEG-lipids having a structure defined by formula (XV), steroids, and nucleic acids as disclosed in U.S. Patent No. 8,034,376 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. The nucleic acid vaccine composition may be formulated using any of the associative complexes described in U.S. Patent No. 8,034,376, the contents of which are incorporated herein by reference in their entirety.

[0338] In some embodiments, the nucleic acid vaccine composition of the present invention can be formulated with a reverse head-base lipid. As a non-limiting example, the nucleic acid vaccine composition can be formulated with a zwitterionic lipid comprising a head-base, wherein a positive charge is located near the acetyl chain region and a negative charge is located at the distal end of the head-base, such as lipids having structure (A) or structure (I) as described in PCT Patent Application Publication No. WO2011056682 issued to Leung et al., the contents of which are incorporated herein by reference in their entirety.

[0339] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in a lipid bilayer carrier. As a non-limiting example, the nucleic acid vaccine composition may be combined with a lipid-cleaner mixture to provide a nucleic acid-lipid-cleaner mixture comprising a lipid mixture of about 5 mol% to about 20 mol% of an anti-aggregating agent, about 0.5 mol% to about 50 mol% of a cationic lipid and a fusion-promoting lipid, and a cleaner; and then the nucleic acid-lipid-cleaner mixture is percolated with a buffered salt solution to remove the cleaner and encapsulate the nucleic acid in the lipid bilayer carrier, and a lipid bilayer-nucleic acid composition is provided, wherein the ionic strength of the buffered salt solution is sufficient to encapsulate about 40% to about 80% of the nucleic acid, as described in PCT Patent Application Publication No. WO1999018933 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.

[0340] In some embodiments, the nucleic acid vaccine composition of the present invention may comprise (a) nucleic acid; (b) 1.0 mol% to 45 mol% of cationic lipid; (c) 0.0 mol% to 90 mol% of another lipid; (d) 1.0 mol% to 10 mol% of a bilayer stabilizing component; (e) 0.0 mol% to 60 mol% of cholesterol; and (f) 0.0 mol% to 10 mol% of cationic polymer lipid, as described in EP1328254 issued to Cullis et al., the contents of which are incorporated herein by reference in their entirety.

[0341] In some embodiments, nucleic acid vaccines may be delivered using smaller LNPs. Such particles can contain diameters from less than 0.1 µm 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. <475 µm, <500 µm, <525 µm, <550 µm, <575 µm, <600 µm, <625 µm, <650 µm, <675 µm, <700 µm, <725 µm, <750 µm, <775 µm, <800 µm, <825 µm, <850 µm, <875 µm, <900 µm, <925 µm, <950 µm, <975 µm.

[0342] In another embodiment, the nucleic acid vaccine may be delivered using smaller LNPs, the diameter of which may 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 50 nm, about 4 ... 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.

[0343] In some embodiments, the nucleic acid vaccine may be formulated in lipid nanoparticles having the following diameters: about 10 nm to about 100 nm, for example (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. 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.

[0344] In some embodiments, the nucleic acid vaccine can be formulated in lipid nanoparticles with a diameter of 10-1000 nm. The nanoparticles may be of the following sizes: 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.

[0345] In some embodiments, the lipid nanoparticles may have a diameter of about 10 to 500 nm.

[0346] In some embodiments, the diameter of the lipid nanoparticles may 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.

[0347] The nucleic acid vaccine composition of the present invention 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 obtained 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® adjuvant obtained from Vical (San Diego, CA), polyglucosamine, cyclodextrin obtained from Calando Pharmaceuticals (Pasadena, CA), dendritic polymers and poly(lactic-co-glycolic acid) (PLGA) polymers, RONDEL™ (RNAi / oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corporation, Pasadena, CA), and pH-responsive coblock copolymers, such as, but not limited to, PHASERX® (Seattle, WA).

[0348] Non-limiting examples of polyglucosamine-based formulations include a positively charged polyglucosamine core and a negatively charged matrix outer portion (U.S. Publication No. US20120258176; the contents of which are incorporated herein by reference in their entirety). Polyglucosamines include, but are not limited to, N-trimethylpolyglucosamine, mono-N-carboxymethylpolyglucosamine (MCC), N-palmolyzedopolyglucosamine (NPCS), EDTA-polyglucosamine, low molecular weight polyglucosamine, polyglucosamine derivatives, or combinations thereof.

[0349] In some embodiments, the polymers used in this invention have been treated to reduce and / or inhibit unwanted substances, such as, but not limited to, bacteria, adhering to the polymer surface. The polymers may be treated by methods known and / or described in this art and / or described in PCT Patent Application Publication No. WO2012150467, the contents of which are incorporated herein by reference in their entirety.

[0350] Non-limiting examples of PLGA-based formulations include, but are not limited to, PLGA-based injectable storage products (such as ELIGARD®, which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder in an aqueous solution and leuprolide. Once injected, the PLGA and leuprolide peptide precipitate into the subcutaneous space. PLGA-based injectable storage products can be long-acting.

[0351] Many such polymer methods have demonstrated efficacy in delivering oligonucleotides in vivo to the cytoplasm (reviewed in FougerollesHum Gene Ther. 2008, 19:125-132; the contents of which are incorporated herein by reference in full). In the case of small interfering RNA (siRNA), two polymer methods have yielded robust in vivo delivery of nucleic acids, namely dynamic multi-conjugates and cyclodextrin-based nanoparticles. The first of these delivery methods uses dynamic multi-conjugates and has been shown to deliver siRNA effectively 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 full). This particular method is a multi-component polymer system characterized by a membrane-active polymer covalently coupled to a nucleic acid (in this case, siRNA) via disulfide bonds, wherein PEG (for charge masking) and N-acetylglucosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (see again Rozema et al., Proc Natl Acad Sci US A. 2007, 104:12982-12887). Upon binding to hepatocytes and entering the endosome, the polymer complex decomposes in a low pH environment, exposing its positive charge, leading to endosome escape and cytoplasmic release of siRNA from the polymer. Replacing the N-acetylglucosamine group with a mannose group has been shown to alter targeting from hepatocytes expressing the desialyl glycoprotein receptor to sinusoidal endothelial cells and Kupffer cells. Another polymer approach involves the use of transferrin-targeted cyclodextrin-containing polycationic nanoparticles. These nanoparticles have been shown to target and silence the EWS-FLI1 gene product in Ewing's sarcoma tumor cells that express the transferrin receptor (Hu-Lieskovan et al., Cancer Res. 2005, 65: 8984-8982; incorporated herein by reference in its entirety), and the siRNA formulated in these nanoparticles is well tolerated in non-human primates (Heidel et al., Proc Natl Acad Sci USA 2007, 104:5715-21; incorporated herein by reference in its entirety). Both delivery strategies combine a rational approach using targeted delivery and endosome escape mechanisms.

[0352] Polymer formulations may allow for sustained or delayed release of the nucleic acid vaccine composition (e.g., after intramuscular, subcutaneous, intraparenchymal, intrathecal, or intraventricular administration). Modified release characteristics of the nucleic acid vaccine composition may induce, for example, translation of proteins or peptides over an extended period. Biodegradable polymers have previously been used to protect nucleic acids from degradation and have been shown to induce sustained in vivo release of the effective load (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. 2010, October 1; Chu et al., Acc Chem Res. 2012, January 13; 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 FougerollesHum 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; (the contents of each are incorporated herein by reference in their entirety).

[0353] In some embodiments, the nucleic acid vaccine of the present invention can be formulated for controlled release. One form of controlled-release formulation contains a therapeutic compound or a salt thereof dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer, such as a copolymer (lactic acid / glycolic acid), as described in the inventive work of Kent et al., U.S. Patent No. 4,675,189, the contents of which are incorporated herein by reference in their entirety. The compound or its salt may also be formulated as cholesterol or other lipid matrix pellets, or as a silicone elastomer matrix implant. As a non-limiting example, the nucleic acid vaccine of the present invention can be dispersed or encapsulated in the polymer disclosed in U.S. Patent No. 4,675,189 for controlled release. Another form of controlled-release formulation comprises a solution of a biodegradable polymer, such as a copolymer (lactic acid / glycolic acid) or a block copolymer of lactic acid and PEG, which is administered subcutaneously or intramuscularly to obtain a reservoir formulation for controlled release.

[0354] In some embodiments, the pharmaceutical composition may be a sustained-release formulation. In other embodiments, the sustained-release formulation may 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).

[0355] As a non-limiting example, a nucleic acid vaccine composition can be formulated in PLGA microspheres by preparing PLGA microspheres at an adjustable release rate (e.g., days and weeks) and encapsulating the nucleic acid vaccine composition within the PLGA microspheres while maintaining the integrity of the nucleic acid vaccine composition during the encapsulation process. EVAc is a non-biodegradable biocompatible polymer widely used in preclinical sustained-release implant applications. Poloxamer F-407 NF is a hydrophilic, nonionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene, exhibiting low viscosity at temperatures below 5°C and forming a solid gel at temperatures above 15°C. PEG-based surgical sealants contain two synthetic PEG components mixed in a delivery device that can be prepared in one minute, sealed in three minutes, and reabsorbed within 30 days. GELSITE® and natural polymers can gel in situ at the injection site. They have been shown to provide stabilizing effects through ionic interactions with protein and peptide therapeutic candidates.

[0356] Polymer formulations can also be selectively targeted via different ligands, such ligands as exemplified by, but not limited to, folic acid, transferrin, and N-acetylglucosamine (GalNAc) (Benoit et al., 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; the contents of these references are incorporated herein by reference in their entirety).

[0357] The nucleic acid vaccine composition of the present invention can be formulated with or formulated in a polymeric compound. 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 onto PLL, cationic lipid polymers, biodegradable cationic lipid polymers, polyethyleneimine (PEI), cross-linked branched poly(alkylimine), polyamine derivatives, modified poloxamer, biodegradable polymers, elastic biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester copolymers, biodegradable polyester block copolymers, biodegradable polyester block random copolymers, multiblock copolymers, linear biodegradable copolymers, and poly[α-(4-aminobutyl)-L-glycolic acid]. (PAGA), biodegradable crosslinked cationic multiblock copolymers, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl transbutadiene, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphoradenene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline), acrylic polymers, amine-containing polymers, polydextrose polymers, polydextrose polymer derivatives, or combinations thereof.

[0358] As a non-limiting example, the nucleic acid vaccine composition of the present invention can be formulated with a polymeric compound grafted with PLL and PEG as described in U.S. Patent No. 6,177,274, which is incorporated herein by reference in its entirety. The formulation can be used for in vitro transfection of cells or in vivo delivery of the nucleic acid vaccine composition. In another example, the nucleic acid vaccine composition can be suspended in a solution or medium having a cationic polymer, a dried pharmaceutical composition, or a solution capable of drying as described in U.S. Publications Nos. US20090042829 and US20090042825, the contents of which are each incorporated herein by reference in their entirety.

[0359] As another non-limiting example, the nucleic acid vaccine composition of the present invention can be formulated with PLGA-PEG block copolymers (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, both of which are incorporated herein by reference in their entirety) or PLGA-PEG-PLGA block copolymers (see U.S. Patent No. 6,004,573, both of which are incorporated herein by reference in their entirety). As a non-limiting example, the nucleic acid vaccine composition of the present invention can be formulated with diblock copolymers of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968, both of which are incorporated herein by reference in their entirety).

[0360] In some embodiments, the nucleic acid vaccine composition may be formulated with branched-chain PEG molecules as described in or prepared by the methods described in PCT Patent Application Publication No. WO20180126084, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the branched-chain PEG that can be used in the formulations described herein may have Formulas I, II, III, IV, V, and VI of PCT Patent Application Publication No. WO20180126084, the contents of which are incorporated herein by reference in their entirety.

[0361] Polyamine derivatives can be used to deliver nucleic acids or to treat and / or prevent diseases or to be included in implantable or injectable devices (U.S. 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 invention can be formulated using the polyamine derivatives described in U.S. Publication No. US20100260817; the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, the nucleic acid vaccine composition of the present invention can be delivered using a polyamide polymer, such as, but not limited to, a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a diyne unit comprising an oligoamine (U.S. Patent No. 8,236,280; the contents of which are incorporated herein by reference in their entirety).

[0362] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated with at least one polymer and / or its derivatives as described in PCT Patent Application Publications Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S. Patent Application Publication No. US20120283427, the contents of which are each incorporated herein by reference in their entirety. The nucleic acid vaccine composition of the present invention may be formulated with polymers of formula Z as described in WO2011115862; the contents of which are incorporated herein by reference in their entirety. The nucleic acid vaccine composition may be formulated with polymers of formula Z, Z' or Z'' as described in PCT Patent Application Publications Nos. WO2012082574 or WO2012068187 and U.S. Patent Application Publication No. US2012028342; the contents of which are each incorporated herein by reference in their entirety. The polymer formulated using the nucleic acid vaccine composition of the present invention can be synthesized by the method described in PCT Patent Application Publication No. WO2012082574 or No. WO2012068187, the contents of which are incorporated herein by reference in their entirety.

[0363] The nucleic acid vaccine composition of the present invention can 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 copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof.

[0364] The formulation of the nucleic acid vaccine composition of the present invention may include at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(aminoamine) dendritic polymer or a combination thereof.

[0365] For example, the nucleic acid vaccine composition of the present invention can be formulated in pharmaceutical compounds comprising: poly(ethyleneimine), biodegradable cationic lipid polymers, biodegradable block copolymers, biodegradable polymers or biodegradable random copolymers, biodegradable polyester block copolymers, biodegradable polyester polymers, biodegradable polyester random copolymers, linear biodegradable copolymers, PAGA, biodegradable crosslinked cationic multiblock copolymers or combinations thereof. Biodegradable cationic lipid polymers can be prepared by methods known in this art and / or as described in U.S. Patent No. 6,696,038 and U.S. Publications Nos. US20030073619 and US20040142474, the contents of which are each incorporated herein by reference in their entirety. Poly(ethyleneimine) can be prepared by methods known in this art and / or as described in U.S. 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 be 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 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 contents of which are incorporated herein by reference in their entirety. PAGA polymers can be copolymerized with polymers to form copolymers or block copolymers, such polymers being, but not limited to, poly-L-lysine, polyarginine, polyguanine, histone, avidin, protamine, polylactide, and poly(lactide-co-glycolic acid). Biodegradable crosslinked cationic multiblock copolymers can be prepared by methods known in this art and / or as described in U.S. Patent No. 8,057,821 or U.S. Publication No. US2012009145, the contents of which are each incorporated herein by reference in their entirety. For example, multiblock 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 this art, described herein, or as described in U.S. Publication No. US20100004315 or U.S. Patent Nos. 6,267,987 and 6,217,912, the contents of which are each incorporated herein by reference in their entirety.

[0366] The nucleic acid vaccine composition of the present invention can be formulated with at least one biodegradable polyester that may contain a polycationic side chain. Biodegradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the biodegradable polyester may include PEG-bonded components to form a polyethylene glycol polymer.

[0367] The nucleic acid vaccine composition of the present invention can be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and those described in U.S. Publication No. US20120269761, the contents of which are incorporated herein by reference in their entirety.

[0368] In some embodiments, the polymers described herein may bind to lipid-terminated PEG. As a non-limiting example, PLGA may bind to lipid-terminated PEG to form PLGA-DSPE-PEG. As another non-limiting example, the PEG conjugates used in this invention 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 bound using ligand conjugates, such as, but not limited to, those described in U.S. Patent No. 8,273,363, the contents of which are incorporated herein by reference in their entirety.

[0369] In some embodiments, the nucleic acid vaccine compositions described herein may 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 which are each incorporated herein by reference in their entirety. In some embodiments, the nucleic acid vaccine compositions of the present invention may be combined with conjugates of Formulas 1-122 as described in U.S. Patent Nos. 7,964,578 and 7,833,992, the contents of which are each incorporated herein by reference in their entirety. The nucleic acid vaccine compositions described herein may be combined with metals, such as, but not limited to, gold. (See, for example, 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 composition described herein may be bound to and / or encapsulated in gold nanoparticles (PCT Application Publication No. WO201216269 and U.S. Publication No. US20120302940; the contents of each of these publications are incorporated herein by reference in their entirety).

[0370] As described in U.S. Publication No. US20100004313, the gene delivery composition may include a nucleotide sequence and poloxamer. As a non-limiting example, the nucleic acid vaccine composition of the present invention may be used in the gene delivery composition containing poloxamer described in U.S. Publication No. US20100004313, the contents of which are incorporated herein by reference in their entirety.

[0371] In some embodiments, polymer formulations comprising the nucleic acid vaccine of the present invention may be stabilized by contacting a polymer formulation, which may include a cationic carrier, with a cationic lipid polymer that can be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with the cationic lipid polymer using the method described in U.S. Publication No. US20090042829, the contents of which are incorporated herein by reference in their entirety.

[0372] The cationic carrier may include, but is not limited to, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationic gelatin, dendritic polymer, polyglucosamine, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleo-3-(2-hydroxyethyl)imide Azoline chloride (DOTIM), 2,3-dioleoxy-N-[2-(spermineamino)ethyl]-N,N-dimethyl-1-propylaminotrifluoroacetate (DOSPA), 3B-[N-(N',N'-dimethylaminoethane)-aminomethoxy]cholesterol hydrochloride (DC-cholesterol HCl), heptadecanylaminoglycanylspermine (DOGS), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleoxy-N,N-dimethylammonium chloride (DODAC), and combinations thereof.

[0373] In some embodiments, the nucleic acid vaccine composition of the present invention may be formulated in a polymeric complex of one or more polymers (U.S. Publications Nos. US20120237565 and US20120270927; the contents of each of these disclosures are incorporated herein by reference in their entirety). In some embodiments, the polymeric complex comprises two or more cationic polymers. The cationic polymers may comprise poly(ethyleneimine) (PEI), such as linear PEI.

[0374] The nucleic acid vaccine composition of the present invention may also be formulated into nanoparticles using polymers, lipids and / or other biodegradable agents, such as, but not limited to, calcium phosphate. Components may be combined in core-shell, mixed and / or layer-by-layer structures to allow fine-tuning of nanoparticles, thereby enhancing the delivery of the nucleic acid vaccine composition (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 Pharm. 2011;8(3):774-87; the contents of each of these references are incorporated herein by reference in their entirety). As a non-limiting example, nanoparticles may comprise 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 (PCT Application Publication No. WO20120225129; the contents of which are incorporated herein by reference in their entirety).

[0375] Combinations of biodegradable calcium phosphate nanoparticles with lipids and / or polymers can be used for in vivo delivery of nucleic acid vaccine compositions. In some embodiments, lipid-coated calcium phosphate nanoparticles containing a targeting ligand such as p-methoxybenzoxamide can also be used to deliver the nucleic acid vaccine compositions of the present invention. For example, lipid-coated calcium phosphate nanoparticles were used to efficiently deliver siRNA in a mouse model of metastatic lung disease (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; the contents of these references are each incorporated herein by reference in their entirety). This delivery system combines targeting nanoparticles with calcium phosphate, a component that enhances endosomal escape, to improve siRNA delivery.

[0376] In some embodiments, calcium phosphate having a PEG-polyanionic block copolymer can be used to deliver the nucleic acid vaccine composition of the present invention (Kazikawa et al., J Contr Rel. 2004, 97:345-356; Kazikawa et al., J Contr Rel. 2006, 111:368-370; the contents of these documents are each incorporated herein by reference in their entirety).

[0377] In some embodiments, PEG-charge conversion polymers (Pitella et al., Biomaterials. 2011, 32:3106-3114; the contents of which are incorporated herein by reference in their entirety) can be used to form nanoparticles for delivery of the nucleic acid vaccine compositions of the present invention. PEG-charge conversion polymers can be modified to enhance endosome escape by cleaving into polycations at acidic pH.

[0378] In some embodiments, core-shell nanoparticles can be used to form nanoparticles for delivering the nucleic acid vaccine composition of the present invention. The use of core-shell nanoparticles also focuses on high-throughput methods for synthesizing cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci US A. 2011, 108:12996-13001; the contents of which are incorporated herein by reference in their entirety). The composite, delivery, and internalization of polymerized nanoparticles can be precisely controlled by altering the chemical composition of both the core and shell components of the nanoparticles. For example, core-shell nanoparticles can efficiently deliver nucleic acid vaccine compositions to mouse hepatocytes after cholesterol is covalently linked to the nanoparticles.

[0379] In some embodiments, the nanoparticles described herein may be nanoparticles comprising at least one ligand, and the ligand may be a peptide, a nucleic acid aptamer (which is a small molecular weight (8-13 kDa) single-stranded RNA or DNA with low nemer binding affinity to its target), a peptide aptamer, an antibody, or a small molecule ligand, such as, but not limited to, folic acid, p-methoxybenzoxamide, 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).

[0380] In some embodiments, a hollow lipid core comprising an intermediate PLGA layer and an outer neutral lipid layer containing PEG can be used to deliver the nucleic acid vaccine composition of the present invention. As a non-limiting example, lipid-polymer-lipid hybrid nanoparticles can be used to deliver the nucleic acid vaccine composition described herein (Shi et al., Angew Chem Int Ed. 2011, 50:7027-7031; the contents of which are incorporated herein by reference in their entirety).

[0381] The core-shell nanoparticles used in the nucleic acid vaccine composition of the present invention can be formed by the method described in U.S. Patent No. 8,313,777, the contents of which are incorporated herein by reference in their entirety.

[0382] In some embodiments, the core-shell nanoparticles may comprise a core and a polymer shell of the nucleic acid vaccine composition described herein. The polymer shell may be any of the polymers described herein and known in the art. In another embodiment, the polymer shell may be used to protect the nucleic acid vaccine composition in the core. (See, for example, U.S. Publication No. 20120321719; the contents of which are incorporated herein by reference in their entirety).

[0383] In some embodiments, the polymer used in the formulations described herein may be a modified polymer (such as, but not limited to, modified polyacetal) as described in PCT Application Publication No. WO2011120053, the contents of which are incorporated herein by reference in their entirety.

[0384] In some embodiments, a nucleic acid vaccine composition can be delivered to the cells or cytoplasm of target cells by contacting a cell with a conjugate of a membrane destabilizing polymer and a nucleic acid vaccine composition, a targeting ligand, and, where appropriate, a linker. Non-limiting examples of membrane destabilizing polymers are taught in International PCT Application Publication No. WO2020093061, the contents of which are incorporated herein by reference in their entirety, including, but not limited to, membrane destabilizing polymers of formula XX therein. Excipients

[0385] In some embodiments, the pharmaceutical formulation may additionally contain pharmaceutically acceptable excipients, as used herein, including but not limited to any and all solvents, dispersion media, diluents or other liquid media, dispersants or suspending agents, surfactants, isotonants, thickeners or emulsifiers, preservatives, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, weight molar osmotic concentration adjusters, pH adjusters and the like. Various excipients used to formulate pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of known excipient media is covered within the scope of this invention unless any known excipient media is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a harmful manner with any one or more other components of the pharmaceutical composition, in which case the use of such other components is covered within the scope of this invention.

[0386] In some embodiments, the pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient may be approved by the United States Food and Drug Administration (FDA). In some embodiments, the excipient may be pharmaceutical grade. In some embodiments, the excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0387] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Such excipients may be included in the pharmaceutical composition as appropriate. The composition may also include excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents and / or aroma agents.

[0388] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, etc., and / or combinations thereof.

[0389] Exemplary granulating agents and / or dispersants include, but are not limited to, potato starch, corn starch, cassava starch, sodium glycolate starch, clay, alginic acid, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, crospovidone (cross-linked polyvinylpyrrolidone), sodium carboxymethyl starch (sodium glycolate starch), carboxymethyl cellulose, crospovidone carboxymethyl cellulose (crospovidone carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0390] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, saffron, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and VEEGUM®). Magnesium aluminum silicate), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetyl glycerol monostearate, glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomer (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers and carboxylated vinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol monolaurate (TWEEN®20), polyoxyethylene sorbitol (TWEEN®60), polyoxyethylene sorbitol monooleate (TWEEN®80), sorbitol monopalmitate) SPAN® 40, Sorbitol Monostearate (SPAN® 60), Sorbitol Tristearate (SPAN® 65), Glyceryl Monooleate, Sorbitol Monooleate (SPAN® 80), Polyoxyethylene Esters (e.g., 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 Oleate, Sodium Oleate, Potassium Oleate, Ethyl Oleate, Oleic Acid, Ethyl Laurate, Sodium Lauryl Sulfate, PLUORINC® F 68. POLOXAMER® 188, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and / or combinations thereof.

[0391] Exemplary adhesives include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural and synthetic gums (e.g., gum arabic, sodium alginate, deer antler extract, panwar gum, ghatti gum, isapol husk mucus, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethyl methacrylate; waxes; water; alcohols; and combinations thereof.

[0392] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and / or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. To prevent oxidation, antioxidants may be added to the formulation. Exemplary antioxidants include, but are not limited to, α-tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, ethylenediaminetetraacetic acid, fumaric acid, malic acid, phosphoric acid, sodium ethylenediaminetetraacetate, tartaric acid, and / or trisodium ethylenediaminetetraacetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzyl chloride, benzyl alcohol, bromonitrile glycol, cetrimonium bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, xylene, cresol, ethanol, glycerin, hexetidine, imidazolidinylurea, 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, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcoholic preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and / or phenylethyl 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 methanesulfonate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®!!, NEOLONE™, KATHON™ and / or EUXYL®.

[0393] In some embodiments, the pH of the pharmaceutical solution is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers for controlling 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 ions (including, but not limited to, potassium). Divalent cations may also be used as buffer ions; however, these are not preferred due to complex formation and / or mRNA degradation.

[0394] Exemplary buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium acetopropionate, 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, sodium dihydrogen phosphate, sodium phosphate mixture, thiamethoxam, magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free water, isotropic physiological saline, Ringer's solution, ethanol, and / or combinations thereof.

[0395] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl betaine, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0396] Example oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, juniper, chamomile, canola, caraway, palm wax, castor bean, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, vanillin, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, and summer. Hawaiian walnut, bright lavender, lavender, lemon, litsea cubeba, macadamia nut kernel, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, Atlantic sea bream, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, camellia, peppermint, sea buckthorn, sesame, shea butter, polysiloxane, soybean, sunflower, tea tree, thistle, ailanthus, vetiver, walnut, and wheat germ oil. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, caprylic triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecyl alcohol, oleyl alcohol, polysiloxane oil, and / or combinations thereof.

[0397] Excipients, such as cocoa butter and suppository wax, colorants, coating agents, sweeteners, flavoring agents and / or aroma agents, may be present in the composition, depending on the blender's judgment.

[0398] Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), the addition of chelating agents (such as DTPA or DTPA-bisacylamine) or calcium chelates (e.g., DTPA calcium, CaNaDTPA-bisacylamine), or, where appropriate, calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). In addition, antioxidants and suspending agents may be used.

[0399] In some embodiments of the present invention, the nucleic acid vaccine composition described herein may comprise at least one nucleic acid vaccine formulated in lipid nanoparticles (LNPs) and at least one excipient. As a non-limiting example, the excipient may be a sugar, such as sucrose. Adjuvant

[0400] Adjuvants may also be administered co-administered or in combination with one or more of the nucleic acid vaccines described herein, such as mRNA vaccines. Adjuvants may be used to enhance the immunogenicity of nucleic acid vaccines, alter immune responses, reduce the amount of nucleic acid vaccine required for immunization, reduce the frequency of additional or "boost" immunizations required, or produce improved immune responses in individuals with weakened or impaired immune systems or in the elderly. Adjuvants may be components of formulations containing nucleic acid vaccines, or may be co-administered with nucleic acid vaccine compositions. Co-administration of adjuvants 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).

[0401] In some embodiments, the adjuvant is natural or synthetic. The adjuvant may also be organic or inorganic.

[0402] In some embodiments, the adjuvant for the nucleic acid vaccine is derived 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 mediators, particulate adjuvants (e.g., monolayer liposome mediators, such as virions or structured complexes of saponins and lipids, such as poly(lactic-co-glycolic acid)), microbial derivatives, endogenous human immunomodulators, and tonicotinic compounds. A list of adjuvants that can be used for the nucleic acid vaccines described herein is available in the online vaccine adjuvant database Vaxjo (see, for example, 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).

[0403] The adjuvant used in the nucleic acid vaccine can be selected by a person generally familiar with this technology. The adjuvant may be interferon, TNF-α, TNF-β, chemokines (e.g., CCL21, eosin, HMGB1, SA100-8α, GCSF, GMCSF, granzyme, 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 microparticle vaccine adjuvant, seaweed dextran, Algammulin, aluminum gel, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, potassium aluminum 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 adjuvants, CRL1OO5, CTA1-DD gene fusion protein, DDA adjuvant, DHEA vaccine adjuvant, DL-PGL (poly(DL-lactide-co-glycolic acid)) vaccine adjuvant, DOC / Alum complex, E. coli heat-sensitive toxin, Etx B subunit adjuvant, flagellin, Freund's complete adjuvant, Freund's incomplete adjuvant, γ-inulin, Gerbu adjuvant, GM-CSF, GMDP, imiquimod, immunoliposomes containing co-stimulatory molecule antibodies, ISCOM(s)™, ISCOMA-TRIX®, inactivated Corynebacterium brevis vaccine adjuvant, lipopolysaccharide, liposomes, loxoribin, 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 liposomes, cell wall peptides, cell wall dipeptide adjuvant, murapalmitine, D-murapalmitine, NAGO, nano-emulsion vaccine adjuvant, nonionic surfactant vesicles, cholera toxin non-toxic mutant E112KmCT-E112K, PMMA, Poly(LC), Polygen vaccine adjuvant, protein lipid rolls, 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 protein liposomes, Sendai-containing lipid ultimate, Spector, SPT (antigen formulation), squalene-based adjuvant, stearyltyrosine, Theramide®, hydroxybutylamine acetomolecular dipeptide (TMDP), Titer-Max Gold adjuvant, Ty Particles vaccine adjuvant, and VSA-3 adjuvant.

[0404] In some embodiments, the nucleic acid vaccine described herein can be used as a vaccine and may further include an adjuvant that enables the vaccine to elicit a stronger immune response. As a non-limiting example, the adjuvant may be a submicron oil-in-water emulsion that can elicit a stronger immune response in human pediatric populations (see, for example, adjuvanted vaccines described in U.S. Patent Publication No. US20120027813 and U.S. Patent No. 8,506,966, the contents of which are each incorporated herein by reference in their entirety). Administration and Dosing

[0405] This invention covers the delivery of nucleic acid vaccine compositions by any suitable route taking into account possible developments in drug delivery science, including, for example, COVID-19 nucleic acid vaccines for any therapeutic, preventative, pharmaceutical, diagnostic, or imaging purpose. Delivery may be unpackaged or formulated.

[0406] The nucleic acid vaccine composition of the present invention can be delivered to cells naked. As used herein, "naked" means delivery of a nucleic acid vaccine composition that does not contain reagents that promote transfection. For example, the nucleic acid vaccine composition delivered to cells may be unmodified. The naked nucleic acid vaccine composition can be delivered to cells using the administration routes known in this art and described herein.

[0407] The nucleic acid vaccine compositions of the present invention can 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 penetrants, pharmaceutically acceptable carriers, delivery agents, biocorrosive or biocompatible polymers, solvents, and sustained-release delivery storage products. The formulated nucleic acid vaccine compositions can be delivered to cells using the administration routes known in this art and described herein.

[0408] The nucleic acid vaccine composition can also be formulated for direct delivery to organs or tissues in any of the following ways of the present technology, including but not limited to: direct immersion or bathing, via catheter, by gel, powder, ointment, cream, lotion and / or drops, by using a substrate coated or impregnated with the composition, such as fabric or biodegradable material, and similar methods. The nucleic acid vaccine composition of the present invention can also be selectively colonized into a retroviral replication vector (RRV) and transduced into cells. Administration

[0409] This document also includes methods for administering the nucleic acid vaccines described herein to individuals in need. The precise amount required will vary from individual to individual, depending on the individual's species, age, health and general condition, disease severity, specific composition, administration pattern, activity pattern, and similar factors. For ease of administration and dose uniformity, the composition is typically prepared in unit dosage forms. However, it should be understood that the total daily dosage of the composition will be determined by the attending physician within reasonable medical judgment. The specific therapeutic, prophylactic, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the disease being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in medical technology.

[0410] This invention covers the following dosage levels: about 0.001 to about 500 mg of nucleic acid vaccine (e.g., COVID-19 nucleic acid vaccine, such as 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. mg / kg, 1.0 to about 40 mg / kg, 1.0 to about 30 mg / kg, 10 to about 50 mg / kg body weight. Other examples cover doses of about 0.001-0.010, 0.010-0.050, 0.050-0.100, 0.1-0.5, 0.5-1.0, 1.0-5.0, 5.0-10, 10-50 mg / kg, 10-100 mg / kg body weight. Dosage can be administered approximately hourly, multiple times daily, daily, every other day, weekly, every other week, monthly, every other month, or as needed.

[0411] In some embodiments, the nucleic acid vaccine composition may be sufficient to deliver approximately 0.0001 mg / kg to approximately 100 mg / kg, approximately 0.001 mg / kg to approximately 0.05 mg / kg, approximately 0.005 mg / kg to approximately 0.05 mg / kg, approximately 0.001 mg / kg to approximately 0.005 mg / kg, approximately 0.05 mg / kg to approximately 0.5 mg / kg, approximately 0.01 mg / kg to approximately 50 mg / kg, approximately 0.1 mg / kg to approximately 40 mg / kg, approximately 0.5 mg / kg to approximately 30 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 25 mg / kg, approximately 1 mg / kg to approximately 50 mg / kg, approximately 10 mg / kg to approximately 100 mg / kg, approximately 10 mg / kg to approximately 50 mg / kg. mg / kg body weight / day, administered once or multiple times daily to achieve the desired therapeutic, diagnostic, preventative, or imaging effect. The required dose may be delivered three times a day, twice a day, once a day, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In some embodiments, the required dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When using multiple administrations, fractionated dosing regimens, such as those described herein, may be used.

[0412] In some embodiments, the nucleic acid vaccine composition described herein is sufficient to deliver to an individual a dose level of about 1 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 35 µg, 40 µg, 50 µg, 60 µg, 70 µg, 80 µg, 90 µg or 100 µg of nucleic acid composition.

[0413] In some embodiments, the nucleic acid vaccine may be administered in a fractionated dosing regimen. As used herein, "fractionated dosing" 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. As used herein, "single unit dose" refers to the dose of any therapeutic agent administered in a single dose / single route / single point of contact, i.e., a single administration event. As used herein, "total daily dose" refers to the amount administered or prescribed over a 24-hour period. It may be administered in a single unit dose. In some embodiments, the nucleic acid vaccine described herein is administered to an individual in fractionated doses. The nucleic acid vaccine may be prepared solely in a buffer solution or in a formulation described herein.

[0414] In some embodiments, the nucleic acid vaccine composition described herein may be administered to an individual in two separate phases (initial administration phase and maintenance administration phase). The administration regimen may include an initial higher starting dose of the nucleic acid vaccine given to the individual for the first time at the start of a prevention, mitigation, and / or treatment process, such as a first dose for the prevention of COVID-19, and a lower maintenance dose following the first starting dose. In some embodiments, the starting dose and maintenance dose have the same amount of the nucleic acid vaccine of the present invention. In some embodiments, more than one maintenance dose is administered to the individual. Multiple maintenance doses may 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 cases where vaccination is used to prevent a disease, such as a nucleic acid (e.g., mRNA) vaccine for COVID-19, the maintenance dose of the nucleic acid vaccine may also be referred to as a booster dose. As used herein, a "boost dose" (or "boost injection") is an additional or supplementary dose of vaccine following the initial initiation dose. The amount of nucleic acid vaccine in the booster dose may be the same as the initial starting dose. Alternatively, the amount of nucleic acid vaccine in the booster dose may be less than the original amount of nucleic acid vaccine in the initial dose. In some embodiments, an individual may receive one, two, three, four or more booster doses.

[0415] Such drug delivery can be used for the chronic or acute treatment or prevention of clinically relevant symptoms. The amount of drug that can be combined with a carrier to produce a single dosage form will vary depending on the host being treated and the specific delivery method. Typical formulations will contain about 5% to about 95% of the active compound (w / w). Preferably, such formulations contain about 20% to about 80%, 30% to about 70%, 40% to about 60%, or about 50% of the active compound. In other embodiments, the formulations used in this invention will be about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or greater than 99% of the active ingredient.

[0416] As an individual's symptoms improve, a maintenance dose of the compounds, compositions, or combinations of the present invention may be administered if necessary. Subsequently, the dose or frequency, or both, may be reduced as symptoms improve until the level of symptom relief is achieved to maintain the desired level of symptom improvement, at which point treatment should be discontinued. However, patients may request long-term intermittent treatment in the event of any recurrence of disease symptoms.

[0417] Those familiar with this technique should understand that lower or higher doses than those mentioned above may be required. The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health condition, sex, diet, administration time, excretion rate, drug combination, severity and course of infection, patient management of the infection, and the judgment of the treating physician. Delivery

[0418] In some embodiments, the delivery of the nucleic acid vaccine may be naked or prepared.

[0419] In some embodiments, the nucleic acid vaccines described herein can be delivered to cells naked. As used herein, "naked" means delivery of a nucleic acid vaccine that does not contain reagents that promote transfection. For example, the nucleic acid vaccine delivered to cells may be unmodified. Naked nucleic acid vaccines can be delivered to cells using administration routes known in the art and described herein.

[0420] In some embodiments, the nucleic acid vaccines described herein may be formulated using the methods described herein. The formulation may further include, but is not limited to, cell penetrants, pharmaceutically acceptable carriers, delivery agents, biocorrosive or biocompatible polymers, solvents, and sustained-release delivery stores. The formulated nucleic acid vaccine may be delivered to cells using the administration routes known in this art and described herein.

[0421] The composition can also be formulated for direct delivery to an organ or tissue in any of the following ways of the art, including but not limited to: direct immersion or bathing, via a catheter, by means of gel, powder, ointment, cream, lotion and / or drops, by means of a substrate coated or impregnated with the composition, such as fabric or biodegradable material, and similar methods.

[0422] In some embodiments, the nucleic acid vaccine composition of the present invention can be administered by any route that produces a preventive or therapeutic effect. These include, but are not limited to, enteric (to the intestine), gastrointestinal tract, epidural (to the dura mater), oral (via the mouth), transdermal, epidural, intracerebral (to the brain), intravenous (to the ventricles), epidermal (applied to the skin), intradermal (to the skin itself), subcutaneous (under the skin), nasal administration (via the nose), intravenous (to the vein), intravenous bolus injection, intravenous drip, intra-arterial (to the artery), intramuscular (to the muscle), intracardiac (to the heart), intraosseous infusion (to the bone marrow), intrathecal (to the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (via the eye), and intracavitary injection (to the bone marrow). Pathological administration, intracavitary (to the root of the penis), intravaginal administration, intrauterine administration, extraamniotic administration, percutaneous (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucosa), vaginal administration, inhalation (nasal inhalation), sublingual administration, sublipal administration, enema, eye drops (to the conjunctiva), ear drops, ear canal (in or through the ear), buccal administration (to the cheek), conjunctiva, skin, dental administration (to one or more teeth), electroosmosis, intracervical administration, intrasinus administration, intratracheal administration, extracorporeal administration, hemodialysis administration, infiltration, interstitium, intraperitoneal administration, intraamniotic, intraarticular administration, intrabiliary administration, intrabronchial administration, intracystic administration, intracartilaginous administration (in the equina), intracerebral administration (in the cisternae), intracerebral administration (in the large... (Within the medullary cistern), cornea (within the cornea), dental cornea, coronary artery (within the coronary artery), corpus cavernosum (within the expandable space of the corpus cavernosum), intervertebral disc (within the intervertebral disc), canal (within the glandular duct), duodenum (within the duodenum), dura mater (within or below the dura mater), epidermis (to the epidermis), esophagus (to the esophagus), stomach (within the stomach), gingiva (within the gingiva), ileum (within the distal part of the small intestine), lesion (within the local lesion or directly introduced into the local lesion), lumen (within the lumen), lymphatic vessel (within the lymph), medullary cavity (within the bone marrow cavity), meninges (within the meninges). Intraocular (within the eye), intraovarian (within the ovary), intracardiac (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate), intrapulmonary (within the lung or its bronchi), intrasinus (within the sinuses or periorbital sinuses), intravertebral (within the spine), intrasynovial (within the synovial fluid cavity of a joint), intratendinous (within the tendon), intratesticular (within the testis), intrasheath (within the cerebrospinal fluid at any level of the brain-spinal axis), intrapleural (within the pleural cavity), intracanal (within the tubules of an organ), intratumoral (within the tumor), tympanic cavity (within the middle layer of the ear), intravascular (within one or more blood vessels), intraventricular (within the ventricle), iontophoresis (using electric current),The composition can be administered via various methods, including: 1) migration of soluble salt ions into body tissues; 2) irrigation (rinsing or flushing open wounds or body cavities); 3) larynx (directly in the larynx); 4) nasogastric (via the nose and into the stomach); 5) occlusal dressing techniques; 6) transocular (to the external eye); 7) oropharynx (directly to the oropharynx); 8) non-intestinal; 9) percutaneous; 10) percutaneous; 11) perarticular; 12) percutaneous; 13) percutaneous; 14) percutaneous; 15) percutaneous; 16) percutaneous; 17) percutaneous; 18) percutaneous; 19) percutaneous; 10) percutaneous; 11) percutaneous; 11) percutaneous; 12) percutaneous; 13) percutaneous; 14) percutaneous; 15) percutaneous; 16) percutaneous; 17) percutaneous; 18) percutaneous; 19) percutaneous; 10 ...1) percutaneous; 11) percutaneous; 11) percutaneous; 11) percutaneous; 11)

[0423] The nucleic acid vaccines described herein can be delivered to individuals over extended periods, such as from one week to one year, by a controlled-release system containing sufficient active ingredient for a sustained release period using a single dose. For this purpose, various controlled-release systems can be utilized, such as monolithic or reservoir-type microcapsules, reservoir-type implants, polymeric hydrogels, osmotic pumps, vesicles, micelles, liposomes, percutaneous patches, iontophoresis devices, and alternative injectable formulations. Targeting the site where the active ingredient needs to be delivered is an additional feature of some controlled-release devices, which has proven beneficial for treating certain conditions.

[0424] In some embodiments, the nucleic acid vaccines described herein can be administered intranasally, similar to the administration of live vaccines. In another embodiment, polynucleotides can be administered intramuscularly or intradermally, similar to the administration of inactivated vaccines known in this art.

[0425] In some embodiments of percutaneous delivery, electrodes (e.g., iontophoresis), electroporation or application of short high-voltage pulses to the skin, radiofrequency, ultrasound (e.g., fluoroscopy), microprojection (e.g., microneedles), jet injectors, thermal ablation, magnetophoresis, laser, velocity, or photomechanical waves are used to enhance delivery across the skin barrier. The drug may be contained in a single-layer drug-eluting adhesive, a multilayer drug-eluting adhesive, a reservoir, a matrix, or a vapor patch, or patchless techniques may be used. Encapsulation, skin lipid fluidizers, or hollow or solid microstructured percutaneous systems (MTS, such as MTS manufactured by 3M), jet injectors may also be used to enhance delivery across the skin barrier. Formula additives used to aid in the penetration of therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell-penetrating peptides, penetration enhancers, encapsulation techniques, enzymes, enzyme inhibitors, gels, nanoparticles, and peptide or protein-associated proteins.

[0426] Other slow-release, reservoir-type implants, or injectable formulations will be apparent to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., 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 herein by reference in its entirety.

[0427] A mixture of the nucleic acid vaccine described herein with a polymeric formulation is suitable for obtaining a formulation with an extremely long duration of action, the polymeric formulation comprising a biodegradable polymer that can form a reservoir formulation after administration.

[0428] When formulated for nasal administration, absorption across the nasal mucosa can be further enhanced by surfactants such as glycocholic acid, cholic acid, taurine, ethcholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycine-deoxycholic acid, cyclodextrin, and the like, in amounts 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 that exhibit greater efficacy and reduced irritation has been reported to be alkyl maltodextrins, such as tetradecyl maltodextrin (Arnold, JJ et al., J Pharm Sci, 2004, 93: 2205-13; Ahsan, F et al., Pharm Res, 2001, 18:1742-46) and their references, all of which are hereby incorporated in full.

[0429] Pharmaceutical compositions may be in the form of sterile injectable formulations, for example, as sterile injectable aqueous or oily suspensions. These suspensions may be formulated using suitable dispersants or wetting agents (such as Tween 80) and suspending agents according to techniques known in this art. Sterile injectable formulations may also be sterile injectable solutions or suspensions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable mediators and solvents, mannitol, water, Ringer's solution, and isotonic sodium chloride solution may be used. Additionally, sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are suitable for the preparation of injectable formulations, as are naturally pharmaceutically acceptable oils (such as olive oil or castor oil, especially in their polyoxyethylated form). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as Ph. Helv or similar alcohols.

[0430] The pharmaceutical compositions of the present invention can be administered orally 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 corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When administered orally in an aqueous suspension, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners and / or flavoring agents and / or coloring agents may be added.

[0431] The pharmaceutical compositions of the present invention can also be administered rectally as suppositories. These compositions can be prepared by mixing the active ingredient of the present invention with a suitable, non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0432] Topical administration of the pharmaceutical compositions of the present invention is particularly suitable when the desired treatment involves areas or organs easily accessible by local application. For local application to the skin, the pharmaceutical compositions are formulated as suitable ointments containing an active ingredient suspended or dissolved in a carrier. Carriers for local administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the pharmaceutical compositions may be formulated as suitable lotions or creams containing an active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, hexadecyl wax, hexadecyl stearyl alcohol, 2-octyl dodecanol, benzyl alcohol, and water. The pharmaceutical compositions of the present invention may also be applied locally to the lower intestine in the form of rectal suppository formulations or suitable enema formulations. Topical percutaneous patches are also included in the present invention.

[0433] The pharmaceutical compositions of the present invention can be administered by nasal aerosol or inhalation. These compositions are prepared according to techniques well known in pharmaceutical formulation and can be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons and / or other solvents or dispersants known in this art.

[0434] When formulated for inhalation delivery, various formulations offer advantages. Adsorbing the therapeutic agent onto an easily dispersible solid, such as diketopiperazine (e.g., Technosphere particles (Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv 2:1097-1106) or a similar structure yields a formulation that allows for rapid initial absorption of the therapeutic compound. Lyophilized powders containing the therapeutic compound and excipients, particularly glassy particles, 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 Forms

[0435] The pharmaceutical compositions described herein can be formulated into the dosage forms described herein, such as topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous) dosage forms. Liquid dosage forms

[0436] Liquid dosage forms for non-enteric administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in this technology, including but not limited to water or other solvents, solubilizers, and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In some embodiments for non-enteric administration, the composition may be mixed with solubilizers such as CREMO-PHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. Injectable Form

[0437] Injectable formulations can be formulated according to known techniques, such as sterile injectable aqueous or oily suspensions, and may include suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable formulations may be sterile injectable solutions, suspensions, and / or emulsions contained in non-toxic, non-enteric-acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents include, but are not limited to, water, Ringer's solution, USP, and isotonic sodium chloride solution. It is conventional to use sterile, non-volatile oils as solvents or suspension media. For this purpose, any mild, non-volatile oil may be used, including synthetic monoglycerides or diglycerides. Injectable formulations may be prepared using fatty acids such as oleic acid.

[0438] Injectable formulations may be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporation of a sterilizing agent, in the form of a sterile solid composition, which may be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0439] To prolong the action of the active ingredient, it may be necessary to slow down the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material 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 crystal form. Alternatively, the absorption of a nucleic acid vaccine administered orally can be delayed by dissolving or suspending it in an oily medium. Injectable accumulation formulations are prepared by forming a microcapsule matrix of the nucleic acid vaccine in a biodegradable polymer (such as polylactide-polyglycolic acid). The release rate of the polynucleotide can be controlled depending on the ratio of the nucleic acid vaccine to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include, but are not limited to, poly(orthoester) and poly(anhydride). Injectable accumulation formulations can be prepared by encapsulating the nucleic acid vaccine in liposomes or microemulsions that are compatible with body tissues.

[0440] The formulations described herein are suitable for pulmonary delivery and can also be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration may be a coarse powder containing an active ingredient and having an average particle size of about 0.2 pm to 500 pm. Such formulations may be administered by nasal inhalation, for example, by rapidly inhaling the powder from a container close to the nose through the nostrils.

[0441] 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 the form of buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges prepared using known methods, and may, for example, contain about 0.1% to 20% (w / w) of the active ingredient, the remainder of which may comprise an orally soluble and / or biodegradable composition, and, where appropriate, one or more of the additional ingredients described herein. Alternatively, formulations suitable for intrabuccal administration may contain a powder and / or aerosolized and / or nebulized solution and / or suspension 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 contain any one or more additional components described herein.

[0442] General considerations in the formulation and / or manufacture of pharmaceutical preparations can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005. Solid dosage forms: coating or shell.

[0443] Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in pharmaceutical compounding techniques. They may contain emulsifiers and may have a composition that releases the active ingredient only or preferentially in a portion of the intestine, and, where appropriate, in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can 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 glycol and its analogues. Characteristics of Pharmaceutical Compositions

[0444] The nucleic acid vaccine pharmaceutical compositions descr...

Claims

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

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

7.

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

7.

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

7.

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

2.

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

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

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

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

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

48.

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

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

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

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

15. A pharmaceutical composition comprising a nucleic acid vaccine as claimed in claim 14 and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition of claim 15, comprising about 0.2 mg / mL of mRNA as claimed in claim 9.

17. The pharmaceutical composition of claim 16 is suitable for intramuscular (IM) injection.

18. The pharmaceutical composition of claim 16, which is packaged in a 3 mL glass vial with 2 mL filler.

19. A method for inducing an immune response in an individual, comprising administering a nucleic acid vaccine as claimed in any one of claims 13 to 14 or a pharmaceutical composition as claimed in any one of claims 15 to 18.

20. The method of claim 19, wherein the immune response comprises a T-cell response.

21. The method of claim 19, wherein the immune response comprises a B cell response.

22. The method of claim 19, further comprising administering a reinforcing agent after the first administration.

23. The method of claim 19, wherein the individual’s immune response is dose-dependent.

24. The method of claim 23, wherein the dose-dependent immune response comprises one or more of the SARS-CoV-2 spike protein-specific IgG, IgG1, IgG2a, IgG2b, IgM and IgA antibodies induced in the individual.

25. The method of claim 23, wherein the dose-dependent immune response comprises inducing one or more of IL-2+ T cells, IL-4+ T cells and IFN-γ+ T cells.

26. A method of treating an individual with COVID-19, comprising administering a nucleic acid vaccine as claimed in any one of claims 13 to 14 or a pharmaceutical composition as claimed in any one of claims 15 to 18.

27. A method of preventing an individual from contracting COVID-19, comprising administering to the individual a nucleic acid vaccine as claimed in any one of claims 13 to 14 or a pharmaceutical composition as claimed in any one of claims 15 to 18.

28. A method for mitigating or improving the physiological effects or symptoms of COVID-19 in an individual, comprising administering to the individual a nucleic acid vaccine as claimed in any of claims 13 to 14 or a pharmaceutical composition as claimed in any of claims 15 to 18.

29. The method of any one of claims 26 to 28, wherein the dose of the mRNA administered to the individual is about 5 µg to about 100 µg.

30. The method of any one of claims 26 to 28, wherein the dose of the mRNA administered to the individual is about 16 µg.

31. The method of any one of claims 26 to 28, wherein the dose of the mRNA administered to the individual is about 40 µg.

32. The method of any one of claims 26 to 28, wherein the dose of the mRNA administered to the individual is about 100 µg.

33. The method of any one of claims 26 to 28, wherein the volume of the pharmaceutical composition administered to the individual is about 0.025 mL to about 0.5 mL.

34. The method of any one of claims 26 to 28, 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.

35. The method of any one of claims 26 to 34, comprising administering a second dose of the nucleic acid vaccine or pharmaceutical composition about one to about five weeks after the first dose.

36. The method of claim 35, wherein the second dose is administered approximately four weeks after the first dose.

37. The method of any one of claims 26 to 28, wherein anti-spike protein IgG antibodies are detected in the individual up to day 28 after receiving the first dose of the nucleic acid vaccine or pharmaceutical composition.

38. The method of claim 37, wherein, up to day 42 following the administration of a second dose of the nucleic acid vaccine or pharmaceutical composition, the amount of the anti-spike protein IgG antibodies present in the individual increases.

39. The method of claim 37, wherein the anti-spike protein IgG antibody in the individual is more than 10 times the value of anti-spike protein IgG antibody in serum samples from SARS-CoV-2 convalescent patients.

40. A method for inducing the production of SARS-CoV-2 neutralizing antibodies in an individual, comprising administering to the individual a nucleic acid vaccine as claimed in any one of claims 13 to 14 or a pharmaceutical composition as claimed in any one of claims 15 to 18.

41. The method of claim 40, wherein the SARS-CoV-2 neutralizing antibodies can be detected in the individual up to day 28 after administration.

42. The method of claim 41, wherein on day 42 following receipt of a second dose of the nucleic acid vaccine or pharmaceutical composition, the production of the SARS-CoV-2 neutralizing antibody in the individual increases.

43. The method of claim 41, wherein the level of SARS-CoV-2 neutralizing antibodies increases tenfold up to day 42.

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