WO2024073848A1 - Compositions and methods for the prevention and/or treatment of covid-19 - Google Patents

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

Info

Publication number
WO2024073848A1
WO2024073848A1 PCT/CA2023/051314 CA2023051314W WO2024073848A1 WO 2024073848 A1 WO2024073848 A1 WO 2024073848A1 CA 2023051314 W CA2023051314 W CA 2023051314W WO 2024073848 A1 WO2024073848 A1 WO 2024073848A1
Authority
WO
WIPO (PCT)
Prior art keywords
mol
cationic lipid
nucleic acid
lipid
subject
Prior art date
Application number
PCT/CA2023/051314
Other languages
French (fr)
Inventor
Aleksandra Pastrak
Natalia Martin Orozco
Lawrence Reiter
Richard Tran
Anne-Claude Gingras
Reuben SAMSON
Original Assignee
Providence Therapeutics Holdings Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Providence Therapeutics Holdings Inc. filed Critical Providence Therapeutics Holdings Inc.
Publication of WO2024073848A1 publication Critical patent/WO2024073848A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure generally relates to compositions, formulations, methods, and/or uses of nucleic acid vaccines, specifically nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment and/or prevention of COVID-19, including mitigation of physiologic effects of infection and/or symptoms.
  • nucleic acid vaccines e.g., RNA, mRNA, DNA vaccines
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus which began infecting mammals in 2019 in China and has spread to a pandemic. SARS-CoV-2 infection causes coronavirus disease 2019 (termed “COVID- 19”), which affects mammals in different ways including individuals who are asymptomatic to individuals who have a wide range of symptoms that range from mild symptoms to severe illness or death.
  • Vaccines are an effective way to provide prophylactic protection against infective diseases.
  • Treatment of COVID-19 has been limited to the management of symptoms and/or the side effects of the disease.
  • COVID-19 vaccines including formulations for delivering the vaccines to a range of different target T-cells.
  • the present disclosure provides nucleic acid vaccines, compositions and formulations comprising nucleic acid vaccines, and methods of using same for preventing infection of coronavirus, and for the prevention, alleviation and treatment of COVID-19.
  • the nucleic acid vaccines may include polynucleotides which encode at least one antigen protein, fragment or variant thereof of SARS-CoV-2.
  • the SARS-CoV-2 antigen protein is a structural protein of SARS-CoV-2.
  • the structural protein may be the spike protein, the membrane protein, the nucleocapsid phosphoprotein or the envelope protein.
  • Non-limiting examples of the amino acid sequences of these structural proteins are shown in Table 1 (SEQ ID Nos: 1-6 and 15-19).
  • nucleic acid vaccines for COVID-19 for use in a method of vaccinating a subject for COVID-19, wherein the nucleic acid vaccine may include at least one polynucleotide encoding at least one structural protein or a fragment thereof of SARS- CoV-2.
  • the immune response may be, but is not limited to, a T-cell response or a B cell response.
  • the immune response may be produced by a single administration of the nucleic acid vaccines described herein.
  • the immune response may be produced by a booster administration of the nucleic acid vaccines described herein.
  • the administering the pharmaceutical composition may produce a dose-responsive immune response in the subject.
  • the dose-responsive immune response may comprise induction of one or more of SARS-CoV-2 spike protein specific IgG, IgGl, IgG2a, IgG2b, IgM and IgA antibodies in the subject.
  • the dose-responsive immune response may comprise induction of one or more of IL-2+ T-cells, IL-4+ T-cells, and IFN- gamma+ T-cells.
  • the administering the pharmaceutical composition does not induce significant adverse reactions in the subject.
  • compositions and formulations of the nucleic acid vaccines for the treatment and prevention of COVID-19.
  • the nucleic acid vaccines described herein may be formulated in one or more lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
  • the LNP comprises (a) 50-65 mol% of a cationic lipid, (b) a non-cationic lipid comprising 4-10 mol% of phospholipids and 30-40 mol% of cholesterol, and (c) 0.5-2 mol% of a conjugated lipid.
  • the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid. [0017] In some embodiments the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5-50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid.
  • the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid.
  • the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA.
  • the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA.
  • nucleic acid vaccines for COVID-19 comprising about 0.2 mg/mL mRNA, wherein the mRNA comprises a coding region with a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7.
  • the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region with a nucleic acid sequence as set forth in SEQ ID NO: 7.
  • the nucleic acid vaccines may be formulated as a 2 mL fill in a 3 mL glass vial.
  • the nucleic acid vaccines may be formulated as a 2.4 mL fill in a 6 ml glass vial.
  • administering the nucleic acid vaccines to a subject comprises administering about 5 pg to about 100 pg of the mRNA to the subject.
  • the methods may comprise administering about 16 pg of the mRNA to the subject.
  • the methods may comprise administering about 40 pg of the mRNA to the subject.
  • the methods may comprise administering about 100 pg of the mRNA to the subject.
  • administering the nucleic acid vaccines to a subject comprises administering about 0.025 mL to about 0.5 mL of the nucleic acid vaccine to the subject.
  • the methods may comprise administering about 0.025 mL of the nucleic acid vaccine to the subject, about 0.05 mL of the nucleic acid vaccine to the subject, about 0.08 mL of the nucleic acid vaccine to the subject, about 0.2 mL of the nucleic acid vaccine to the subject, or about 0.5 mL of the nucleic acid vaccine to the subject.
  • the administering comprises an intramuscular (IM) injection of the nucleic acid vaccine to the subject.
  • IM intramuscular
  • the nucleic acid vaccines may be administered to a subject in a first dose of the nucleic acid vaccine followed by a second dose of the nucleic acid vaccine after between about 1 and about 5 weeks. In some embodiments, the second dose of the nucleic acid vaccine is administered about 4 weeks after the first dose. [0033] In some embodiments, anti-Spike protein IgG antibodies are detected in the subject by day 28 after receiving a first dose of the nucleic acid vaccine.
  • anti-Spike protein IgG antibodies are detected in the subject by day 28 after receiving a first dose of the nucleic acid vaccine and are enhanced after receiving a second dose of the nucleic acid vaccine in the subject by day 42.
  • anti-Spike protein IgG antibodies in the subject are increased to 10-fold higher than the average values of anti-Spike protein IgG antibodies from serum samples from SARS-CoV-2 convalescent patients.
  • the SARS-CoV-2 neutralizing antibodies are detected in the subject by day 28 after a first dose of the nucleic acid vaccine.
  • the SARS-CoV-2 neutralizing antibodies are enhanced after a second dose of the nucleic acid vaccine in the subject by day 42.
  • the subject produces neutralizing antibodies to SARS- CoV-2 for at least about 1 days post initial administration.
  • the subject produces neutralizing antibodies to SARS- CoV-2 for about 21 days post initial administration.
  • the subject produces neutralizing antibodies to SARS- CoV-2 for about 28 days post initial administration.
  • the subject produces neutralizing antibodies to SARS- CoV-2 for about 42 days post initial administration.
  • the subject produces neutralizing antibodies to SARS- CoV-2 for at least about 42 days post initial administration.
  • the subject produces neutralizing antibodies to SARS- CoV-2 for about 1 year post initial administration.
  • the amount of neutralizing antibody in a subject increased by at least about 4-fold relative to levels prior to administration of the nucleic acid vaccine. [0045] In some embodiments, the amount of neutralizing antibody in a subject increased by at least about 5-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by at least about 6-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by at least about 7-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by more than 4-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by more than 5-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by more than 6-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by more than 7-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by about 100-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the amount of neutralizing antibody in a subject increased by about 200-fold relative to levels prior to administration of the nucleic acid vaccine.
  • the nucleic acid vaccine is administered as a booster vaccine.
  • the nucleic acid vaccine may be administered as the original vaccination or another vaccine may be administered as the original vaccination to the subject.
  • the disclosure provides nucleic acid vaccines for COVID-19 for use in a method of vaccinating a subject for COVID-19, wherein the nucleic acid vaccine comprises about 0.2 mg/mL mRNA, wherein the mRNA comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7, and wherein the nucleic acid vaccine is formulated for intramuscular (IM) injection and formulated in a lipid nanoparticle (LNP).
  • IM intramuscular
  • LNP lipid nanoparticle
  • FIG. 1 A shows a histogram of solicited TEAEs of systemic events and local reactions reported in the Safety Population.
  • FIG. IB shows a histogram of solicited TEAEs (systemic events and local reactions) reported in the Safety Population (left graph) and evaluable immunogenicity population (right graph) At each time point (Dose 1 or Dose 2) the left histogram bar corresponds to subjects administered PTX-B vaccine, the right histogram bar corresponds to subject administered COMIRNATY®.
  • FIG. 1C shows a histogram of solicited systemic events in the Safety Population within 7 days post each dose.
  • FIG. ID shows a histogram of solicited local events in the Safety Population within 7 days post each dose.
  • FIG. 2 shows results from Nab titer assays in the EIP group. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
  • FIG. 3 shows results from Nab titer assays in the post-hoc EIP group with high baseline Nab titers removed.
  • the left box-plot corresponds to subjects administered PTX-B vaccine
  • the right box plot corresponds to subject administered COMIRNATY®.
  • FIG. 4 shows the results of administering either PTX-B (grey bar) or COMIRNATY® (black bar) on Nab levels (as fold increase).
  • FIG. 5 shows results from Nab titer assays in the mITT group with high baseline Nab titers removed. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
  • FIG. 6A shows results from Nab titer assays in the full mITT. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
  • Fig. 6B shows a histogram where PTX-B was non-inferior versus COMIRNATY®.
  • Fig. 6C shows a histogram where PTX-B was non-inferior and numerically higher versus COMIRNATY®.
  • Fig. 6D shows histograms for the mITT population and the seronegative mITT population where was non-inferior and numerically higher versus COMIRNATY®.
  • Fig. 6E shows a histogram for Interferon-y (left graph) and Interleukin-5 (right graph) for PTX-B and COMIRNATY®.
  • Fig. 6F shows the saliva antibody response for PTX-B and COMIRNATY®.
  • Fig. 6G shows a histogram for Cellular Immunity (Interferon-y) during the 6 months after primary vaccination.
  • FIG. 7 shows the results from Nab titer assays in subjects with a baseline titer greater than 100 (> 100) after PTX-B administration.
  • FIG. 8 shows the anti-RBD and anti-spike IgG at day 42 as determined by MSD.
  • FIG. 9 shows the anti-RBD and anti-spike IgG up to six months post vaccination by ELISA.
  • FIG. 10 shows the anti-spike IgG up to 42 post vaccination determined by ELISA.
  • FIG. 11 shows the anti-RBD and anti-spike IgA up to six months post vaccination by ELISA.
  • FIG. 12 shows the pseudovirus neutralization titers for PTX-B vaccinated cohorts.
  • FIG. 13 shows the correlation of the Lentivirus and rVSV Neutralization Assay (Day 28 and Day 42).
  • FIG. 14 shows that PTX-B induces neutralizing antibodies against variants of concern (VOCs).
  • nucleic acid vaccines comprising polynucleotides encoding one or more antigen proteins, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19.
  • the antigen protein may be a structural protein of SARS-CoV-2.
  • the structural protein may be the spike(S) protein, the membrane(M) protein, the nucleocapsid(N) phosphoprotein or the envelope(E) protein.
  • At least one component of the nucleic acid vaccine is a polynucleotide encoding at least one of the antigen proteins or the fragments or variants of the antigen proteins of SARS-CoV-2.
  • the antigen protein may be a structural protein of SARS-CoV-2.
  • the polynucleotide may be a RNA polynucleotide such as an mRNA polynucleotide.
  • the nucleic acid vaccine includes at least one mRNA polynucleotide encoding at least one of the structural proteins or the fragments or variants of the structural proteins of SARS-CoV-2.
  • the polynucleotide may be designed to encode one or more polypeptides of interest from SARS-CoV-2, or fragments or variants thereof.
  • polypeptide of interest of SARS-CoV-2 may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides or variants of polypeptides, which independently may be encoded by one or more regions or parts or the whole of a polynucleotide from SARS-CoV-2.
  • the term “polypeptides of interest” refer to any polypeptide which is selected to be encoded within, or whose function is affected by, the polynucleotides described herein. Any of the peptides or polypeptides described herein may be antigenic (also referred to as immunogenic).
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
  • the term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function, or origin.
  • the polypeptides of interest are antigens encoded by the polynucleotides as described herein.
  • polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long.
  • polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked.
  • polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
  • variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, or at least about 85%, more preferably at least about 90%, even more preferably at least about 95% identical (homologous) to a native or reference sequence.
  • variant mimics are provided.
  • the term “variant mimic” is one which contains one or more amino acids which would mimic an activated sequence.
  • glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine.
  • variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
  • “Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gap and penalties introduced in the calculation.
  • homologs as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
  • Analogs is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
  • the present disclosure contemplates several types of compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives.
  • derivative is used synonymously with the term “variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.
  • sequence tags or amino acids can be added to the peptide sequences described herein (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support.
  • amino acids e.g., C-terminal or N-terminal residues
  • substitutional variants when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position.
  • the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine.
  • substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
  • nonconservative substitutions include the substitution of a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
  • Insertional variants when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. “Immediately adjacent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.
  • “Deletional variants” when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
  • Covalent derivatives when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant hosT-cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
  • polypeptides are defined as distinct amino acid sequence-based components of a molecule.
  • Features of the polypeptides encoded by the polynucleotides described herein include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof.
  • surface manifestation refers to a polypeptide-based component of a protein appearing on an outermost surface.
  • local conformational shape means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
  • fold refers to the resultant conformation of an amino acid sequence upon energy minimization.
  • a fold may occur at the secondary or tertiary level of the folding process.
  • secondary level folds include beta sheets and alpha helices.
  • tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
  • the term “turn” as it relates to polypeptide conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
  • loop refers to a structural feature of a polypeptide which may serve to reverse the direction of the backbone of a peptide or polypeptide. Where the loop is found in a polypeptide and only alters the direction of the backbone, it may comprise four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Bio., 1266 (4): 814- 830; 1997). Loops may be open or closed. Closed loops or “cyclic” loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties.
  • Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having disulfide bridges or alternatively bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
  • Cys-Cys cysteine-cysteine bridge
  • bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
  • half-loop refers to a portion of an identified loop having at least half the number of amino acid resides as the loop from which it is derived. It is understood that loops may not always contain an even number of amino acid residues. Therefore, in those cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of amino acids of the loop/2+/-0.5 amino acids).
  • domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
  • sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
  • site as it pertains to amino acid-based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.”
  • a site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide-based molecules described herein.
  • terminal refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
  • the polypeptide-based molecules described herein may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins described herein are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers).
  • any of the features may be modified such that they begin or end, as the case may be, with a non-polypeptide-based moiety such as an organic conjugate.
  • any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating.
  • manipulation of features may result in the same outcome as a modification to the molecules described herein. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full-length molecule would.
  • modification refers to a modification as compared to the canonical set of 20 amino acids.
  • the modifications may be various distinct modifications.
  • the regions may contain one, two, or more (optionally different) modifications.
  • Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis or a priori incorporation during chemical synthesis.
  • the resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
  • the polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation.
  • a “consensus” sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest.
  • any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical to a reference protein.
  • the protein fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids in length.
  • any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids, or more, which are about 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 can be utilized in accordance with the nucleic acid vaccines described herein.
  • a polypeptide to be utilized in accordance with the nucleic acid vaccines 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.
  • polynucleotides of the present disclosure encode peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the peptide or polypeptide sequences disclosed herein.
  • the polynucleotides may also contain substitutions, insertions and/or additions, deletions and covalent modifications with respect to the polynucleotide reference sequences.
  • Reference molecules may share a certain identity with the designed molecules (polypeptides or polynucleotides).
  • identity refers to a relationship between the sequences of two or more peptides, polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleosides. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”).
  • Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, N.Y., 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, N.Y, 1991; and Carillo et al., SIAM J. Applied Math. 48: 1073; 1988).
  • the encoded polypeptide variant may have the same or a similar activity as the reference polypeptide.
  • the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide.
  • variants of a particular polynucleotide or polypeptide described herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • Such tools for alignment include those of the BLAST suite (Stephen F. Altschul et al. duplicate Gapped BLAST and PSLBLAST: a new generation of protein database search programs, Nucleic Acids Res. 1997, 25:3389-3402.) Other tools are described herein, specifically in the definition of “Identity.”
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus which causes coronavirus disease 2019 termed “COVID-19.”
  • COVID-19 affects mammals in different ways including individuals who are asymptomatic to individuals who have a wide span of symptoms that range from mild symptoms to severe illness or death. To date, about 80% of COVID-19 patients have mild to moderate symptoms whereas about 20% may develop complications such as sever 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 difficulty breathing, fatigue, body aches, muscle aches, headaches, sore throat, congestion or runny nose, nausea and/or vomiting, diarrhea, and a new loss of taste or smell.
  • SARS-CoV-2 encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and nonstructural proteins (named nspl to nspl6) and accessory proteins.
  • the viral surface spike protein is generally the leading mediator for viral entry into cells.
  • SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases.
  • hACE2 human ACE2
  • RBD receptor-binding domain
  • SARS-CoV-2 Another feature of the spike protein of SARS-CoV-2 is that the protein has a functional furin cleavage site at the S1-S2 boundary (SI is the receptor binding unit and S2 is the membrane fusion unit), which can preactivate the entry of many viruses including SARS-CoV-2.
  • SARS-CoV-2 spike has been used as a protective antigen that elicits neutralizing antibodies in various vaccine developing strategies.
  • the membrane protein and the envelope protein are for viral assembly.
  • the envelop protein (E) can forms a homopentameric cation channel that is important for virus 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 has 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 herein incorporated by reference in their entirety). Additionally, the nucleocapsid protein in SARS-CoV-2 can modulate the host cell machinery and may be included in regulatory roles in the viral life cycle.
  • RNP helical ribonucleocapsid
  • 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.
  • RBD amino acids There are six RBD amino acids have been shown to be critical for binding to ACE2 receptors and the SARS- CoV-2 genome appears to have a RBD that has a high affinity binding to ACE2 for 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 herein incorporated by reference in their entirety).
  • the polynucleotides of the nucleic acid vaccine described herein encode the full-length polypeptide of a structural protein, or a fragment or variant of the structural protein of SARS-CoV-2, such as the spike protein, the nucleocapsid protein, the envelop protein or the membrane protein.
  • the polynucleotides of the nucleic acid vaccine described herein encode more than one fragment or variant of a structural protein of SARS-CoV-2, such as the spike protein, the nucleocapsid protein, the envelop membrane and/or the membrane protein.
  • the polynucleotides of the nucleic acid vaccine described herein encode a mutated variant of one of the structural proteins, or a fragment of the mutated variant of the structural proteins of SARS-CoV-2.
  • the variant may be a single amino acid change from Aspartic Acid to Glycine in one of the structural proteins of SARS-CoV-2.
  • the polynucleotides of the nucleic acid vaccine described herein encode a full-length polypeptide of the spike protein, or a fragment, or a variant of the spike protein of SARS-CoV-2.
  • the variant may be a single amino acid change from Aspartic Acid to Glycine in the spike protein of SARS-CoV-2.
  • the variant may be a single amino acid change from Aspartic Acid to Glycine at position 614 (D614G) in the spike protein of SARS-CoV-2 (Korber et al.;
  • the nucleic acid vaccine described herein may encode one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2.
  • proteins, peptides, fragments or variants of the structural proteins of SARS-CoV-2 are provided in Table 1. In the table, the NCBI reference number is also provided if known. Table 1. Structural Protein Sequences of SARS-CoV-2
  • the nucleic acid vaccine described herein may encode at least one structural protein with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1.
  • the nucleic acid vaccine may be an mRNA vaccine that, when translated, produces one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2.
  • the polynucleotides of the mRNA vaccine are mRNA polynucleotides encoding one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2.
  • the coding sequences of mRNA vaccines described herein may be based on the coding sequence of the spike(S) protein from the genome of SARS- CoV-2 Wuhan-Hu-1 isolate (GenBank: NM908947.3, complete genome sequence).
  • a change of the code for a single amino acid change from D614 to G614 is introduced to match the amino acid of the current dominant circulating strains.
  • Non-limiting examples of a RNA sequence encoding proteins, peptides, fragments or variants of the structural proteins of SARS-CoV-2 are provided in Table 2.
  • the mRNA sequence encoding the spike protein with D614G mutation of SARS-CoV-2 comprises the coding region of SEQ ID NO: 7, or a fragment or variant thereof.
  • the mRNA sequence encoding the spike protein with D614G mutation of SARS-CoV-2 comprises SEQ ID NO: 27, or a fragment or variant thereof.
  • the nucleic acid vaccines may comprise a region encoding any of the sequences listed in Table 1 or a fragment or variant thereof.
  • the nucleic acid vaccines may comprise hybrid or chimeric regions, or mimics or variants.
  • the nucleic acid vaccines may comprise any of the polynucleotide sequences listed in Table 3.
  • the nucleic acid vaccine described herein encodes a protein or fragment 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 a protein provided by an amino acid sequence in Table 1.
  • the terms “identical” or percent “identity” in the context of two or more polypeptide sequences refer to two or more sequences that are the same. The percent identity between polypeptide sequences may be performed using algorithms known in the art, such as BLAST and CLUSTAL.
  • sequence of the SARS-CoV-2 protein or fragment or variant thereof may be obtained from any source.
  • sequence of the SARS-CoV-2 protein or fragment or variant thereof is from a strain that is capable of or at risk of infecting human subjects.
  • sequence of the SARS-CoV-2 protein or fragment or variant thereof may be modified or optimized (such as codon optimized) for expression in a particular cell or host organism.
  • the nucleic acid vaccine described herein may be a multivalent vaccine.
  • the multivalent vaccine may include polynucleotides that encodes at least two different one or more proteins, peptides, fragments or variants thereof of SARS- CoV-2.
  • the polynucleotides may encode the same or a different structural protein.
  • the polynucleotides may encode the same structural protein but different variants of the structural protein.
  • the nucleic acid vaccine encodes the full-length S protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a fragment of the S protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the 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 a S protein sequence of SARS-CoV-2 (e.g., full-length, fragment or variant) where the S protein has a mutated furin cleavage site.
  • RBD receptor binding domain
  • the S protein furin cleavage site mutant will remove or disable the furin cleavage site(s) in S protein (e.g., between the SI and S2 boundary). In some viral envelope proteins, disruption of a furin cleavage site was found to enhance expression and stability.
  • the nucleic acid vaccine encodes a S protein sequence of SARS-CoV-2 (e.g., full-length, fragment or variant) where the S protein includes the D614G mutation.
  • the nucleic acid vaccine encoding the S protein of SARS-CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
  • linker e.g., GSG linker
  • the nucleic acid vaccine encodes the full-length M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a fragment of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the topological domain (e.g., virion surface or intravirion region) of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the M protein (e.g., full-length protein or fragment) of SARS-CoV-2.
  • the nucleic acid vaccine encoding the M protein of SARS- CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
  • linker e.g., GSG linker
  • 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 the dimerization domain of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the N protein (e.g., full-length protein or fragment) of SARS-CoV-2.
  • the nucleic acid vaccine encoding the N protein of SARS-CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
  • linker e.g., GSG linker
  • 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 E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the topological domain (e.g., virion surface or intravirion region) of the E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the E protein (e.g., full-length protein or fragment) of SARS-CoV-2.
  • the nucleic acid vaccine encoding the E protein of SARS- CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
  • linker e.g., GSG linker
  • the nucleic acid vaccine encodes two different structural proteins of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and a M protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and a N protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and an E protein, fragment or variant thereof of SARS-CoV-2.
  • the nucleic acid vaccine encodes a M protein, fragment or variant thereof of SARS-CoV-2 and a N protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a M protein, fragment or variant thereof of SARS-CoV-2 and an E protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a N protein, fragment or variant thereof of SARS-CoV-2 and an E protein, fragment or variant thereof of SARS-CoV-2.
  • the nucleic acid vaccine encoding two different structural proteins, fragment or variant thereof of SARS-CoV-2 may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
  • the sequences encoding the two different structural proteins or fragments or variants thereof of SARS- Cov-2 of the nucleic acid vaccine are constructed as a single polynucleotide.
  • the nucleic acid vaccine encodes at least three different sequences of the structural proteins fragment or variant thereof for SARS-CoV-2.
  • the nucleic acid vaccine encodes two different S proteins, fragments or variants sequences for SARS-CoV-2 and a M protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S proteins, fragments or variants sequences for SARS-CoV-2 and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S proteins, fragments or variants sequences for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2.
  • the nucleic acid vaccine encodes two different M proteins, fragments or variants sequences for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N proteins, fragments or variants sequences for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E proteins, fragments or variants sequences for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2.
  • the nucleic acid vaccine encodes two different M proteins, fragments or variants sequences for SARS-CoV-2 and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M proteins, fragments or variants sequences for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N proteins, fragments or variants sequences for SARS-CoV-2 and a M protein, fragment or variant sequence for SARS-CoV-2.
  • the nucleic acid vaccine encodes two different N proteins, fragments or variants sequences for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E proteins, fragments or variants sequences for SARS-CoV-2 and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant sequence for SARS-CoV-2, a M protein, fragment or variant sequence for SARS-CoV-2, and a N protein, fragment or variant sequence for SARS-CoV-2.
  • the nucleic acid vaccine encodes a S protein, fragment or variant sequence for SARS-CoV-2, a M protein, fragment or variant sequence for SARS-CoV-2, and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant sequence for SARS-CoV-2, a N protein, fragment or variant sequence for SARS-CoV-2, and an E protein, fragment or variant sequence for SARS- CoV-2. In some embodiments, the nucleic acid vaccine encodes a M protein, fragment or variant sequence for SARS-CoV-2, a N protein, fragment or variant sequence for SARS- CoV-2, and an E protein, fragment or variant sequence for SARS-CoV-2.
  • the nucleic acid vaccine encoding at least three different sequences of the structural proteins fragment or variant thereof for SARS-CoV-2 may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
  • a linker e.g., GSG linker
  • SARS-CoV-2 is a member of the large coronavirus family of viruses. Multiple variants (sometimes referred to as “strains” or “lineages”) of SARS-CoV-2 have been identified globally. The nomenclature for SARS-CoV-2 variants used in this description is consistent with the PANGO nomenclature for new virus lineages (Rambaut, Andrew, et al., A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nature microbiology, 2020, 5:: 1403-1407, the contents of which are incorporated herein by reference in their entirety). Near real-time data relating to PANGO SARS-CoV-2 lineages or variants can be accessed online using Nextstrain’s SARs-CoV-2 analysis user interface (nextstrain.org/ncov/global).
  • PANGO lineage variants of SARS-CoV-2 have been identified, including the following (number in parentheses represents number of cases per each submitted PANGO lineage: A (37); A.l (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.l (1); AE.2 (2); AE.4 (1); AE.5 (1); AE.7 (1); AE.8 (1); AG.
  • 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. l.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
  • variants are typically categorized as Variants of Interest (VOIs), Variants of Concern (VOCs), and Variants of High Consequence (VOHCs).
  • VOIs Variants of Interest
  • VOCs Variants of Concern
  • VOHCs Variants of High Consequence
  • VOIs may have certain genetic markers associated with changes to receptor binding, reduced neutralization by antibodies generated against previous infection or vaccination, reduced efficacy of treatments, potential diagnostic impact, or predicted increase in transmissibility or disease severity. In some instances, VOIs have specific genetic markers that are predicted to affect transmission, diagnostics, therapeutics, or immune escape, or cause an increased proportion of cases or unique outbreak clusters.
  • SARS-CoV-2 VOIs include, for example, PANGO lineage B. 1.1.7 (Alpha), B.1.351 (Beta); B.1.427/429 (Epsilon); B.1.526 (Iota); B.1.525 (Eta); B.1.617.1 (Kappa);
  • VOCs may include variants for which there is evidence of an increase in transmissibility, more severe disease (increased hospitalizations or deaths), significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures.
  • VOCs have evidence of impact on diagnostics, treatments, and vaccines, widespread interference with diagnostic test targets, evidence of substantially increased resistance to one or more class of therapies, evidence of significant decreased neutralization by antibodies generated during previous infection or vaccination, evidence of reduced vaccine-induced protection from severe disease, evidence of increased transmissibility, or evidence of increased disease severity.
  • SARS-CoV-2 VOCs may include, for example, PANGO lineage B.l.1.7 (Alpha), P.l (Gamma), B.1.351(Beta), B.1.427and B.1.429 ((Epsilon), B.1.526 (Iota), B.1.525 (Eta), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), and C37 (Lamba).
  • VOHCs may have clear evidence that prevention measures or medical countermeasures (MCMs) have significantly reduced effectiveness relative to previously circulating variants.
  • MCMs Medical Countermeasures
  • VOHCs have impact on Medical Countermeasures (MCM), demonstrated failure of diagnostics, evidence to suggest a significant reduction in vaccine effectiveness, a disproportionately high number of vaccine breakthrough cases, very low vaccine-induced protection against severe disease, significantly reduced susceptibility to multiple Emergency Use Authorization (EUA) or approved therapeutics, more severe clinical disease and increased hospitalizations.
  • MCM Medical Countermeasures
  • the nucleic acid vaccines disclosed herein may encode one or more polypeptides, e.g., one or more proteins, peptides, fragments or variants thereof, of any of the SARS-CoV-2 variants described herein.
  • the nucleic acid vaccines disclosed herein may encode one or more polypeptides, e.g., one or more proteins, peptides, fragments or variants thereof, of a SARS-CoV-2 VOI, VOC, and/or VOHC.
  • the nucleic acid vaccines encode a polypeptide comprising the specific mutation called D614G.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.526 SARS-CoV-2 variant (i.e., Iota variant), such as one or more of Spike protein substitutions L5F, T95I, D253G, S477N, E484K, D614G, and/or A701 V; ORFla substitutions L3201P, T265I, and/or A3675/3677; ORFlb substitutions P314L and/or Q1011H; ORF3a substitutions P42L, Q57H; ORF8 substitution Ti ll; and/or 5’UTR substitution R81C.
  • Iota variant such as one or more of Spike protein substitutions L5F, T95I, D253G, S477N, E484K, D614G, and/or A701 V; ORFla substitutions L3201P, T265I, and/or A3675/3677; OR
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.525 SARS-CoV-2 variant (i.e., Eta variant), such as one or more of Spike protein substitutions A67V, A69/70, A144, E484K, D614G, Q677H and/or F888L; ORFlb substitution P314F; ORFla substitution T2007I; M protein substitution I82T; N protein substitutions A12G and/or T205I; and/or 5’UTR substitution R81C.
  • Eta variant such as one or more of Spike protein substitutions A67V, A69/70, A144, E484K, D614G, Q677H and/or F888L
  • ORFlb substitution P314F ORFla substitution T2007I
  • M protein substitution I82T M protein substitution I82T
  • N protein substitutions A12G and/or T205I and/or 5’UTR substitution R81C.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the P.2 SARS- CoV-2 variant, such as one or more of: Spike protein substitutions E484K, D614G, and/or V1176F; ORFla substitutions L3468V and/or L3930F; ORFlb substitution P314L; N protein substitutions Al 19S, R203K, G204R, and/or M234I; 5’UTR substitution R81C.
  • Spike protein substitutions E484K, D614G, and/or V1176F such as one or more of: Spike protein substitutions E484K, D614G, and/or V1176F; ORFla substitutions L3468V and/or L3930F; ORFlb substitution P314L; N protein substitutions Al 19S, R203K, G204R, and/or M234I; 5’UTR substitution R81C.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.l.1.7 SARS-CoV-2 variant (i.e., Alpha variant), such as one or more of: Spike protein substitutions A69/70, A144Y, E484K, S494P, N501Y, A570D, D614G, and/or P681H.
  • SARS-CoV-2 variant i.e., Alpha variant
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the P.l SARS- CoV-2 variant (i.e., Gamma variant), such as one or more of: Spike protein substitutions K417N/T, E484K, N501Y, and/or D614G.
  • P.l SARS- CoV-2 variant i.e., Gamma variant
  • Spike protein substitutions K417N/T i.e., E484K, N501Y, and/or D614G.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.351 SARS-CoV-2 variant (i.e., Beta variant), such as one or more of: Spike protein substitutions K417N, E484K, N501Y, and/or D614G.
  • the B.1.351 variant is also referred to as the South African variant, as it first originated in South Africa.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.427 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions L452R and/or D614G.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.429 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions SI 31, W152C, L452R, and/or D614G.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.617.1 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions G142D, E154K, L452R, E484Q, D614G, P681R, and/or Q1071H.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant (i.e., Delta variant), such as one or more of: Spike protein substitutions T19R, T95I, G142D, A156/157, R158G, L452R, T478K, D614G, P681R, and/or D950N.
  • SARS-CoV-2 variant i.e., Delta variant
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more further mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions V70F, A222V, W258L, and/or K417N.
  • the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.617.3 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions T19R, G142D, L452R, E484Q, D614G, P681R, and/or D950N.
  • the nucleic acid vaccines encode a SARS-CoV-2 Spike protein, e.g., protein, peptide, fragment, or variant, comprising one or more substitutions and/or deletions selected from: A570D, A67V, A701V, D253G, D614G, E484K, F888L, K417N/T, L452R, L5F, N501Y, P681H, Q677H, S13I, S477N, S494P, T95I, V1176F, W152C, A144, A144Y, and A69/70.
  • SARS-CoV-2 Spike protein e.g., protein, peptide, fragment, or variant, comprising one or more substitutions and/or deletions selected from: A570D, A67V, A701V, D253G, D614G, E484K, F888L, K417N/T, L452R, L5F, N501Y, P681H,
  • the nucleic acid vaccines encode a SARS-CoV-2 ORF la comprising one or more substitutions and/or deletions selected from: L3201P, T265I, T2007I, L3468V, A3675-3677, and L3930F.
  • the nucleic acid vaccines encode a SARS-CoV-2 ORF lb comprising one or more substitutions selected from: P314F, P314L, and Q1011H.
  • the nucleic acid vaccines encode a SARS-CoV-2 ORF3a comprising one or more substitutions selected from: P42L and Q57H. [0166] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 ORF8 comprising a Ti ll substitution.
  • the nucleic acid vaccines encode a SARS-CoV-2 5’UTR comprising a R81C substitution.
  • the nucleic acid vaccines encode a SARS-CoV-2 M protein, e.g., protein, peptide, fragment, or variant, comprising I82T substitution.
  • the nucleic acid vaccines encode a SARS-CoV-2 N protein, e.g., protein, peptide, fragment, or variant, comprising one or more substitutions selected from: A12G, A119S, R203K, G204R, T205I, and M234I.
  • the polynucleotides described herein encode at least one polypeptide of interest, e.g., one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2.
  • the proteins, peptides, fragments or variants thereof of SARS-CoV-2 of the present disclosure may be wild type where they are derived from the infectious agent, or modified (e.g., the structural proteins or fragments and variants thereof are engineered, designed or artificial). They may have any combination of the features described herein.
  • 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, or fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19.
  • these peptides or polypeptides may serve as an antigen or antigenic molecule (also preferred to as immunogenic molecule).
  • the term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.
  • nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'- amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol
  • IVT polynucleotides encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 of the present disclosure.
  • the nucleic acid vaccines may include “chimeric polynucleotides” which have portions or regions which differ in size and/or encoded protein (e.g., structural protein of SARS-CoV-2).
  • a “chimera” is an entity having two or more incongruous or heterogeneous parts or regions.
  • a “part” or “region” of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide.
  • the nucleic acid vaccine includes polynucleotides from about 30 to about 100,000 nucleotides in length(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 500 to 2,000, from 500 to 3,000, from
  • the length of a region encoding at least one peptide or polypeptide of interest of the polynucleotides of the nucleic acid vaccine is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 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).
  • the polynucleotides of the nucleic acid vaccine is or functions as a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the term “messenger RNA (mRNA)” refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide or polypeptide of interest in vitro, in vivo, in situ or ex vivo.
  • the shortest length of a region of the polynucleotide of the nucleic acid vaccine can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide.
  • the length may be sufficient to encode a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids.
  • the length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g., no longer than35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.
  • the region of the polynucleotide of the nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19 may be greater than about 30 nucleotides in length.
  • the length may be, but is not limited to, at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,
  • the region includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000
  • the nucleic acid vaccines described herein may be an mRNA vaccine.
  • the mRNA vaccine includes at least one mRNA molecule which, when translated, produce at least one peptide or polypeptide of interest for the prevention, alleviation and/or treatment of COVID-19.
  • 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
  • the mRNA includes a region to initiate translation.
  • This region may include any translation initiation sequence or signal including a Start codon.
  • the region includes a Start codon.
  • the Start codon may be “ATG,” “ACG,” “AGG,” “ATA,” “ATT,” “CTG,” “GTG,” “TTG,” “AUG,” “AUA,” “AUU,” “CUG,” “GUG,” or “UUG”.
  • the mRNA includes a region to stop translation.
  • This region may include any translation termination sequence or signal including a Stop codon.
  • the region includes a Stop codon.
  • the Stop codon may be “TGA,” “TAA,” “TGA,” “TAG,” “UGA,” “UAA,” “UGA” or “UAG.”
  • the regions to initiate or terminate translation may independently range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. Additionally, these regions may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
  • a masking agent may be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.
  • the start codon may be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon which is not the start codon. Translation of the polynucleotide may begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon.
  • the polynucleotide sequence where the start codon is removed may further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide.
  • the coding region of the polynucleotide of the nucleic acid vaccine may encode at least one peptide or polypeptide of interest.
  • Non-limiting examples of peptides or polypeptides of interest include one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19.
  • mRNA Components Untranslated Region
  • the polynucleotides of the nucleic acid vaccines described herein may comprise one or more regions or parts which act or function as an untranslated region (UTR). Wild type UTRs of a gene are transcribed but not translated. In mRNA, the 5 'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, UTRs may have a role in terms of stability and translation of the nucleic acid molecule and translation. Variants of UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
  • the UTRs of the polynucleotide of the nucleic acid vaccine may range independently from 15-1,000 nucleotides in length (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).
  • 15-1,000 nucleotides in length 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, 900, and 1,000 nucleotides.
  • Wild type 5' UTRs include features which play roles in translation initiation as these 5’ UTRs include sequences such as Kozak sequences which are known to be involved in how the ribosome initiates translation of many genes. 5' UTRs also have been known to form secondary structures which are involved in elongation factor binding. Other non-UTR sequences (e.g., introns or portions of intron sequences) may also be used as regions or subregions which may increase protein production as well as polynucleotide levels.
  • Natural or wild type 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides of the nucleic acid vaccines.
  • AREs 3' UTR AU rich elements
  • the UTR from any gene may be incorporated into the regions of the polynucleotides of the nucleic acid vaccines.
  • artificial UTRs which are not variants of wild type regions, may also be used in the polynucleotides of the nucleic acid vaccines.
  • These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs from a different parental sequence.
  • flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property.
  • polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the 5’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the 5’UTR is directly 5’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine.
  • the 5’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 5’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine; e.g., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 5’UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine.
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO:
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the 3’UTR is directly 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine.
  • the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine; e.g., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 3’UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine.
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO:
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the 3’UTR is directly 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine.
  • the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine; e.g., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 3’UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine.
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA).
  • mRNA Components Cap and IRES Sequences
  • the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’ cap structure.
  • the 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5' proximal introns removal during mRNA splicing.
  • the 5’ terminal capping region of the polynucleotide of the nucleic acid vaccine may comprise a single cap or a series of nucleotides forming the cap.
  • the capping region may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length.
  • the capping region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.
  • the cap is absent.
  • cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs may be used in the nucleic acid vaccines.
  • Cap analogs which may be chemically (e.g., non-enzymatically) or enzymatically synthesized, differ from natural (e.g., endogenous, wild-type or physiological) 5'-caps in their chemical structure, but they retain cap function.
  • the 5' terminal caps of the polynucleotides of the nucleic acid vaccines may include endogenous caps or cap analogs.
  • 5' terminal caps may comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine (mlG), 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • 5' capping can be generated via enzymatic or other synthetic processes. Endogenous mRNA molecules are 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. This 5 '-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or ante-terminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2'-O-methylated.
  • 5 '-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • Polynucleotides, e.g., mRNAs, of the nucleic acid vaccine described herein may be modified to include a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp- 5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction.
  • a vaccinia virus capping enzyme available from, e.g., New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 '-ppp-5' cap.
  • Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (e.g., non-enzymatically) or enzymatically synthesized and linked to a nucleic acid molecule, such as an mRNA molecule.
  • the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5 '-triphosphate-5 '- guanosine (m7G-3'mppp-G; which may equivalently be designated 3' O-Me- m7G(5')ppp(5')G).
  • the 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA).
  • the N7- and 3 '-O-methlyated guanine provide the terminal moiety of the capped nucleic acid molecule (e.g., mRNA).
  • mCAP which is similar to ARCA but has a 2'-O- methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5 '-triphosphate-5 '- guanosine, m7Gm-ppp-G).
  • cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of cap analogs from endogenous 5 '-cap structures may lead to reduced translational competency and reduced cellular stability.
  • polynucleotides e.g., mRNAs
  • mRNAs can be capped post-transcriptionally, using enzymes.
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'- terminal nucleotide of the mRNA contains a 2'-O-methyl.
  • the Cap 1 structure is termed the Cap 1 structure.
  • the Cap 1 structure provides a higher translational- competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art.
  • Cap structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')NlmpNp (Cap 1), and 7mG(5')-ppp(5')NlmpN2mp (Cap 2).
  • the polynucleotide of the nucleic acid vaccine described herein comprises a Cap 1 structure.
  • polynucleotides e.g., mRNA
  • the polynucleotides may be capped post- transcriptionally, and because this process is more efficient, up to 100% of the polynucleotides, e.g., mRNA, may be capped. This is in contrast to -80% when a cap analog is linked to an mRNA in the course of an in vitro transcription reaction.
  • the polynucleotides of the nucleic acid vaccines may contain an internal ribosome entry site (IRES) sequence. While not wishing to be bound by theory, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site or may serve as one of multiple ribosome binding sites of an mRNA.
  • IRES internal ribosome entry site
  • the polynucleotide of the nucleic acid vaccine e.g., the mRNA includes a tailing region.
  • Non-liming examples of a tailing region include a poly-A sequence, a poly-C sequence, and/or a polyA-G quartet.
  • the mRNA includes a chain terminating nucleoside.
  • chain terminating nucleosides include 2'-0 methyl, F and locked nucleic acids (LNA).
  • the sequence of the tailing region of the polynucleotide of the nucleic acid vaccine may range from absent to 500 nucleotides in length (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 or as a function of poly-A Binding Protein binding.
  • poly-A tails may also be added after the construct is exported from the nucleus.
  • a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecule during RNA processing in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA.
  • polyadenylation adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
  • the length of a poly-A tail when present, is greater than 30 nucleotides in length (e.g., at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
  • the poly-A tail region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 1,000 to 1,500, from 1,000 to
  • the poly-A tail is approximately 99 nucleotides in length (SEQ ID NO: 44).
  • the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design may be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.
  • the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof.
  • the poly-A tail may also be designed as a fraction of the polynucleotides to which it belongs.
  • the poly- A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail.
  • engineered binding sites and conjugation of polynucleotides for Poly-A binding protein may enhance expression.
  • the polynucleotides of the nucleic acid vaccines may also encode additional features which may facilitate the trafficking of the polypeptides to therapeutically relevant sites.
  • One such feature which aids in protein trafficking is the signal sequence.
  • a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3- 60 amino acids) in length which is incorporated at the 5' terminus of the coding region or the N-terminus polypeptide encoded, respectively.
  • addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways.
  • the polynucleotides of the nucleic acid vaccines described herein include a signal sequence comprising SEQ ID NO: 45 (DNA) or SEQ ID NO: 49 (RNA).
  • the polynucleotides of the nucleic acid vaccines, their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include, but are not limited to, match codon frequencies in target and host organisms to ensure proper folding, alter GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g.
  • Codon optimization tools, algorithms and services are known in the art, non-limiting examples include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods.
  • the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 4.
  • the nucleic acid vaccine is vectorized after codon optimization.
  • vectors include, but are not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • Nucleic acid vaccines of the present disclosure may include one or more modifications.
  • modification or, as appropriate, “modified” refer to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties.
  • modification refers to a modification as compared to the canonical set of 20 amino acids.
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • organic base e.g., a purine or pyrimidine
  • nucleotide is defined as a nucleoside including a phosphate group or other backbone linkage (intemucleoside linkage).
  • modifications may be various distinct modifications.
  • the coding region(s), the untranslated region(s), the flanking region(s), and/or the terminal or tailing regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • nucleic acid vaccines of the present disclosure comprise one or more modifications which render the nucleic acid molecules, when introduced to a cell, more resistant to degradation in the cell and/or more stable in the cell as compared to unmodified polynucleotides.
  • the polynucleotides of the nucleic acid vaccines described herein can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone).
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted, for example, with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or groups.
  • modifications are present in each of the sugar and the internucleoside linkage.
  • Modifications according to the present disclosure may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional modifications are described herein.
  • the modifications include 2’-O-Methyl-modified or 2’- O-Methoxyethyl-modified nucleotides (2’-0Me and 2’-M0E modifications, respectively).
  • the polynucleotides of the nucleic acid vaccines described herein may comprise at least one modification described herein.
  • polynucleotides of the nucleic acid vaccines described herein can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage.
  • RNA polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • Modifications of polynucleotides 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, modification or deletion of nucleotides (or codons) encoding one or more N- linked glycosylation site in a translated polypeptide.
  • Modifications that are useful in the vaccines of the present disclosure may also comprise any modifications as described in PCT Publication WO2018200892, the contents of which are incorporated herein by reference in their entirety.
  • the vaccines of the present disclosure may further comprise features or modifications as described in PCT patent application publications W02020255063, WO2020182869, W02016011222, W02016011226, W02016005004, W02016000792, WO2015176737, WO2015085318, WO2015048744, and WO2015034925, and United States patent application publications US20200254086, US20200206362, US20180311336 and US20180303929; the contents of each of which are incorporated herein by reference in their entireties.
  • the polynucleotides, including the mRNA molecules of the nucleic acid vaccines described herein can include modifications as follows.
  • the intemucleoside linkages of the polynucleotides may be partially or fully modified.
  • the polynucleotides may comprise modifications to one or more nucleobases.
  • the polynucleotides may comprise 5-methylcytosines in place of all cytosine nucleobases/cytidine nucleotides.
  • the polynucleotides may have one or more modifications to one or more of the sugar subunits of a nucleoside.
  • the sugar modification can be one or more locked nucleic acids (LNAs) or 2’-O-Methoxyethyl-modified (“2’-M0E”) modifications.
  • LNAs locked nucleic acids
  • 2’-M0E 2’-O-Methoxyethyl-modified
  • the polynucleotides can be designed with a patterned array of sugar, nucleobase or linkage modifications.
  • the polynucleotides can comprise modifications to maximize stability.
  • the modified nucleosides and nucleotides can include a modified nucleobase.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil.
  • nucleobases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine.
  • the modified nucleobase is a modified uracil.
  • exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (y), pyridin- 4-one ribonucleoside, 5 -aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3- methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (y), pseudouridine (
  • the modified nucleobase is a modified cytosine.
  • exemplary nucleobases and nucleosides having a modified cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f’C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5- halo-cytidine (e.g., 5 -iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2- thio-5-methyl-cytidine, 4-thio-pseu
  • the modified nucleobase is a modified adenine.
  • exemplary nucleobases and nucleosides having a modified adenine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6- halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza- adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyl -ad enosine (nfA), 2-methyl-adenine (m 2 A), N6-methyl
  • the modified nucleobase is a modified guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1- methyl-inosine (m 1 !), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (02yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl- queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-d
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog.
  • the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine.
  • nucleotide modifications may be introduced at various positions in a polynucleotide described herein.
  • nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.
  • the polynucleotides of the present disclosure may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, T/U or C) or any intervening percentage e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 100%, and from 95% to 100%
  • the polynucleotides of the nucleic acid vaccines described herein may be modified to be a circular nucleic acid.
  • the termini of the polynucleotides may be linked by chemical reagents or enzymes, producing circular polynucleotides that have no free ends.
  • Circular polynucleotides are expected to be more stable than linear counterparts and to be resistant to digestion with exonucleases.
  • Circular polynucleotides may further comprise other structural and/or chemical modifications with respect to A, G, T/U or C ribonucleotides/deoxyribonucleotides.
  • the polynucleotides are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the polynucleotides are at least 75% modified, e.g., at least 75% of the nucleotides are modified. It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linkage) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification.
  • the polynucleotides are at least 10% modified in only one component of the nucleotide, with such component being the nucleobase, sugar, or linkage between nucleosides.
  • modifications may be made to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleobases, sugars, or linkages of a polynucleotide described herein.
  • the uracil nucleosides of the polynucleotide of the nucleic acid vaccine are all modified.
  • the modifications may be the same or different.
  • the guanine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified.
  • the modifications may be the same or different.
  • the guanine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified.
  • the modifications may be the same or different.
  • the cytosine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified.
  • the modifications may be the same or different.
  • the adenine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified. The modifications may be the same or different.
  • the polynucleotide of the nucleic acid vaccine is modified to comprise Nl-methyl-pseudouri dine nucleotides.
  • modified nucleosides and nucleotides which may be incorporated into polynucleotides (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents.
  • substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted Cl -6 alkyl; optionally substituted Cl-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-Cl-6 alkoxy, optionally substituted Cl-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20,
  • the polynucleotide such as the mRNA of the nucleic acid vaccine described herein comprises at least one sugar modification.
  • RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
  • modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multi cyclic forms (e.g., tri cyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (GNA) (GN
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • polynucleotide molecules as described herein, including mRNAs can include nucleotides containing, e.g., arabinose, as the sugar.
  • Nonlimiting examples of the sugar modification may include the modifications provided in Table 5.
  • the polynucleotides of the present disclosure can have one or more nucleotides carrying a modification as provided in Table 5.
  • each of the nucleotides of a polynucleotide described herein carries any one of the modifications as provided in Table 5, or none of the modifications as provided in Table 5.
  • At least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -OMe, referred to as 2’ -OMe.
  • at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -F, referred to as 2’-F.
  • the polynucleotides of the present disclosure can include any modification to the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone).
  • the phrases “phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonates phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both nonlinking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • the a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • Phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • a modified nucleoside includes an alpha-thio- nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O- (l-thiophosphate)-pseudouridine).
  • alpha-thio- nucleoside e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O- (l-thiophosphate)-p
  • the polynucleotides comprise at least one phosphorothioate linkage or methylphosphonate linkage between nucleotides.
  • the polynucleotides comprise at least one 5 ’-(E)- vinylphosphonate (5’- -VP), a phosphate mimic, as a modification.
  • the polynucleotide (e.g., mRNA) of the nucleic acid vaccine for CO VID-19 may be modified.
  • Nucleic acid vaccines of the present disclosure may vary in their valency. “Valency” refers to the number of antigenic components in the nucleic acid vaccine or the polynucleotide of the nucleic acid vaccines. The antigenic components of the nucleic acid vaccine may be on the same polynucleotide or they may be on different polynucleotides. In some embodiments, the nucleic acid vaccine may be monovalent. In some embodiments, the nucleic acid vaccine may be divalent. In some embodiments, the nucleic acid vaccine may be trivalent.
  • the nucleic acid vaccine may be multivalent which may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 antigens or antigenic moi eties such as, but not limited to, antigenic peptides.
  • antigenic peptides may be one or more fragments or variants of the structural proteins of SARS-CoV-2.
  • cDNA encoding the polynucleotides of the nucleic acid vaccines described herein may be transcribed using an in vitro transcription (IVT) system.
  • the system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
  • NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
  • the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
  • the polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and polymerase variants.
  • the DNA template is removed from the IVT reaction, using a DNase I enzyme.
  • the digested DNA and nucleotides are then removed during oligo dT purification of the mRNA.
  • This purification method is based on affinity of the poly-A tail of the mRNA to the poly-dT column bed. Centrifugation may be used but may not be required to remove the digested DNA and nucleotides.
  • a reverse phase column e.g., SDVB
  • ultrafiltration may be utilized, followed by one or more filtration steps.
  • RNA polymerases Any number of RNA polymerases or variants may be used in the synthesis of the polynucleotides of the nucleic acid vaccine described herein. RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence.
  • Polynucleotide or nucleic acid synthesis reactions may be carried out by enzymatic methods utilizing polymerases.
  • Polymerases catalyze the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain.
  • DNA polymerase I polymerase I
  • a polymerase family including the KI enow fragments of E. Coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among the best studied of these families.
  • DNA polymerase a or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from phages T4 and RB69. Although they employ similar catalytic mechanism, these families of polymerases differ in substrate specificity, substrate analogincorporating efficiency, degree and rate for primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity against inhibitors.
  • polynucleotides of the nucleic acid vaccines described herein may be manufactured in whole or in part using solid phase techniques.
  • Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer-controlled solidphase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids.
  • automated solid-phase synthesis is used where the chain is synthesized in 3' to 5' direction.
  • the hydroxyl group in the 3' end of a nucleoside is tethered to a solid support via a chemically cleavable or light-cleavable linker.
  • Activated nucleoside monomers such as 2'-deoxynucleosides (dA, dC, dG and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, are added to the support-bound nucleoside sequentially.
  • a cleaving agent such as ammonia or ammonium hydroxide is added to remove all the protecting groups and release the polynucleotide chains from the solid support.
  • Light may also be applied to cleave the polynucleotide chain.
  • the product can then be further purified with high pressure liquid chromatography (HPLC) or electrophoresis.
  • HPLC high pressure liquid chromatography
  • the synthesis of polynucleotides of the nucleic acid vaccines described herein by the sequential addition of monomer building blocks may be carried out in a liquid phase.
  • a covalent bond is formed between the monomers or between a terminal functional group of the growing chain and an incoming monomer.
  • Functional groups not involved in the reaction must be temporarily protected.
  • the reaction mixture has to be purified before adding the next monomer building block.
  • the functional group at one terminal of the chain has to be deprotected to be able to react with the next monomer building blocks.
  • a liquid phase synthesis is labor- and timeconsuming and cannot not be automated. Despite the limitations, liquid phase synthesis is still useful in preparing short polynucleotides in a large scale. Because the system is homogenous, it does not require a large excess of reagents and is cost- effective in this respect.
  • the polynucleotides of the nucleic acid vaccines described herein may be quantified in exosomes or when derived from one or more bodily fluid.
  • bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl
  • exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
  • the exosome quantification method a sample of not more than 2 mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • the level or concentration of a polynucleotide may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.
  • the assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
  • Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.
  • the polynucleotide may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
  • UV/Vis ultraviolet visible spectroscopy
  • Anon-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.).
  • the quantified polynucleotide may be analyzed in order to determine if the polynucleotide may be of proper size, check that no degradation of the polynucleotide has occurred.
  • Degradation of the polynucleotide may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
  • Purification of the polynucleotides of the nucleic acid vaccines described herein may include, but is not limited to, polynucleotide clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGEN- COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNATM oligo-T capture probes (EX- IQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
  • AGEN- COURT® beads Beckman Coulter Genomics, Danvers, Mass.
  • poly-T beads poly-T beads
  • LNATM oligo-T capture probes EX- IQON® Inc, Vedbaek, Denmark
  • HPLC based purification methods such
  • purified when used in relation to a polynucleotide such as a “purified polynucleotide” refers to one that is separated from at least one contaminant.
  • a “contaminant” is any substance which makes another unfit, impure or inferior.
  • a purified polynucleotide e.g., DNA and RNA
  • a quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
  • nucleic acid vaccines described herein may be used as therapeutic or prophylactic agents.
  • the present disclosure provides pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and a nucleic acid vaccine, i.e., a nucleic acid vaccine for COVID-19.
  • the pharmaceutical compositions comprising the nucleic acid vaccine described herein can be used for preventing, alleviating and/or treating COVID-19.
  • nucleic acid vaccines and pharmaceutical compositions thereof which may be used in combination with one or more pharmaceutically acceptable excipients.
  • Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances.
  • Pharmaceutical compositions of the nucleic acid vaccines described herein may be sterile and/or pyrogen-free.
  • compositions are administered to humans, human patients or subjects.
  • active ingredient generally refers to the nucleic acid vaccines or the polynucleotides contained therein, e.g., polynucleotides encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19, to be delivered as described herein.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., nonhuman mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated 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 birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD,
  • any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • the formulations described herein may contain at least one nucleic acid vaccine composition, e.g., nucleic acid vaccine for COVID-19, e.g., one mRNA vaccine for CO VID-19.
  • the formulations may contain 1, 2, 3, 4 or 5 nucleic acid vaccine compositions with different sequences, e.g., 1, 2, 3, 4 or 5 mRNA vaccine compositions with different sequences.
  • the formulation contains at least two nucleic acid vaccine (e.g., mRNA vaccine) compositions with different sequences.
  • the formulation contains at least three nucleic acid vaccine (e.g., mRNA vaccine) compositions with different sequences.
  • 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.
  • the nucleic acid vaccine compositions of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the nucleic acid vaccine composition); (4) alter the biodistribution (e.g., target the nucleic acid vaccine composition to specific tissues or cell types); (5) increase the translation of encoded protein in vivo, and/or (6) alter the release profile of encoded protein in vivo.
  • excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the nucleic acid vaccine composition); (4) alter the biodistribution (e.g., target the nucleic acid vaccine composition to specific tissues or cell types); (5) increase the translation of encoded protein in vivo, and/or (6) alter the release profile of encoded protein in vivo.
  • excipients of the present disclosure can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with nucleic acid vaccine compositions (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
  • the formulations of the present disclosure can include one or more excipients, each in an amount that together increases the stability of the nucleic acid vaccine compositions and/or increases cell transfection by the nucleic acid vaccine compositions.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated using selfassembled nucleic acid nanoparticles.
  • Pharmaceutically acceptable carriers, excipients, and delivery agents for nucleic acids that may be used in the formulation with the nucleic acid vaccine compositions of the present disclosure are disclosed in PCT Patent Application Publication WO 2013/090648, the contents of which are incorporated herein by reference in their entirety.
  • nucleic acid vaccine compositions of the disclosure can be formulated using one or more lipidoids.
  • nucleic acid vaccines include nucleic acid vaccines.
  • Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the nucleic acid vaccine compositions following the injection of a lipidoid formulation via localized and/or systemic routes of administration.
  • Lipidoid complexes containing nucleic acid vaccine compositions can be administered by various means including, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), or intracerebroventricular (ICV) administration.
  • nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are herein incorporated by reference in their entirety).
  • particle size Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are herein incorporated by reference in their entirety.
  • small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy.
  • Formulations with the different lipidoids including, but not limited to penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 2010, 401 :61 ; the contents of which are herein incorporated by reference in their entirety), Cl 2-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
  • TETA-5LAP penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride
  • Cl 2-200 including derivatives and variants
  • MD1 can be tested for in vivo activity.
  • the lipidoid referred to herein as “C 12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010, 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010, 669-670; the contents of both of which are herein incorporated herein by reference in their entirety.
  • the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to the nucleic acid vaccine compositions.
  • formulations with certain lipidoids include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 alkyl chain length).
  • formulations with certain lipidoids include, but are not limited to, Cl 2-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
  • nucleic acid vaccine compositions formulated with a lipidoid for systemic intravenous administration can result in the distribution of the formulation to be greater than 90% to the liver, (see, Akinc et al., Mol Ther.
  • an intravenous formulation using a C12-200 lipidoid may have a molar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipid to nucleic acid and a mean particle size of 80 nm may be effective to deliver nucleic acid vaccine compositions (see, Love et al., Proc Natl Acad Sci USA. 2010, 107: 1864-1869, the contents of which are herein incorporated by reference herein in their entirety).
  • an MD1 lipidoid-containing formulation may be used to effectively deliver nucleic acid vaccine compositions to hepatocytes in vivo.
  • the characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther.
  • lipidoid-formulated nucleic acid vaccine compositions to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly sizelimited.
  • Use of lipidoid formulations to deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and endothelium 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.
  • lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of nucleic acid vaccine compositions for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
  • the component molar ratio may include, but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011, 29: 1005-1010; the contents of which are herein incorporated by reference in their entirety).
  • the use of lipidoid formulations for the localized delivery of nucleic acids to cells via either subcutaneous or intramuscular delivery may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and nucleic acid vaccine compositions.
  • nucleic acid vaccine compositions of the disclosure can be formulated using one or more liposomes.
  • compositions of nucleic acid vaccine compositions include liposomes.
  • Liposomes are artificially prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, poly dispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • compositions comprising the nucleic acid vaccines described herein may include, without limitation, liposomes such as those formed from 1,2-di oleyloxy -A, A-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), SMARTICLES®/NOV340 (Marina Biotech, Bothell), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US Patent Application Publication US20100324120; the contents of which are herein incorporated by reference in their entirety), neutral DOPC (l,2-dioleoyl-sn-glycero-3- phosphocholine) based liposomes (e.
  • DODMA A-di
  • compositions comprising the nucleic acid vaccines described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (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.
  • liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (
  • the liposome formulations may be composed of 3 to 4 lipid components in addition to the nucleic acid vaccine compositions.
  • a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%
  • certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-A,A-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or
  • the nucleic acid-lipid particle may comprise a cationic lipid comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; a noncationic lipid comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and a conjugated lipid that inhibits aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle as described in W02009127060 to Maclachlan et al; the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid- lipid particle may be any nucleic acid-lipid particle disclosed in US2006008910 to Maclachlan et al.; the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid-lipid particle may comprise a cationic lipid of Formula I, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
  • the liposome may contain a sugar-modified lipid disclosed in US Pat. No.; US5595756 to Bally et al., the contents of which are incorporated herein by reference in their entirety.
  • the lipid may be a ganglioside and cerebroside in an amount of about 10 mol percent.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a liposome comprising a cationic lipid.
  • the liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phosphates in the nucleic acid vaccine compositions (N:P ratio) of between 1 : 1 and 20: 1 as described in PCT Patent Application Publication No. W02013006825, the contents of which are herein incorporated by reference in their entirety.
  • the liposome may have a N:P ratio of greater than 20: 1 or less than 1 : 1.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid-polycation complex.
  • the formation of the lipidpolycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, the contents of which are herein incorporated by reference in their entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in PCT Patent Application Publication No. WO2012013326; the contents of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size.
  • the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA.
  • the pharmaceutical compositions may be formulated with any amphoteric liposome disclosed in PCT Patent Application Publication No.: WO 2008043575 to Panzner and US Pat. No.: US 8,580,297 to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety.
  • the amphoteric liposome may comprise a mixture of lipids including a cationic amphiphile, an anionic amphiphile and optional one or more neutral amphiphiles.
  • the amphoteric liposome may comprise amphoteric compounds based on amphiphilic molecules, the head groups of which being substituted with one or more amphoteric groups.
  • the pharmaceutical compositions may be formulated with an amphoteric lipid comprising one or more amphoteric groups having an isoelectric point between 4 and 9, as disclosed in US Patent Application Publication No.: US20140227345 to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety.
  • the pharmaceutical composition may be formulated with liposomes comprising a sterol derivative as disclosed in US Pat. No.: US7312206 to Panzner et al. (Novosom), the contents of which are incorporated herein by reference in their entirety.
  • the pharmaceutical composition may be formulated with amphoteric liposomes comprising at least one amphipathic cationic lipid, at least one amphipathic anionic lipid, and at least one neutral lipid, or liposomes comprise at least one amphipathic lipid with 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 US Pat. No.
  • the pharmaceutical composition may be formulated with liposomes comprising a serum-stable mixture of lipids taught in US Patent Application Publication No.: US 20110076322 to Panzner et al, the contents of which are incorporated herein by reference in their entirety, capable of encapsulating the nucleic acid vaccine compositions of the present disclosure.
  • the lipid mixture comprises phosphatidylcholine and phosphatidylethanolamine in a ratio in the range of about 0.5 to about 8.
  • the lipid mixture may also include pH sensitive anionic and cationic amphiphiles, such that the mixture is amphoteric, being negatively charged or neutral at pH 7.4 and positively charged at pH 4.
  • the drug/lipid ratio may be adjusted to target the liposomes to particular organs or other sites in the body.
  • liposomes loaded with the nucleic acid vaccine compositions of the present disclosure as cargo are prepared by the method disclosed in US Patent Application Publication No.: US 20120021042 to Panzner et al., the contents of which are incorporated herein by reference in their entirety.
  • the method comprises steps of admixing an aqueous solution of a polyanionic active agent and an alcoholic solution of one or more amphiphiles and buffering said admixture to an acidic pH, wherein the one or more amphiphiles are susceptible of forming amphoteric liposomes at the acidic pH, thereby to form amphoteric liposomes in suspension encapsulating the active agent.
  • nucleic acid vaccine compositions of the disclosure can be formulated using one or more lipoplexes.
  • the nucleic acid vaccine compositions may be formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and nontargeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008, 68:9788-9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and nontargeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008, 68:9788
  • LNPs Lipid Nanoparticles
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid nanoparticle (LNP).
  • LNPs can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space.
  • LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.
  • LNPs may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.
  • LNPs useful herein are known in the art and generally comprise cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from nonspecific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP-forming composition.
  • cholesterol saids in stability and promotes membrane fusion
  • a phospholipid which provides structure to the LNP bilayer and also may aid in endosomal escape
  • PEG polyethylene glycol
  • ionizable lipid complexes negatively charged RNA and enhances endosomal escape
  • the components of the LNP may be selected based on the desired target, tropism, cargo, size, or other desired feature or property.
  • the LNP may be the lipid nanoparticles described in PCT Patent Application Publication No. W02012170930, the contents of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a LNP that comprises at least one cationic lipid.
  • the cationic lipid which may be used in formulations of the present disclosure may be selected from, but not limited to, a cationic lipid described in PCT Patent Application Publication Nos. W02012040184, WO2011153120, WO201 1149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865 and W02008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No.
  • the cationic lipid may be also selected from, but not limited to, formula A described in PCT Patent Application Publication Nos. W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO201 1022460, WO2012061259, WO2012054365 and WO2012044638; the contents of each of which are herein incorporated by reference in their entirety.
  • the cationic lipid may be selected from, but not limited to, formula CLLCLXXIX of PCT Patent Application No. W02008103276, formula CLLCLXXIX of US Patent No.
  • the cationic lipid may be a multivalent cationic lipid such as the cationic lipid disclosed in US 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 including two quaternary amine groups and a hydrophobic portion including four hydrocarbon chains as described in US Patent No. 7,223,887 to Gaucheron et al..
  • the cationic lipid may be biodegradable as the biodegradable lipids disclosed in US Patent Application Publication No.: US20130195920 to Maier et al., the contents of which are incorporated herein by reference in their entirety.
  • the cationic lipid may have one or more biodegradable groups located in a lipidic moiety of the cationic lipid as described in formula I-IV in US20130195920 to Maier et al..
  • the cationic lipid may also be the cationic lipids disclosed in US20130156845 to Manoharan et al.
  • the cationic lipid may be selected from (20Z,23Z)- N,N-dimethylnonacosa-20,23 -dien- 10-amine, ( 17Z,20Z)-N,N-dimemylhexacosa- 17,20- dien-9-amine, (lZ,19Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)-N,N- dimethyldocosa- 13,16-dien-5-amine, (12Z, 15Z)-N,N-dimethylhenicosa- 12, 15-dien-4- amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N- dimethyltetracosa- 15,18-dien-7-amine, ( 18Z,2 lZ)-N,N-dimethylheptacosa
  • a lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, at least one additional LNP functional component, or any combination thereof.
  • a lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component.
  • the LNP may be comprised of at least one cationic lipid, at least one noncationic lipid, and at least one sterol.
  • the LNP may be comprised of at least one cationic lipid, at least one non-cationic lipid, and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one non-cationic lipid. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one sterol. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one additional LNP functional component.
  • the LNP may be comprised of at least one non-cationic lipid and at least one sterol. In some embodiments, the LNP may be comprised of at least one non-cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one sterol and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one cationic lipid. In some embodiments, the LNP may be comprised of at least one noncationic lipid. In some embodiments, a LNP may be comprised of a sterol. In some embodiments, the LNP may be comprised of a additional LNP functional component.
  • the at least one cationic lipid may comprise any of at least one ionizable cationic lipid, at least one amino lipid, at least one saturated cationic lipid, at least one unsaturated cationic lipid, at least one zwitterionic lipid, at least one multivalent cationic lipid, or any combination thereof.
  • the LNP may be essentially devoid of the at least one cationic lipid. In some embodiments, the LNP may contain no amount of the at least one cationic lipid.
  • At least one cationic lipid may be selected from, but not limited to, at least one of l,3-Bis-(l,2-bis-tetradecyloxy-propyl-3- dimethylethoxyammoniumbromide)-propan-2-ol ((R)-PLC-2), 2-(Dinonylamino)ethan-l- ol (17-10), 2-(Didodecylamino)ethan-l-ol (17-11), 3-(Didodecylamino)propan-l-ol (17- 12), 4-(Didodecylamino)butan-l-ol (17-13), 2-(Hexyl((9Z,12Z)-octadeca-9,l 2-dien- 1- yl)amino)ethan- 1 -ol (17-2), 2-(Nonyl((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)
  • the at least one non-cationic lipid comprises at least one phospholipid, at least one fusogenic lipid, at least one anionic lipid, at least one helper lipid, at least one neutral lipid, or any combination thereof.
  • the LNP may be essentially devoid of the at least one non-cationic lipid. In some embodiments, the LNP may contain no amount of the at least one non-cationic lipid.
  • At least one non-cationic lipid may be selected from, but is not limited to, at least one of l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), DSPCbutwith3unsaturateddoublebondspertail (18:3 PC), Acylcamosine (AC), l-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), N-oleoyl-SPM (C 18 :1), N-lignocerylSPM (C24:0), N-nervacylC (C24:l), carbamoyl]cholesterol (Cet-P), cholesterolhemisuccinate (CHEMS), cholesterol (Choi), Cholesterolhemidodecanedicarboxylicacid (Chol-C12), 12- Cholesteryloxycarbonylaminododecanoicacid (Chol-C 13
  • the LNP comprises at least one of the cationic lipids described in International Patent Publication No. WO2018118102 (the contents of which are herein incorporated reference in its entirety) such as, but not limited to ATX-2, ATX- 43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, and ATX-88.
  • Methods of making these cationic lipids are also provided in International Patent Publication No. WO2018118102 and the contents of which are herein incorporated by reference in its entirety.
  • the cationic lipid in the LNP is ATX-88.
  • the LNP comprises about 20-60% of ATX-2, ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, or ATX-88, about 5-25% of a noncationic lipid, about 25-55% of sterol and about 0.5-15% of a PEG-modified lipid.
  • the LNP comprises at least one of the cationic lipids described in International Patent Publication No. WO2018119163 (the contents of which are herein incorporated reference in its entirety) such as, but not limited to ATX-0002, ATX-0043, ATX-0057, ATX-0061, ATX-0063, ATX-0058, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0086, ATX-0087, ATX-0088, ATX-0091, ATX-0092, ATX-0093, ATX-0094, ATX-0095, ATX-0096, ATX-0097, ATX-0098, ATX-0100, ATX-0101, ATX-0102, ATX-0106, ATX-0107, ATX-0108, ATX-0109, ATX-0110, ATX-0111, ATX-0114, ATX-0115, ATX-0117, ATX-0118
  • cationic lipids are also provided in International Patent Publication No. WO2018119163 and the contents of which are herein incorporated by reference in its entirety.
  • the cationic lipid in the LNP is ATX-88.
  • the LNP comprises about 20-60% of ATX-0002, ATX-0043, ATX-0057, ATX-0061, ATX-0063, ATX-0058, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0086, ATX-0087, ATX-0088, ATX-0091, ATX-0092, ATX-0093, ATX-0094, ATX-0095, ATX-0096, ATX-0097, ATX-0098, ATX-0100, ATX-0101, ATX-0102, ATX-0106, ATX-0107, ATX-0108, ATX-0109, ATX-0110, ATX-0111, ATX-0114, ATX-0115, ATX-0117, ATX-0118, ATX-0121, ATX-0122, ATX-0123, ATX-0124, ATX-0125, ATX-0126, ATX-0129, ATX-0132, or ATX
  • the LNP comprises at least one of the cationic lipids described in International Patent Publication Nos. W02012019168. W02012045082.
  • the LNP comprises about 20-60% of a cationic lipid, about 5-25% of a non-cationic lipid, about 25-55% of sterol and about 0.5-15% of a PEG-modified lipid.
  • the LNP comprises an ionizable lipid or lipid-like material.
  • the ionizable lipid may be C12-200, CKK-E12, 5A2- SC8, BAMEA-016B, or 7C1.
  • Other ionizable lipids are known in the art and are useful herein.
  • the LNP comprises a phospholipid.
  • the phospholipid helper
  • the LNP comprises a PEG derivative.
  • the PEG derivative may be a lipid-anchored such as PEG is C14-PEG2000, C14- PEG1000, C14-PEG3000, C14-PEG5000, C12-PEG1000, C12-PEG2000, C12-PEG3000, C12-PEG5000, C16-PEG1000, C16-PEG2000, C16-PEG3000, C16-PEG5000, C18- PEG1000, C18-PEG2000, C18-PEG3000, or C18-PEG5000.
  • PEG is C14-PEG2000, C14- PEG1000, C14-PEG3000, C14-PEG5000, C12-PEG1000, C12-PEG2000, C12-PEG3000, C12-PEG5000, C16-PEG1000, C16-PEG2000, C16-PEG3000, C16-PEG5000, C18- PEG1000, C18-PEG2000, C18-PEG3000, or C18-PEG
  • the at least one sterol comprises at least one cholesterol or cholesterol derivative.
  • the LNP may be essentially devoid of an at least one sterol. In some embodiments, the LNP may contain no amount of the at least one sterol.
  • the at least one additional LNP functional component comprises at least one component that reduced aggregation of particles, at least one component that decreases clearing of the LNP from circulation in a subject, at least component that increases the LNP’s ability to traverse mucus layers, at least one component that decreases a subjects immune response to administration of the LNP, at least one component that modifies membrane fluidity of the LNP, at least one component that contributes to the stability of the LNP, or any combination thereof.
  • the LNP may be essentially devoid of the at least one additional LNP functional component.
  • the LNP may contain no amount of the at least one additional LNP functional component.
  • the additional LNP functional component may be comprised of a polymer.
  • the polymer comprising the additional LNP functional component may be comprised of at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.
  • the average molecular weight of the polymer moiety may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 0,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000, 9,000 to 20,000, 9,500 to0,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to0,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to0,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000
  • the polymer e.g., PEG
  • the lipid conjugated to the polymer comprised of at least one neutral lipid, at least one phospholipid, at least one anionic lipid, at least one cationic lipid, at least one cholesterol, at least one cholesterol derivative, or any combination thereof.
  • the lipid conjugated to the polymer may be selected from, but is not limited to, at least one of the cationic, non-cationic, or sterol lipids listed previously.
  • the at least one PEG-lipid conjugate may be selected from, but is not limited to at least one of Siglec-IL-PEG-DSPE, R)-2,3- bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S- DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG Cl 4, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k
  • the amounts and ratios of LNP components may be varied by any amount dependent on the desired form, structure, function, cargo, target, or any combination thereof.
  • the amount of each component may be expressed in various embodiments as percent of the total molar mass of all lipid or lipid conjugated components accounted for by the indicated component (mol%),
  • the amount of each component may be expressed in various embodiments as the relative ratio of each component based on molar mass (Molar Ratio).
  • the amount of each component may be expressed in various embodiments as the weight of each component used to formulate the LNP prior to fabrication (mg or equivalent).
  • the amount of each component may be expressed in various embodiments by any other method known in the art.
  • any formulation given in one representation of component amounts (“units”) is expressly meant to encompass any formulation expressed in different units of component amounts, wherein those representations are effectively equivalent when converted into the same units.
  • “effectively equivalent” means two or more values within about 10% of one another.
  • the LNP comprises at least one cationic lipid in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 20 to 60 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 50 to 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of more than about 60 mol% or about 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 95 mol% or less.
  • the LNP comprises a cationic lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the LNP comprises at least one cationic lipid in an amount from about 20 to 30 mol%, 20 to 35 mol%, 20 to 40 mol%, 20 to 45 mol%, 20 to 50 mol%, 20 to 55 mol%, 20 to 60 mol%, 20 to 65 mol%, 20 to 70 mol%, 20 to 75 mol%, 20 to 80 mol%, 20 to 85 mol%, 20 to 90 mol%, 25 to 35 mol%, 25 to 40 mol%, 25 to 45 mol%, 25 to 50 mol%, 25 to 55 mol%, 25 to 60 mol%, 25 to 65 mol%, 25 to 70 mol%, 25 to 75 mol%, 25 to 80 mol%, 25 to 85 mol%, 25 to 90 mol%, 30 to 40 mol%, 30 to 45 mol%, 30 to 50 mol%, 30 to 55 mol%, 30 to 60 mol%, 30 to 65 mol%, 30 to 70 mol%, 30 to 75 mol%, 30 to 40 mol%
  • the LNP comprises at least one non-cationic lipid in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one non-one cationic lipid in an amount of about 5 to 35 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 5 to 25 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of less than about 5 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of more than about 25 mol% or about 35 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 95 mol% or less.
  • the LNP comprises at least one non-cationic lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the LNP comprises at least one non-cationic lipid in an amount from about 5 to 15 mol%, 5 to 25 mol%, 5 to 35 mol%, 5 to 45 mol%, 5 to 55 mol%, 10 to 20 mol%, 10 to 30 mol%, 10 to 40 mol%, 10 to 50 mol%, 15 to 25 mol%, 15 to 35 mol%, 15 to 45 mol%, 20 to 30 mol%, 20 to 40 mol%, 20 to 50 mol%, 25 to 35 mol%, 25 to 45 mol%, 30 to 40 mol%, 30 to 50 mol%, and 35 to 45 mol%.
  • the LNP comprises at least one sterol in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 20 to 45 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 25 to 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of more than about 45 mol% or about 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 95 mol% or less.
  • the LNP comprises at least one sterol in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the LNP comprises at least one sterol in an amount from about 10 to 20 mol%, 10 to 30 mol%, 10 to 40 mol%, 10 to 50 mol%, 10 to 60 mol%, 15 to 25 mol%, 15 to 35 mol%, 15 to 45 mol%, 15 to 55 mol%, 15 to 65 mol%, 20 to 30 mol%, 20 to 40 mol%, 20 to 50 mol%, 20 to 60 mol%, 25 to 35 mol%, 25 to 45 mol%, 25 to 55 mol%, 25 to 65 mol%, 30 to 40 mol%, 30 to 50 mol%, 30 to 60 mol%, 35 to 45 mol%, 35 to 55 mol%, 35 to 65 mol%, 40 to 50 mol%, 40 to 60 mol%, 45 to 55 mol%, 45 to 65 mol%, 50 to 60 mol%, and 55 to 65 mol%.
  • the LNP comprises at least one additional LNP functional component in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 0.5 to 15 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 15 to 40 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of less than about 0.1 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of more than about 15 mol% or about 40 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 95 mol% or less.
  • the LNP comprises at least one additional LNP functional component in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
  • the LNP comprises at least one additional LNP functional component in an amount from about 0.1 to 1 mol%, 0.1 to 2 mol%, 0.1 to 3 mol%, 0.1 to 4 mol%, 0.1 to 5 mol%, 0.1 to 6 mol%, 0.1 to 7 mol%, 0.1 to 8 mol%, 0.1 to
  • the LNP is comprised of about 30-60 mol% of at least one cationic lipid, about 0-30 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one sterol (e.g., cholesterol), and about 0- 10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 35-55 mol% of at least one cationic lipid, about 5-25 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mol% of at least one sterol (e.g., cholesterol), and about 0-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • the LNP is comprised of about 35-45 mol% of at least one cationic lipid, about 25-35 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 20-30 mol% of at least one sterol (e.g., cholesterol), and about 0-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 45-65 mol% of at least one cationic lipid, about 5-10 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 25-40 mol% of at least one sterol (e.g., cholesterol), and about 0.5-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 40-60 mol% of at least one cationic lipid, about 5-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 35-45 mol% of at least one sterol (e.g., cholesterol), and about 0.5-3 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 30-60 mol% of at least one cationic lipid, about 0-30 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 15-50 mol% of at least one sterol (e.g., cholesterol), and about 0.01- 10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 10-75 mol% of at least one cationic lipid, about 0.5-50 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 5-60 mol% of at least one sterol (e.g., cholesterol), and about 0.1-20 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 50-65 mol% of at least one cationic lipid, about 3-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mol% of at least one sterol (e.g., cholesterol), and about 0.5-2 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 50-85 mol% of at least one cationic lipid, about 3-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mol% of at least one sterol (e.g., cholesterol), and about 0.5-2 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 25-75 mol% of at least one cationic lipid, about 0.1-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 5-50 mol% of at least one sterol (e.g., cholesterol), and about 0.5-20 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 50-65 mol% of at least one cationic lipid, about 5-10 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 25-35 mol% of at least one sterol (e.g., cholesterol), and about 5-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the LNP is comprised of about 20-60 mol% of at least one cationic lipid, about 5-25 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 25-55 mol% of at least one sterol (e.g., cholesterol), and about 0.5-15 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
  • a non-cationic lipid e.g., a phospholipid
  • sterol e.g., cholesterol
  • additional LNP functional component e.g., a PEGylated lipid
  • the nucleic acid vaccine described here is formulated in a lipid nanoparticle comprising a cationic lipid, a phospholipid, a sterol and a PEGylated lipid as described herein.
  • the LNP may comprise about 20-70% (mole) of a cationic lipid in the formulation , e.g., about 30-60%, or about 30-50%, or 50%.
  • the LNP may comprise about 5-20% (mole) of a phospholipid in the formulation, e.g., 5-10%, 5-15%, 10-15% or about 10% of the phospholipid.
  • the LNP may comprise about 20-45% (mole) of a sterol in the formulation, e.g., 20-40%, 30-40%, or about 38% of the sterol.
  • the LNP may comprise about 0.5 to 5.0% of a PEGylated lipid, e.g., about 0.5-2.0%, 1.0-3.0%, 1.5-2.0% or about 2.0% of the PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, a phospholipid, a sterol and a PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, DSPC, a sterol and a PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, a phospholipid, a sterol and PEG-DMA.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, a phospholipid, cholesterol and PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, DSPC, a sterol and a PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, a phospholipid, cholesterol and a PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, a phospholipid, a sterol and PEG- DMA.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, a phospholipid, cholesterol and PEG-DMA.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, DSPC, a sterol and PEG- DMA.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, DSPC, cholesterol and a PEGylated lipid.
  • the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, DSPC, cholesterol and PEG- DMA.
  • the nucleic acid vaccine described here is formulated in a lipid nanoparticle comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 1.6% of a PEGylated lipid in the formulation.
  • the cationic lipid, sterol, phospholipid and PEGylated lipid is 3D-P-DMA, DSPC, Cholesterol and PEG-DMA, respectively.
  • the nucleic acid vaccine described here is formulated in a lipid nanoparticle comprising about 53% (by weight) of 3D-P-DMA, about 14% (by weight of DSPC, about 26% (by weight) of cholesterol and about 7% (by weight) of a PEG-DMA in the formulation.
  • the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 27 that is formulated in a LNP comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 2.0 % of a PEGylated lipid.
  • the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 27 that is formulated in a LNP comprising about 50% (by mole) of 3D-P-DMA, about 10% (by mole) of DSPC, about 38% (by mole) of cholesterol and about 2.0 % of a PEG-DMA.
  • the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 43 that is formulated in a LNP comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 2.0 % of a PEGylated lipid.
  • the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 43 that is formulated in a LNP comprising about 50% (by mole) of 3D-P-DMA, about 10% (by mole) of DSPC, about 38% (by mole) of cholesterol and about 2.0 % of a PEG-DMA.
  • the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 39 or SEQ ID NO: 41 that is formulated in a LNP comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 2.0 % of a PEGylated lipid.
  • the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 39 or SEQ ID NO: 41 that is formulated in a LNP comprising about 50% (by mole) of 3D-P-DMA, about 10% (by mole) of DSPC, about 38% (by mole) of cholesterol and about 2.0 % of a PEG-DMA.
  • the LNPs can be characterized by their shape.
  • the LNPs are essentially spherical.
  • the LNPs are essentially rod-shaped (i.e., cylindrical).
  • the LNPs are essentially disk shaped.
  • the LNPs can be characterized by their size.
  • the size of an LNP can be defined as the diameter of its largest circular cross section, referred to herein simply as its diameter.
  • the LNPs may have a diameter between 30 nm to about 150 nm.
  • the LNP may have diameters ranging between about 40 to 150 nm 50 to 150 nm, 60 to 150 nm, about 70 to 150 nm, or 80 to 150 nm, 90 to 150 nm, 100 to nm, 110 to 150 nm, 120 to 150 nm, 130 to 150 nm, or 140 to 150 nm.
  • a population of LNPs such as those resulting from the same formulation, may be characterized by measuring the uniformity of size, shape, or mass of the particles in the population, uniformity may be expressed in some embodiments as the poly dispersity index (PI) of the population. In some embodiments uniformity may be expressed in some embodiments as the disparity (D) of the population.
  • PI poly dispersity index
  • D disparity
  • polydispersity index and “disparity” are understood herein to be equivalent and may be used interchangeably.
  • a population of LNPs resulting from a given formulation will have a PI of between about 0.1 and 1.
  • a population of LNPs resulting from a giving formulation will have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, a population of LNPs resulting from a given formulation will have a PI of between about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1.
  • the LNP may fully or partially encapsulate a cargo, such as nucleic acid constructs of the present disclosure.
  • a cargo such as nucleic acid constructs of the present disclosure.
  • essentially 0% of the cargo present in the final formulation is exposed to the environment outside of the LNP (i.e., the cargo is fully encapsulated.
  • the cargo is associated with the LNP but is at least partially exposed to the environment outside of the LNP.
  • the LNP may be characterized by the% of the cargo not exposed to the environment outside of the LNP, e.g., the encapsulation efficiency.
  • an encapsulation efficiency of about 100% refers to an LNP formulation where essentially all the cargo is fully encapsulated by the LNP, while an encapsulation rate of about 0% refers to an LNP where essential none of the cargo is encapsulated in the LNP, such as with an LNP where the cargo is bound to the external surface of the LNP.
  • an LNP may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%.
  • an LNP may have an encapsulation efficiency of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%,
  • the at least one targeting agent may be presented on the external surface of the nanoparticle. In some embodiments, the at least one targeting agent may be conjugated to a lipid-component of the nanoparticle. In some embodiments, the at least one targeting agent may be conjugated to a polymer component of the nanoparticle. In some embodiments, the at least one targeting agent may be anchored to the nanoparticle via hydrophobic ad hydrophilic interactions among the at least one targeting agent, the nanoparticle membrane, and the aqueous environments inside or outside the nanoparticle. In some embodiments, the at least one targeting agent is conjugated to a peptide/protein component of the nanoparticle membrane. In some embodiments, the at least one targeting agent is conjugated to a suitable linker moiety which is conjugated to a component of the nanoparticle membrane. In some embodiments, any combination of forces and bonds can result in the targeting agent being associated with the nanoparticle.
  • the LNPs described herein may be formed using techniques known in the art.
  • an organic solution containing the lipids is mixed together with an acidic aqueous solution containing the nucleic acid compositions in a microfluidic channel resulting in the formation of targeting system (delivery vehicle and the nucleic acid vaccine).
  • the lipid compositions are described according to the respective molar ratios of the component lipids in the formulation.
  • the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol- %.
  • the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%.
  • the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%.
  • the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%.
  • the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol- %.
  • the ionizable lipid mol-% of the transfer vehicle batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol-%.
  • transfer vehicle variability between lots will be less than 15%, less than 10% or less than 5%.
  • the mol-% of the helper lipid may be from about 1 mol- % to about 50 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 5 mol-% to about 30 mol-%.
  • the mol-% of the helper lipid may be from about 10 mol-% to about 20 mol- %. In some embodiments, the helper lipid mol-% of the transfer vehicle batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol-%. [0372] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%.
  • the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%. In some embodiments, the structural lipid mol-% of the transfer vehicle batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol-%.
  • the mol-% of the PEG modified lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be about 1.5 mol-%.
  • the PEG modified lipid mol-% of the transfer vehicle batch will be ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 2.5% of the target mol-%.
  • a lipid nanoparticle formulation may be prepared by the methods described in International Publication Nos. WO2011127255 or W02008103276, the contents of each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, the contents of which is herein incorporated by reference in its entirety.
  • a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. WO2020237227, the contents of each of which is herein incorporated by reference in their entirety.
  • lipid nanoparticle formulations may be as described in International Publication No. WO2020237227, the contents of which is herein incorporated by reference in its entirety.
  • the lipid may be a cleavable lipid such as those described in PCT Patent Application Publication No. WO2012170889, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles described herein may comprise at least one cationic polymer described herein and/or known in the art.
  • the cationic lipid may be synthesized by methods known in the art and/or as described in PCT Patent Application Publication Nos.
  • the pharmaceutical compositions of the nucleic acid vaccine compositions may include at least one of the PEGylated lipids described in PCT Patent Application Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.
  • the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
  • LNP formulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol.
  • the LNP formulations of the nucleic acid vaccine compositions may contain PEG-c-DOMG at 3% lipid molar ratio.
  • the LNP formulations of the nucleic acid vaccine compositions may contain PEG-c-DOMG at 1.5% lipid molar ratio.
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
  • PEG-DSG 1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol
  • PEG-DPG 1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene 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, C 12-200 and DLin-KC2-DMA.
  • the LNP formulation may contain PEG-DMG 2000 (1,2- dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(poly ethylene glycol)-2000), a cationic lipid known in the art.
  • the LNP formulation may contain PEG-DMG 2000and at least one other component.
  • the LNP formulation may contain PEG-DMG 2000, DSPC and cholesterol.
  • the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.
  • the LNP formulation may contain PEG- DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS, 2012, 109(36): 14604-14609; herein incorporated by reference in its entirety).
  • nucleic acid vaccine compositions described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Patent Application Publication No. US20120207845; the contents of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a plurality of cationic lipids, such as a first and a second cationic lipid as described in US 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 can be selected on the basis of a first property and the second cationic lipid can be selected on the basis of a second property, where the properties may be determined as outlined in US20130017223.
  • the first and second properties are complementary.
  • nucleic acid vaccine compositions described herein may be formulated with a lipid particle comprising one or more cationic lipids and one or more second lipids, and one or more nucleic acids, wherein the lipid particle comprises a solid core, as described in US Patent Publication No. US20120276209 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be complexed with a cationic amphiphile in an oil-in-water (o/w) emulsion such as described in European Publication No.: EP2298358 to Satishchandran et al., the contents of which are incorporated herein by reference in their entirety.
  • the cationic amphiphile may be a cationic lipid, modified or unmodified spermine, bupivacaine, or benzalkonium chloride and the oil may be a vegetable or an animal oil.
  • nucleic acid-cationic amphiphile complex is in the oil phase of the oil-in-water emulsion (see e.g., the complex described in. EP2298358 to Satishchandran et al.), the contents of which are incorporated herein by reference in its entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a composition comprising a mixture of cationic compounds and neutral lipids.
  • the cationic compounds may be 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
  • the neutral lipid may be selected from the group consisting of diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide and sphingomyelin.
  • the lipid formulation may comprise a cationic lipid of formula A, a neutral lipid, a sterol and a PEG or PEG-modified lipid disclosed in US Patent Publication No.: US 20120101148 to Akinc et al., the contents of which are incorporated herein by reference in their entirety.
  • the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or W02008103276.
  • the nucleic acid vaccine compositions of the present disclosure may be encapsulated in any of the lipid nanoparticle (LNP) formulations described in WO201 1127255 and/or W02008103276; the contents of each of which are herein incorporated by reference in their entirety.
  • LNP lipid nanoparticle
  • the LNP formulations described herein may comprise a polycationic composition.
  • the polycationic composition may be selected from formula 1-60 of US Patent Publication No. US20050222064; the contents of which are herein incorporated by reference in their entirety.
  • the LNP formulations comprising a polycationic composition may be used for the delivery of the nucleic acid vaccine compositions described herein in vivo and/or in vitro.
  • the LNP formulations described herein may additionally comprise a permeability enhancer molecule.
  • a permeability enhancer molecule are described in US Patent Publication No. US20050222064; the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid vaccine composition (e.g., a nucleic acid vaccine for COVID-19).
  • the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., PCT Patent Application Publication No. W02012109121; the contents of which are herein incorporated by reference in their entirety).
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • 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 a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain.
  • the internal ester linkage may replace any carbon in the lipid chain.
  • the nucleic acid vaccine compositions is formulated as a solid lipid nanoparticle.
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm.
  • SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8): 1696-1702; the contents of which are herein incorporated by reference in their entirety).
  • formulations comprising the nucleic acid vaccine compositions described herein may also be constructed or altered such that they passively or actively are directed to 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.
  • One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3 -DMA-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010, 18: 1357-1364; the contents of which are herein incorporated by reference in their entirety).
  • Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011, 8: 197-206; Musacchio and T orchilin, 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.
  • the nucleic acid vaccine compositions of the present disclosure can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a therapeutic outcome.
  • the nucleic acid vaccine compositions may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • encapsulate means to enclose, surround, or encase. As it relates to the formulation of the compositions of the disclosure, encapsulation may be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulated” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition of the disclosure using fluorescence and/or electron micrograph. 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 disclosure are encapsulated in the delivery agent.
  • the nucleic acid vaccine compositions may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EV Ac), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc., Deerfield, IL), PEG- based sealants, and COSEAL® (Baxter International, Inc., Deerfield, IL).
  • the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject.
  • the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
  • the formulations comprising the 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 ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
  • the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable 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.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a targeting lipid with a targeting moiety such as the targeting moieties disclosed in US Patent Application Publication No.: US20130202652 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety of formula I of US 20130202652 to Manoharan et al. may be selected in order to favor the lipid being localized with a desired organ, tissue, cell, cell type or subtype, or organelle.
  • Non-limiting targeting moieties that are contemplated in the present disclosure include transferrin, anisamide, an RGD peptide, prostate specific membrane antigen (PSMA), fucose, an antibody, or an aptamer.
  • the nucleic acid vaccine compositions of the present disclosure may be encapsulated in a therapeutic nanoparticle.
  • Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, PCT Patent Application Publication Nos. W02010005740, W02010030763, W02010005721, W02010005723, and WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286 and US20120288541 and US Pat. No.
  • Therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which are herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle may be formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the nucleic acid vaccine compositions of the present disclosure (see PCT Patent Application Publication No. W02010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, the contents of each of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticles may be formulated to be target specific.
  • the therapeutic nanoparticles may include a corticosteroid (see PCT Patent Application Publication No. WO2011084518; the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticles may be formulated to be cancer specific.
  • the therapeutic nanoparticles may be formulated in nanoparticles described in PCT Patent Application Publication No. WO2008121949, W02010005726, W02010005725, and WO2011084521, and US Pub No. US20100069426, US20120004293 and US20100104655, the contents of each of which are herein incorporated by reference in their entirety.
  • the nanoparticles of the present disclosure may comprise a polymeric matrix.
  • the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, poly acrylates, polymethacrylates, poly cyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the therapeutic nanoparticle comprises a diblock copolymer.
  • the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates
  • the therapeutic nanoparticle comprises a PLGA- PEG block copolymer (see US Pub. No. US20120004293 and US Pat. No. 8,236,330, each of which is herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat. No 8,246,968 and PCT Patent Application Publication No. WO2012166923, the contents of each of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle may comprise a multiblock copolymer such as, but not limited to the multiblock copolymers described in U.S. Pat. Nos. 8,263,665 and 8,287,910; the contents of each of which are herein incorporated by reference in their entirety.
  • the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer.
  • a polyion complex comprising a non-polymeric micelle and the block copolymer.
  • the nanoparticles for delivery of the nucleic acid vaccines described herein include block co-polymers.
  • block copolymers include those of formula I, formula II, formula III, formula IV, formula V, formula VI and formula VII of PCT Patent Application Publication No. W02015017519, the contents of which are herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle 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 methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No. 8,287,849; the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
  • amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No. 8,287,849; the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable 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.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the therapeutic nanoparticle may include a conjugation of at least one targeting ligand.
  • the targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006, 66:6732-6740; the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see PCT Patent Application Publication No. WO2011084513 and US Pub No. US20110294717, the contents of each of which are herein incorporated by reference in their entirety).
  • the nucleic acid vaccine compositions may be encapsulated in, linked to and/or associated with synthetic nanocarriers.
  • Synthetic nanocarriers include, but are not limited to, those described in PCT Patent Application Publication Nos. W02010005740, W02010030763, W0201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, W02012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US Pub. Nos.
  • the synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in PCT Patent Application Publication Nos. W02010005740, W02010030763 and W0201213501and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US2012024422, the contents of each of which are herein incorporated by reference in their entirety.
  • the synthetic nanocarrier formulations may be lyophilized by methods described in PCT Patent Application Publication Pub. No. WO2011072218 and US Pat. No. 8,211,473; the contents of each of which are herein incorporated by reference in their entirety.
  • the synthetic nanocarriers may contain reactive groups to release the nucleic acid vaccine compositions described herein (see PCT Patent Application Publication No. WO20120952552 and US Pub No. US20120171229, the contents of each of which are herein incorporated by reference in their entirety).
  • the synthetic nanocarriers may be formulated for targeted release.
  • the synthetic nanocarrier may be formulated to release the nucleic acid vaccine compositions at a specified pH and/or after a desired time interval.
  • the synthetic nanoparticle may be formulated to release the nucleic acid vaccine compositions after 24 hours and/or at a pH of 4.5 (see PCT Patent Application Publication Nos. W02010138193 and W02010138194 and US Pub Nos. US20110020388 and US20110027217, the contents of each of which are herein incorporated by reference in their entireties).
  • the synthetic nanocarriers may be formulated for controlled and/or sustained release of the nucleic acid vaccine compositions described herein.
  • the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in PCT Patent Application Publication No. W02010138192 and US Pub No. US20100303850, the contents each of which are herein incorporated by reference in their entirety.
  • the nanoparticle may be optimized for oral administration.
  • the nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof.
  • the nanoparticle may be formulated by the methods described in U.S. Pub. No. US20120282343; the contents of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a modular composition such as described in US Pat. No. US 8,575,123 to Manoharan et al., the contents of which are herein incorporated by reference in their entirety.
  • the modular composition may comprise a nucleic acid, e.g., the nucleic acid vaccine compositions of the present disclosure, at least one endosomolytic component, and at least one targeting ligand.
  • the modular composition may have a formula such as any formula described in US 8,575,123 to Manoharan et al..
  • the nucleic acid vaccine compositions of the present disclosure may be encapsulated in the lipid formulation to form a stable nucleic acid-lipid particle (SNALP) such as described in US Pat. No. US8,546,554 to de Fougerolles et al., the contents of which are incorporated here by reference in their entirety.
  • SNALP stable nucleic acid-lipid particle
  • the lipid may be cationic or non-cationic.
  • the lipid to nucleic acid ratio (mass/mass ratio) (e.g., lipid to nucleic acid vaccine compositions ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from 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.
  • the SNALP includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (Lipid A), 10% di oleoylphosphatidylcholine (DSPC), 40% cholesterol, 10% polyethylene glycol (PEG)-C- DOMG (mole percent) with a particle size of 63.0 ⁇ 20 nm and a 0.027 nucleic acid/lipid ratio.
  • Lipid A 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DSPC di oleoylphosphatidylcholine
  • cholesterol 10% polyethylene glycol (PEG)-C- DOMG (mole percent) with a particle size of 63.0 ⁇ 20 nm and a 0.027 nucleic acid/lipid ratio.
  • nucleic acid vaccine compositions of the present disclosure may be formulated with a nucleic acid-lipid particle comprising an endosomal membrane destabilizer as disclosed in US Pat. No. US 7,189,705 to Lam et al., the contents of which are incorporated herein by reference in their entirety.
  • the endosomal membrane destabilizer may be a Ca 2+ ion.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with formulated lipid particles (FLiPs) disclosed in US Pat. No. US 8,148,344 to Akinc et al., the contents of which are herein incorporated by reference in their entirety.
  • FLiPs may comprise at least one of a single or double-stranded oligonucleotide, where the oligonucleotide has been conjugated to a lipophile and at least one of an emulsion or liposome to which the conjugated oligonucleotide has been aggregated, admixed or associated.
  • the nucleic acid vaccine compositions of the present disclosure may be delivered to a cell using a composition comprising an expression vector in a lipid formulation as described in US Pat. No. US 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 comprising first and second inverted repeated sequences from an adeno associated virus (AAV), a rep gene from AAV, and a nucleic acid fragment.
  • AAV adeno associated virus
  • the expression vector in Tam is complexed with lipids.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a lipid formulation disclosed in US Pub. No. US 20120270921 to de Fougerolles et al., the contents of which are incorporated herein by reference in their entirety.
  • the lipid formulation may include a cationic lipid having the formula A described in US 20120270921.
  • the compositions of exemplary nucleic acid-lipid particles disclosed in Table A of US20120270921 may be used with the nucleic acid vaccine compositions of the present disclosure.
  • the nucleic acid vaccine compositions of the present disclosure may be fully encapsulated in a lipid particle disclosed in US Pub. No. US 20120276207 to Maurer et al., the contents of which are incorporated herein by reference in their entirety.
  • the particles may comprise a lipid composition comprising preformed lipid vesicles, a charged therapeutic agent, and a destabilizing agent to form a mixture of preformed vesicles and therapeutic agent in a destabilizing solvent, wherein the destabilizing solvent is effective to destabilize the membrane of the preformed lipid vesicles without disrupting the vesicles.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a conjugated lipid.
  • the conjugated lipid may have a formula such as described in US Pub. No. US 20120264810 to Lin et al., the contents of which are incorporated herein by reference in their entirety.
  • the conjugate lipid may form a lipid particle which further comprises a cationic lipid, a neutral lipid, and a lipid capable of reducing aggregation.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a neutral liposomal formulation such as disclosed in US Pub. No.
  • neutral liposomal formulation refers to a liposomal formulation with a near neutral or neutral surface charge at a physiological pH.
  • Physiological pH can be, e.g., about 7.0 to about 7.5, or, e.g., about 7.5, or, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or, e.g., 7.3, or, e.g., 7.4.
  • An example of a neutral liposomal formulation is an ionizable lipid nanoparticle (iLNP).
  • a neutral liposomal formulation can include an ionizable cationic lipid, e.g., DLin-KC2-DMA.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a charged lipid or an amino lipid.
  • charged lipid is meant to include those lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amino head group.
  • the quaternary amine carries a permanent positive charge.
  • the head group can optionally include an ionizable group, such as a primary, secondary, or tertiary amine that may be protonated at physiological pH.
  • a charged lipid is referred to as an "amino lipid.”
  • the amino lipid may be any amino lipid described in US Pub. No. US20110256175 to Hope et al., the contents of which are incorporated herein by reference in their entirety.
  • the amino lipids may have the structure disclosed in Tables 3-7 of Hope, such as structure (II), DLin-K-C2-DMA, DLin-K2-DMA, DLin-K6-DMA, etc.
  • the resulting pharmaceutical preparations may be lyophilized according to Hope.
  • the amino lipids may be any amino lipid described in US 20110117125 to Hope et al., the contents of which are incorporated herein by reference in their entirety, such as a lipid of structure (I), DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA, etc.
  • the amino lipid may have the structure (I), (II), (III), or (IV), or 4-(R)-DLin-K-DMA (VI), 4-(S)-DLin-K-DMA (V) as described in PCT Patent Application Publication No. W02009132131 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.
  • the charged lipid used in any of the formulations 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.
  • the nucleic acid vaccine composition s of the present disclosure may be formulated with an association complex.
  • the association complex comprises one or more compounds each having a structure defined by formula (I), a PEG-lipid having a structure defined by formula (XV), a steroid and a nucleic acid disclosed in US Pat. No. US8,034,376 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid vaccine compositions may be formulated with any association complex described in US Pat. No. US8,034,376., the contents of which are herein incorporated by reference in its entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with reverse head group lipids.
  • the nucleic acid vaccine compositions may be formulated with a zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the head group, such as a lipid having structure (A) or structure (I) described in PCT Patent Application Publication No. WO201 1056682 to Leung et al., the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid bilayer carrier.
  • the nucleic acid vaccine compositions may be combined with a lipid-detergent mixture comprising a lipid mixture of an aggregation-preventing agent in an amount of about 5 mol% to about 20 mol%, a cationic lipid in an amount of about 0.5 mol% to about 50 mol%, and a fusogenic lipid and a detergent, to provide a nucleic acid-lipid-detergent mixture; and then dialyzing the nucleic acid-lipid-detergent mixture against a buffered salt solution to remove the detergent and to encapsulate the nucleic acid in a lipid bilayer carrier and provide a lipid bilayer-nucleic acid composition, wherein the buffered salt solution has an ionic strength sufficient to encapsulate of from about 40 % to about 80 % of the nucleic acid, described in PCT Patent
  • the nucleic acid vaccine compositions of the present disclosure may comprise (a) a nucleic acid; (b) 1.0 mole % to 45 mole % of a cationic lipid; (c) 0.0 mole % to 90 mole % of another lipid; (d) 1.0 mole % to 10 mole % of a bilayer stabilizing component; (e) 0.0 mole % to 60 mole % cholesterol; and (f) 0.0 mole % to 10 mole % of cationic polymer lipid as described in EP1328254 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid vaccine may be delivered using smaller LNPs.
  • Such particles may comprise a diameter from below 0.1 pm up to 100 nm such as, but not limited to, less than 0.1 pm, less than 1.0 pm, less than 5 pm, less than 10 pm, less than 15 pm, less than 20 pm, less than 25 pm, less than 30 pm, less than 35 pm, less than 40 pm, less than 50 pm, less than 55 pm, less than 60 pm, less than 65 pm, less than 70 pm, less than 75 pm, less than 80 pm, less than 85 pm, less than 90 pm, less than 95 pm, less than 100 pm, less than 125 pm, less than 150 pm, less than 175 pm, less than 200 pm, less than 225 pm, less than 250 pm, less than 275 pm, less than 300 pm, less than 325 pm, less than 350 pm, less than 375 pm, less than 400 pm, less than 425 pm, less than 450 pm, less than 475 pm, less than 500 pm, less than 525 pm, less than
  • nucleic acid vaccine may be delivered using smaller LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about
  • the nucleic acid vaccine may be formulated in lipid nanoparticles having a diameter from about 10 nm to about 100 nm such as, but not limited to, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about
  • the nucleic acid vaccine may be formulated in lipid nanoparticles having a diameter from 10-1000 nm.
  • the nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,
  • the lipid nanoparticles may have a diameter from about 10 to 500 nm.
  • the lipid nanoparticle may have a diameter 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.
  • the nucleic acid vaccine compositions of the disclosure can be formulated using natural and/or synthetic polymers.
  • polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (PHASERX®, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.
  • chitosan-based formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No. US20120258176; the contents of which are herein incorporated by reference in their entirety).
  • Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N- carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.
  • the polymers used in the present disclosure have undergone processing to reduce and/or inhibit the attachment of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer.
  • the polymer may be processed by methods known and/or described in the art and/or described in PCT Patent Application Publication No. WO2012150467; the contents of which are herein incorporated by reference in their entirety.
  • PLGA based formulations include, but are not limited to, PLGA-based injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space.
  • PLGA-based injectable depots may be long- acting.
  • the first of these delivery approaches uses dynamic poly conjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887; the contents of which are herein incorporated by reference in their entirety).
  • This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and A-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (See again, Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887).
  • PEG for charge masking
  • A-acetylgalactosamine for hepatocyte targeting
  • the polymer formulation can permit the sustained or delayed release of nucleic acid vaccine compositions (e.g., following intramuscular, subcutaneous, intraparenchymal, intrathecal, intracerebroventricular administration).
  • the altered release profile for the nucleic acid vaccine compositions can result in, for example, translation of an encoded protein, or polypeptide or peptide over an extended period of time.
  • Biodegradable polymers have been previously used to protect nucleic acids from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887; Sullivan et al., Expert Opin Drug Deliv.
  • the nucleic acid vaccines of the present disclosure may be formulated for controlled release.
  • One form of controlled-release formulation contains the therapeutic compound or its salt dispersed or encapsulated in a slowly degrading, nontoxic, non-antigenic polymer such as copoly(lactic/glycolic) acid, as described in the pioneering work of Kent et al., US Patent No. 4,675,189, the contents of which are incorporated by reference herein in their entirety.
  • the compounds, or their salts may also be formulated in cholesterol or other lipid matrix pellets, or silastomer matrix implants.
  • the nucleic acid vaccines of the present disclosure may be dispersed or encapsulated in the polymers disclosed in US Patent No.
  • An additional form of controlled-release formulation comprises a solution of biodegradable polymer, such as copoly(lactic/gly colic acid) or block copolymers of lactic acid and PEG, which is injected subcutaneously or intramuscularly to achieve a depot formulation for controlled release.
  • biodegradable polymer such as copoly(lactic/gly colic acid) or block copolymers of lactic acid and PEG
  • the pharmaceutical compositions may be sustained release formulations.
  • the sustained release formulations may be for subcutaneous delivery.
  • Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EV Ac), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
  • nucleic acid vaccine compositions may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the nucleic acid vaccine compositions in the PLGA microspheres while maintaining the integrity of the nucleic acid vaccine compositions during the encapsulation process.
  • EV Ac are non-biodegradable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications.
  • Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxy ethylene-polyoxypropylene-polyoxy ethylene having a low viscosity at temperatures less than 5°C and forms a solid gel at temperatures greater than 15°C.
  • PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days.
  • GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.
  • Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N- acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011, 12:2708-2714; Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature ,2010 464: 1067-1070; the contents of each of which are herein incorporated by reference in their entirety).
  • GalNAc N- acetylgalactosamine
  • the nucleic acid vaccine compositions of the disclosure may be formulated with or in a polymeric compound.
  • the polymeric compound may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobutyl)-L-glycolic acid) (PAGA), biodede
  • the nucleic acid vaccine compositions of the disclosure may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its entirety.
  • the formulation may be used for transfecting cells in vitro or for in vivo delivery of the nucleic acid vaccine compositions.
  • the nucleic acid vaccine compositions may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. US20090042829 and US20090042825; the contents of each of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions of the disclosure may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, herein incorporated by reference in their entireties) or PLGA-PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, herein incorporated by reference in its entirety).
  • the nucleic acid vaccine compositions of the disclosure may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).
  • the nucleic acid vaccines compositions may be formulated with branched PEG molecules as described in or made by the methods described in PCT Patent Application Publication No. W020180126084; the contents of which are herein incorporated by reference in their entirety.
  • the branched PEG which may be used in the formulations described herein may have the formula I, formula II, formula III, formula IV, formula V, formula VI of PCT Publication No. WO20180126084, the contents of which are herein incorporated by reference in their entirety.
  • a polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. US20100260817; the contents of which are herein incorporated by reference in their entirety).
  • the nucleic acid vaccine compositions of the present disclosure may be formulated using the polyamine derivative described in U.S. Pub. No. US20100260817; the contents of which are incorporated herein by reference in their entirety.
  • the nucleic acid vaccine compositions of the present disclosure may 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 dialkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; the contents of which are herein incorporated by reference in their entirety).
  • 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 dialkyne unite comprising oligoamines
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with at least one polymer and/or derivatives thereof described in PCT Patent Application Publication Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No. US20120283427, the contents of each of which are herein incorporated by reference in their entireties.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated with a polymer of formula Z as described in WO2011115862; the contents of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions may be formulated with a polymer of formula Z, Z’ or Z” as described in PCT Patent Application Publication Nos.
  • the polymers formulated with the nucleic acid vaccine compositions of the present disclosure may be synthesized by the methods described in PCT Patent Application Publication Nos. WO2012082574 or WO2012068187, the contents of each of which are herein incorporated by reference in their entireties.
  • the nucleic acid vaccine compositions of the disclosure may be formulated with at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), poly cyanoacrylates and combinations thereof.
  • Formulations of nucleic acid vaccine compositions of the disclosure may include at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof.
  • the nucleic acid vaccine compositions of the disclosure may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof.
  • the biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S.
  • the poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. US20100004315, which is herein incorporated by reference in its entirety.
  • the biodegradable polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos.
  • the linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886; the contents of which are each incorporated herein by reference in their entirety.
  • the PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,217,912; the contents of which are herein incorporated by reference in their entirety.
  • the PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co- glycolides).
  • the biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. US2012009145; the contents of each of which are herein incorporated by reference in their entireties.
  • the multi -block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines.
  • LPEI linear polyethyleneimine
  • the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. US20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912; the contents of each of which are herein incorporated by reference in their entireties.
  • the nucleic acid vaccine compositions of the disclosure may be formulated with at least one degradable polyester which may contain polycationic side chains.
  • Degradable 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.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the nucleic acid vaccine compositions of the disclosure may be formulated with at least one crosslinkable polyester.
  • Crosslinkable polyesters include those known in the art and described in US Pub. No. US20120269761; the contents of which herein are incorporated by reference in their entirety.
  • the polymers described herein may be conjugated to a lipid-terminating PEG.
  • PLGA may be conjugated to a lipid- terminating PEG forming PLGA-DSPE-PEG.
  • PEG conjugates for use with the present disclosure include those described in PCT Patent Application Publication No. W02008103276; the contents of which are herein incorporated by reference in their entirety.
  • the polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363; the contents of which are herein incorporated by reference in their entirety.
  • the nucleic acid vaccine compositions described herein may be conjugated with another compound.
  • conjugates are described in US Pat. Nos. 7,964,578 and 7,833,992; the contents of each of which are herein incorporated by reference in their entireties.
  • the nucleic acid vaccine compositions of the present disclosure may be conjugated with conjugates of formula 1-122 as described in US Pat. Nos. 7,964,578 and 7,833,992; the contents of each of which are herein incorporated by reference in their entireties.
  • the nucleic acid vaccine compositions described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Journ. Amer.
  • nucleic acid vaccine compositions described herein may be conjugated and/or encapsulated in gold-nanoparticles (PCT Application Publication No.
  • a gene delivery composition may include a nucleotide sequence and a poloxamer.
  • the nucleic acid vaccine compositions of the present disclosure may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. US20100004313; the contents of which are each incorporated herein by reference in their entirety.
  • the polymer formulations comprising the nucleic acid vaccines of the present disclosure may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups.
  • the polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. US20090042829; the contents of which are herein incorporated by reference in their entirety.
  • the cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycosidepolyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N- [l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3- dio
  • the nucleic acid vaccine compositions of the disclosure may be formulated in a polyplex of one or more polymers (U.S. Pub. Nos. US20120237565 and US20120270927; the contents of each of which are herein incorporated by reference in their entirety).
  • the polyplex comprises two or more cationic polymers.
  • the cationic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
  • the nucleic acid vaccine compositions of the disclosure can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
  • Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so delivery of the nucleic acid vaccine compositions may be enhanced (Wang et al., Nat Mater. 2006, 5:791-796; Fuller et al., Biomaterials. 2008, 29: 1526-1532;
  • the nanoparticle 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 herein incorporated by reference in their entirety).
  • Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers may be used to deliver nucleic acid vaccine compositions in vivo.
  • a lipid coated calcium phosphate nanoparticle which may also contain a targeting ligand such as anisamide, may be used to deliver the nucleic acid vaccine compositions of the present disclosure.
  • a targeting ligand such as anisamide
  • This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
  • calcium phosphate with a PEG-polyanion block copolymer may be used to delivery nucleic acid vaccine compositions of the disclosure (Kazikawa et al., J Contr Rel. 2004, 97:345-356; Kazikawa et al., J Contr Rel. 2006, 111 :368-370; the contents of each of which are herein incorporated by reference in their entirety).
  • a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011, 32:3106-3114; the contents of which are herein incorporated by reference in their entirety) may be used to form a nanoparticle to deliver the nucleic acid vaccine compositions of the present disclosure.
  • the PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
  • a core-shell nanoparticle may be used to form a nanoparticle to deliver the nucleic acid vaccine compositions of the present disclosure.
  • the use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci U SA. 2011, 108: 12996-13001; the contents of which are herein incorporated by reference in their entirety).
  • the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
  • the core-shell nanoparticles may efficiently deliver nucleic acid vaccine compositions to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
  • the nanoparticles described herein may be nanoparticles which include 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 nanomolar binding affinities toward their targets, a peptide aptamer, an antibody, a small molecule ligand such as, but not limited to, folate, anisamide, and galactose. (Leng et al. Journal of Drug Delivery. 2017, 17, Article ID 6971297; the contents of which are herein incorporated by reference in their entirety).
  • a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of the nucleic acid vaccine compositions of the present disclosure.
  • the lipid- polymer-lipid hybrid nanoparticle may be used to deliver the nucleic acid vaccine compositions described herein (Shi et al, Angew Chem Int Ed. 2011, 50:7027-7031; the contents of which are herein incorporated by reference in their entirety).
  • Core-shell nanoparticles for use with the nucleic acid vaccine compositions of the present disclosure may be formed by the methods described in U.S. Pat. No.
  • the core-shell nanoparticles may comprise a core of the nucleic acid vaccine compositions described herein and a polymer shell.
  • the polymer shell may be any of the polymers described herein and are known in the art.
  • the polymer shell may be used to protect the nucleic acid vaccine compositions in the core, (see, e.g., US Publication No. 20120321719; the contents of which are herein incorporated by reference in their entirety).
  • the polymer used with the formulations described herein may be a modified polymer (such as, but not limited to, a modified polyacetal) as described in PCT Application Publication No. WO2011120053; the contents of which are herein incorporated by reference in their entirety.
  • a modified polymer such as, but not limited to, a modified polyacetal
  • the nucleic acid vaccine compositions may be delivered to the cell or cytosol of a target cell by contacting the cell with a membrane-destabilizing polymer and a conjugate of the nucleic acid vaccine composition, a targeting ligand and an optional linker.
  • a membrane-destabilizing polymer and a conjugate of the nucleic acid vaccine composition, a targeting ligand and an optional linker.
  • membrane-destabilizing polymers are taught in International PCT Application Publication No. W02020093061, the contents of which are herein incorporated by reference in their entirety, such as, but not limited to, the membrane-destabilizing polymers of formula XX therein.
  • pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, anti-oxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, anti-oxidants, osmolality adjusting agents, pH adjusting agents and the like, as
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use.
  • an excipient may be approved by United States Food and Drug Administration.
  • an excipient may be of pharmaceutical grade.
  • an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • the composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents.
  • 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, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cationexchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone cross-linked polyvinylpyrrolidone
  • sodium carboxymethyl starch sodium starch glyco
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chon- drux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, 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.
  • natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chon- drux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g. bentonite (alum
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene sorbitan monolaurate TWEEN®20
  • polyoxyethylene sorbitan TWEEN®60
  • polyoxyethylene sorbitan monooleate TWEEN®80
  • sorbitan monopalmitate SPAN®40
  • sorbitan monostearate SPAN®60
  • sorbitan tristearate SPAN®65
  • polyoxyethylene esters e.g.
  • polyoxyethylene monostearate MYRJ®45
  • polyoxyethylene hydrogenated castor oil polyethoxylated castor oil
  • polyoxymethylene stearate polyethoxylated castor oil
  • SOLUTOL® sucrose fatty acid esters
  • polyethylene glycol fatty acid esters e.g. CREMOPHOR®
  • polyoxyethylene ethers e.g.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g.
  • natural and synthetic gums e.g. acacia, sodium alginate, extract of
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. In order to prevent oxidation, antioxidants can be added to the formulation.
  • antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenyl ethyl 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.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®1 15, GERMAB EN®! !, NEOLONETM, KATHONTM, and/or EUXYL®.
  • the pH of the pharmaceutical solutions are maintained between pH 5 and pH 8 to improve stability.
  • exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/or sodium malate.
  • the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions; however, these are not preferred due to complex formation and/or mRNA degradation.
  • Exemplary buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/ or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • nucleic acid vaccine compositions described herein may comprise at least one nucleic acid vaccine that is formulated in a lipid nanoparticle (LNP) and at least one excipient.
  • the excipient may be a sugar such as sucrose.
  • Adjuvants may also be administered with or in combination with one or more of the nucleic acid vaccines described herein, e.g., the mRNA vaccine.
  • Adjuvants may be used to enhance the immunogenicity of the nucleic acid vaccine, modify the immune response, reduce the amount of nucleic acid vaccine needed for immunization, reduced the frequency of additional or “booster” immunizations needed or to create an improved immune response in those with weakened or immunocompromised immune systems or the elderly.
  • the adjuvants may be a component of the formulation containing the nucleic acid vaccine or they may be co-administered with the nucleic acid vaccines compositions. Coadministration of the adjuvant may be any method known in the art or described herein such as, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC) or intradermal (ID).
  • the adjuvant is natural or synthetic.
  • the adjuvants may also be organic or inorganic.
  • the adjuvant used with the nucleic acid vaccine is 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 emulsion, surfactant based emulsion, purified saponin, and oil-in water emulsion), inert vehicles, particulate adjuvants (e.g., unilamellar liposomal vehicles such as virosomes or a structured complex of saponions and lipids such as polylactide coglycolide (PLG)), microbial derivatives, endogenous human immunomodulators, and tensoactive compounds.
  • 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
  • Adjuvants may be selected for use with the nucleic acid vaccines by one of ordinary skill in the art.
  • Adjuvants may be interferons, TNF-alpha, TNF-beta, chemokines (e.g., CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD40L, CD28 agonists, PD1, soluble PD1, PDL1, PDL2) or interleukins (e.g., IL1, IL2, IL4, IL6, IL7, IL10, IL12, IL13, IL15, IL17, IL18, IL21, and IL23).
  • chemokines e.g., CCL21, eotaxin, HMGB1, SA100-8alpha
  • GCSF e.g., eotaxin, HMGB1, SA100-8alpha
  • GMCSF
  • Nonlimiting examples of adjuvants include Abisco-100 vaccine adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, AdjumerTM, AF03, Albumin-heparin microparticles vaccine adjuvant, Algal Glucan, Algammulin, alhydrogel, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, Aluminum vaccine adjuvant, amorphous aluminum hydroxyphosphate sulfate adjuvant, Arlacel A, ASO, 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 Liposomal Vaccine Adjuvant, cationic liposome-DNA complex JVRS- 100, Cholera toxin, Cholera toxin B subunit
  • the nucleic acid vaccines described herein may be used as a vaccine and may further comprise an adjuvant which may enable the vaccine to elicit a higher immune response.
  • the adjuvant could be a sub-micron oil-in-water emulsion which can elicit a higher immune response in human pediatric populations (see e.g., the adjuvanted vaccines described in US Patent Publication No.
  • nucleic acid vaccine compositions including, for example, nucleic acid vaccine for COVID-19 for any therapeutic, prophylactic, pharmaceutical, diagnostic or imaging use by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
  • the nucleic acid vaccine compositions of the present disclosure may be delivered to a cell naked.
  • naked refers to delivering nucleic acid vaccine compositions free from agents which promote transfection.
  • the nucleic acid vaccine compositions delivered to the cell may contain no modifications.
  • the naked nucleic acid vaccine compositions may be delivered to the cell using routes of administration known in the art and described herein.
  • the nucleic acid vaccine compositions of the present disclosure may be formulated, using the formulation components and methods described herein.
  • the formulations may contain nucleic acid vaccine compositions which may be modified and/or unmodified.
  • the formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot.
  • the formulated nucleic acid vaccine compositions may be delivered to the cell using routes of administration known in the art and described herein.
  • the nucleic acid vaccine compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
  • the nucleic acid vaccine compositions of the present disclosure may also be cloned into a retroviral replicating vector (RRV) and transduced to cells.
  • RRV retroviral replicating vector
  • the pharmaceutical composition may be packaged as a percentage of fill of the capacity of a glass vial.
  • the fill may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% of the capacity of the glass vial.
  • the pharmaceutical composition may be packaged as a 2.4 mL fill in a 6 mL glass vial.
  • the pharmaceutical composition may be packaged as a 2 mL fill in a 3 mL glass vial.
  • compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • nucleic acid vaccine e.g., nucleic acid vaccine for COVID-19, e.g., mRNA vaccine for COVID-19
  • body weight per day about 0.001 and about 200 mg/kg, about 0.001 and about 100 mg/kg, 0.01 and about 100 mg/kg, preferably between about 0.005 and about 50 mg/kg, 0.01 and about 50 mg/kg, 0.01 and about 40 mg/kg, 0.01 and about 30 mg/kg, 0.01 and about 10 mg/kg, 0.05 and about 50 mg/kg, 0.05 and about 30 mg/kg, 0.05 and about 10 mg/kg, 0.05 and about 5 mg/kg, 0.1 and about 50 mg/kg, 0.1 and about 30 mg/kg, 0.1 and about 10 mg/kg, 0.1 and about 1 mg/kg, 1.0 and about 50 mg/kg, 1.0 and about 40 mg/kg, 1.0 to about 30 mg/kg, 10 to about 50mg/kg
  • the dosages may be administered about hourly, multiple times per day, daily, every other day, weekly, every other week, monthly, every other month, or on an as-needed basis.
  • compositions of the nucleic acid vaccines may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 1 mg/kg to about 50 mg/kg, from about 10 mg/kg to about 100 mg/kg, from about 10 mg/kg to about 50 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic
  • the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • multiple administrations e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
  • split dosing regimens such as those described herein may be used.
  • compositions of the nucleic acid vaccines described herein may be administered at dosage levels sufficient to deliver to a subject, about 1 pg, lOpg. 15pg, 20pg, 25pg, 30pg, 35pg, 40pg, 50pg , 60pg, 70pg, 80pg, 90pg, or lOOpg of the nucleic acid composition.
  • the nucleic acid vaccines may be administered in splitdose regimens.
  • a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose.
  • a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • a “total daily dose” is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose.
  • the nucleic acid vaccines described herein are administered to a subject in split doses.
  • the nucleic acid vaccines may be formulated in buffer only or in a formulation described herein.
  • the nucleic acid vaccine compositions described herein may be administered to a subject in two separate phases, a loading dosing phase and a maintenance dosing phase.
  • the dosing regimen may comprise an initial higher loading dose of the nucleic acid vaccine that is given to the subject first time at the beginning of a course of prevention, alleviation and/or treatment, e.g., first dose for preventing COVID- 19, and a lower maintenance dose following the first loading dose.
  • the loading dose and the maintenance dose have the same amount of the nucleic acid vaccines of the present disclosure.
  • more than one maintenance doses are administered to the subject.
  • the multiple maintenance doses may be administered biweekly, every three weeks, every four weeks, monthly, bimonthly, every three months, every four months, every five months, or every six months.
  • the maintenance doses of the nucleic acid vaccines may also be referred to as booster doses.
  • a “booster dose” (or “booster shot) is an extra or supplemental dose of a vaccine after an initial primer dose.
  • the booster dose may have the same amount of the nucleic acid vaccine as the initial loading dose.
  • the booster dose has an amount of the nucleic acid vaccine that is smaller than the original amount of the nucleic acid vaccine in the initial dose.
  • the subject may receive one, two, three, four or more booster doses.
  • Such administration can be used as a chronic or acute treatment or prevention of a clinic-concerning condition.
  • the amount of drug that may be combined with the carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • such preparations contain from about 20% to about 80%, 30% to about 70%, 40% to about 60%, or about 50% active compound.
  • the preparations used in the present disclosure 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.
  • a maintenance dose of a compound, composition or combination of the present disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • the delivery of the nucleic acid vaccines may be naked or formulated.
  • the nucleic acid vaccines described herein may be delivered to a cell naked.
  • naked refers to delivering nucleic acid vaccines free from agents which promote transfection.
  • the nucleic acid vaccines delivered to the cell may contain no modifications.
  • the naked nucleic acid vaccines may be delivered to the cell using routes of administration known in the art and described herein.
  • the nucleic acid vaccines described herein may be formulated, using the methods described herein.
  • the formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot.
  • the formulated nucleic acid vaccines may be delivered to the cell using routes of administration known in the art and described herein.
  • compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
  • the nucleic acid vaccine compositions of the present disclosure may be administered by any route which results in a prophylactic or therapeutically effective outcome. These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal,
  • Delivery of the nucleic acid vaccines described herein to a subject over prolonged periods of time, for example, for periods of one week to one year, may be accomplished by a single administration of a controlled release system containing sufficient active ingredient for the desired release period.
  • a controlled release system containing sufficient active ingredient for the desired release period.
  • Various controlled release systems such as monolithic or reservoir-type microcapsules, depot implants, polymeric hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms may be utilized for this purpose. Localization at the site to which delivery of the active ingredient is desired is an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders.
  • the nucleic acid vaccines described herein may be administered intranasally similar to the administration of live vaccines.
  • the polynucleotide may be administered intramuscularly or intradermally similarly to the administration of inactivated vaccines known in the art.
  • Electrodes e.g., iontophoresis
  • electroporation or the application of short, high-voltage electrical pulses to the skin, radiofrequencies, ultrasound (e.g. sonophoresis), microprojections (e.g. microneedles), jet injectors, thermal ablation, magnetophoresis, lasers, velocity, or photomechanical waves.
  • the drug can be included in single-layer drug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, or vapor style patches, or could utilize patchless technology.
  • Delivery across the barrier of the skin could also be enhanced using encapsulation, a skin lipid fluidizer, or a hollow or solid microstructured transdermal system (MTS, such as that manufactured by 3M), jet injectors.
  • Additives to the formulation to aid in the passage of therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell penetrating peptides, permeation enhancers, encapsulation technologies, enzymes, enzyme inhibitors, gels, nanoparticles and peptide or protein chaperones.
  • nucleic acid vaccines described herein with a polymeric formulation comprising biodegradable polymers that can form a depot formulation upon administration, is suitable to achieve very long duration of action formulations.
  • the absorption across the nasal mucous membrane may be further enhanced by surfactants, such as, for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cycledextrins and the like in an amount in the range of between about 0.1 and 15 weight percent, between about 0.5 and 4 weight percent, or about 2 weight percent.
  • surfactants such as, for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cycledextrins and the like in an amount in the range of between about 0.1 and 15 weight percent, between about 0.5 and 4 weight percent, or about 2 weight percent.
  • absorption enhancers reported to exhibit greater efficacy with decreased irritation is the class of alkyl maltosides, such as tetradecylmaltoside (Arnold, JJ et al., J Pharm Set, 2004, 93: 2205-13; Ahsan, F et al., Pharm Res, 2001,18: 1742-46) and references therein, all of which are hereby incorporated by reference in their entirety.
  • alkyl maltosides such as tetradecylmaltoside (Arnold, JJ et al., J Pharm Set, 2004, 93: 2205-13; Ahsan, F et al., Pharm Res, 2001,18: 1742-46) and references therein, all of which are hereby incorporated by reference in their entirety.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol.
  • suitable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.
  • the pharmaceutical compositions of the present disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions.
  • compositions of present disclosure may also be administered in the form of suppositories for rectal administration.
  • compositions can be prepared by mixing the active ingredient the present disclosure with a suitable nonirritating excipient that is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable nonirritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • Topical administration of the pharmaceutical compositions of the present disclosure is especially useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of the present disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of the present disclosure may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included in the present disclosure.
  • compositions of the present disclosure may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous.
  • Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art including, but not limited to,
  • compositions may be mixed with solubilizing agents such as CREMO- PHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • solubilizing agents such as CREMO- PHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3 -butanediol.
  • the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • nucleic acid vaccine In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the nucleic acid vaccine then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered nucleic acid vaccine may be accomplished by dissolving or suspending the nucleic acid vaccine in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the nucleic acid vaccine in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of nucleic acid vaccine to polymer and the nature of the particular polymer employed, the rate of polynucleotides release can be controlled.
  • biodegradable polymers examples include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the nucleic acid vaccine in liposomes or microemulsions which are compatible with body tissues.
  • Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, e.g., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
  • a formulation suitable for buccal administration may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • nucleic acid vaccine pharmaceutical compositions described herein may be characterized using one or more of bioavailability, therapeutic window, volume of distribution, biological effect and detection of polynucleotides by mass spectrometry. Bioavailability
  • the nucleic acid vaccines when formulated into a composition with a delivery agent as described herein, can exhibit an increase in bioavailability as compared to a composition lacking a delivery agent as described herein.
  • bioavailability refers to the systemic availability of a given amount of nucleic acid vaccines administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration of the unchanged form of a compound following administration of the compound to a mammal. AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis).
  • the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modem Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, N.Y, Inc., 1996, herein incorporated by reference in its entirety.
  • the Cmax value is the maximum concentration of the compound achieved in the serum or plasma of a mammal following administration of the compound to the mammal.
  • the Cmax value of a particular compound can be measured using methods known to those of ordinary skill in the art.
  • the phrases “increasing bioavailability” or “improving the pharmacokinetics,” as used herein mean that the systemic availability of a first nucleic acid vaccine, measured as AUC, Cmax, or Cmin, in a mammal is greater, when co-administered with a delivery agent as described herein, than when such coadministration does not take place.
  • the bioavailability of the nucleic acid vaccines can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • liquid formulations of nucleic acid vaccines may have various in vivo half-life, requiring modulation of doses to yield a prophylactic or therapeutic effect.
  • nucleic acid vaccine formulations may be designed to improve bioavailability and/or prophylactic or therapeutic effect during repeat administrations. Such formulations may enable sustained release of nucleic acid vaccines and/or reduce nucleic acid vaccine degradation rates by nucleases.
  • suspension formulations are provided comprising nucleic acid vaccines, water immiscible oil depots, surfactants and/or co-surfactants and/or co-solvents. Combinations of oils and surfactants may enable suspension formulation with nucleic acid vaccines.
  • nucleic acid vaccines in a water immiscible depot may be used to improve bioavailability through sustained release of polynucleotides from the depot to the surrounding physiologic environment and/or prevent polynucleotide degradation by nucleases.
  • cationic nanoparticles comprising combinations of divalent and monovalent cations may be formulated with nucleic acid vaccines. Such nanoparticles may form spontaneously in solution over a given period (e.g. hours, days, etc.). Such nanoparticles do not form in the presence of divalent cations alone or in the presence of monovalent cations alone.
  • the delivery of nucleic acid vaccines in cationic nanoparticles or in one or more depot comprising cationic nanoparticles may improve nucleic acid vaccine bioavailability by acting as a long-acting depot and/or reducing the rate of degradation by nucleases.
  • nucleic acid vaccines when formulated into a composition with a delivery agent as described herein, can exhibit an increase in the therapeutic window of the administered nucleic acid vaccine composition as compared to the therapeutic window of the administered nucleic acid vaccine composition lacking a delivery agent as described herein.
  • therapeutic window refers to the range of plasma concentrations, or the range of levels of therapeutically active substance at the site of action, with a high probability of eliciting a prophylactic or therapeutic effect.
  • the therapeutic window of the nucleic acid vaccines when co-administered with a delivery agent as described herein can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • the nucleic acid vaccines when formulated into a composition with a delivery agent as described herein, can exhibit an improved volume of distribution (Vdist), e.g., reduced or targeted, relative to a composition lacking a delivery agent as described herein.
  • the volume of distribution (Vdist) relates the amount of the drug (e.g., nucleic acid vaccine of the present disclosure) in the body to the concentration of the drug in the blood or plasma.
  • the term “volume of distribution” refers to the fluid volume that would be required to contain the total amount of the drug in the body at the same concentration as in the blood or plasma: Vdist equals the amount of drug in the body/concentration of drug in blood or plasma.
  • the volume of distribution would be 1 liter.
  • the volume of distribution reflects the extent to which the drug is present in the extravascular tissue.
  • a large volume of distribution reflects the tendency of a compound to bind to the tissue components compared with plasma protein binding.
  • Vdist can be used to determine a loading dose to achieve a steady state concentration.
  • the volume of distribution of the nucleic acid vaccines when co-administered with a delivery agent as described herein can decrease at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
  • the biological effect of the nucleic acid vaccine delivered to the animals may be categorized by analyzing the protein expression in the animals.
  • the protein expression may be determined from analyzing a biological sample collected from a mammal administered the nucleic acid vaccine described herein.
  • Mass spectrometry is an analytical technique that can provide structural and molecular mass/concentration information on molecules after their conversion to ions.
  • the molecules are first ionized to acquire positive or negative charges and then they travel through the mass analyzer to arrive at different areas of the detector according to their mass/charge (m/z) ratio.
  • Mass spectrometry is performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis.
  • ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization.
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • FAB fast atom bombardment
  • LIMS liquid secondary ionization
  • MALDI matrix assisted laser desorption/ ionization
  • field ionization field desorption
  • thermospray/plasmaspray ionization and particle beam ionization.
  • the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., m/z).
  • Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers.
  • the ions may be detected using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
  • SIM selective ion monitoring mode
  • MRM multiple reaction monitoring
  • SRM selected reaction monitoring
  • targeted MS methods are peptide sequence-based modes of MS that focus the full analytical capacity of the instrument on tens to hundreds of selected peptides in a complex mixture. By restricting detection and fragmentation to only those peptides derived from proteins of interest, sensitivity and reproducibility are improved dramatically compared to discovery -mode MS methods.
  • This method of mass spectrometry based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples.
  • MRM multiple reaction monitoring
  • the biological sample once obtained from the subject, may be subjected to enzyme digestion.
  • digest means to break apart into shorter peptides.
  • treating a sample to digest proteins means manipulating a sample in such a way as to break down proteins in a sample.
  • enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin.
  • a biological sample may be analyzed for protein using electrospray ionization.
  • Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to aid in the transfer of ions from the solution to the gaseous phase before they are analyzed by mass spectrometry.
  • Samples may be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003, 24(1):3-12; herein incorporated by reference in its entirety).
  • the ionic species contained in solution may be transferred into the gas phase by dispersing a fine spray of charge droplets, evaporating the solvent and ejecting the ions from the charged droplets to generate a mist of highly charged droplets.
  • the mist of highly charged droplets may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers such as, but not limited to, a quadropole mass analyzer.
  • the mass spectrometry method may include a purification step.
  • the first quadrapole may be set to select a single m/z ratio so it may filter out other molecular ions having a different m/z ratio which may eliminate complicated and time-consuming sample purification procedures prior to MS analysis.
  • a biological sample may be analyzed for protein in a tandem ESIMS system (e.g., MS/MS).
  • the droplets may be analyzed using a product scan (or daughter scan) a precursor scan (parent scan) a neutral loss or a multiple reaction monitoring.
  • a biological sample may be analyzed using matrix- assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS).
  • MALDI matrix- assisted laser desorption/ionization
  • MALDIMS matrix- assisted laser desorption/ionization mass spectrometry
  • MALDI provides for the nondestructive vaporization and ionization of both large and small molecules, such as proteins.
  • the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, an ultraviolet absorbing weak organic acid.
  • Non-limiting examples of matrices used in MALDI are a-cyano-4-hy- droxy cinnamic acid, 3, 5 -dimethoxy -4-hydroxy cinnamic acid and 2,5-dihydroxybenzoic acid.
  • Laser radiation of the analyte-matrix mixture may result in the vaporization of the matrix and the analyte.
  • the laser induced desorption provides high ion yields of the intact analyte and allows for measurement of compounds with high accuracy.
  • Samples may be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; the contents of which are herein incorporated by reference in their entirety).
  • mass analyzers used in the MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or a Fourier transform mass analyzer.
  • nucleic acid vaccines described herein may be operably linked to one or more regulatory nucleotide sequences and encoded in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate for a host cell used for expression.
  • Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, transcriptional start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector includes a selectable marker gene to allow the selection of transformed host cells.
  • Certain embodiments include an expression vector encoding a nucleic acid vaccine for COVID-19 sequence operably linked to at least one regulatory sequence. Regulatory sequences for use herein include promoters, enhancers, and other expression control elements.
  • an expression vector is designed considering the choice of the host cell to be transformed, the particular nucleic acid vaccine sequence to be expressed, the vector's copy number, the ability to control that copy number, or the expression of other proteins encoded by the vector, such as antibiotic markers.
  • the nucleic acids described herein may be expressed in microorganisms.
  • the nucleic acid may be expressed in a bacterial system, for example, in Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Caulobacter crescentus, Escherichia coli and their derivatives.
  • Exemplary promoters include the 1-arabinose inducible araBAD promoter (PBAD), the lac promoter, the 1- rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter Pl, and the anhydrotetracycline-inducible tetA promoter/operator.
  • PBAD 1-arabinose inducible araBAD promoter
  • the lac promoter the 1- rhamnose inducible rhaP BAD promoter
  • T7 RNA polymerase promoter the trc and tac promoter
  • the lambda phage promoter Pl the anhydrotetracycline-inducible tetA promoter/operator.
  • the nucleic acids described herein may be expressed in a yeast expression system.
  • Non-limiting examples of promoters which may be used in yeast vectors include the promoters for 3 -phosphoglycer
  • glycolytic enzymes Hess et al., J. Adv. Enzyme Res. 7: 149 (1968); Holland et al., Biochemistry 17:4900 (1978).
  • Others promoters are from, e.g., enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase alcohol oxidase I (A0X1), alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydr
  • Any plasmid vector containing a yeast-compatible promoter and termination sequences, with or without an origin of replication, is suitable.
  • Certain yeast expression systems are commercially available, for example, from Clontech Laboratories, Inc. (Palo Alto, Calif., e.g., Pyex 4T family of vectors for S. cerevisiae), Invitrogen (Carlsbad, Calif., e.g. Ppicz series Easy Select Pichia Expression Kit) and Stratagene (La Jolla, Calif., e.g. ESP.TM. Yeast Protein Expression and Purification System for S. pombe and Pesc vectors for S. cerevisiae).
  • the nucleic acids described herein may be expressed in mammalian expression systems.
  • mammalian promoters include, for example, promoters from the following genes: ubiquitin/S27a promoter of the hamster (WO 97/15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV).
  • ubiquitin/S27a promoter of the hamster WO 97/15664
  • Simian vacuolating virus 40 (SV40) early promoter adenovirus major late promoter
  • mouse metallothionein-I promoter the long terminal repeat region of Rous Sarcoma Virus (RSV)
  • MMTV mouse mamm
  • heterologous mammalian promoters examples include the actin, immunoglobulin or heat shock promoter(s).
  • a yeast alcohol oxidase promoter is used.
  • promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published
  • Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters.
  • the early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers et al., Nature 273: 113-120 (1978).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. Greenaway, P. J. et al., Gene 18: 355-360 (1982). The foregoing references are incorporated by reference in their entirety.
  • the nucleic acids described herein may be expressed in insect cell expression systems.
  • Eukaryotic expression systems employing insect cell hosts may rely on either plasmid or baculoviral expression systems.
  • Typical insect host cells are derived from the fall army worm (Spodoptera frugiperdct).
  • Spodoptera frugiperdct For expression of a foreign protein these cells are infected with a recombinant form of the baculovirus Autographa californica nuclear polyhedrosis virus which has the gene of interest expressed under the control of the viral polyhedron promoter.
  • Other insects infected by this virus include a cell line known commercially as "High 5" (Invitrogen) which is derived from the cabbage looper (Trichoplusia ni).
  • Baculovirus sometimes used is the Bombyx mori nuclear polyhedorsis virus which infect the silkworm (Bombyx mori).
  • Numerous baculovirus expression systems are commercially available, for example, from Thermo Fisher (Bac-N- BlueTMk or BAC-TO-BACTM Systems), Clontech (BacPAKTM Baculovirus Expression System), Novagen (Bac Vector SystemTM), or others from Pharmingen or Quantum Biotechnologies.
  • Another insect cell host is the common fruit fly, Drosophda melanogaster, for which a transient or stable plasmid-based transfection kit is offered commercially by Thermo Fisher (The DESTM System).
  • cells are transformed with vectors that express a nucleic acid described herein. Transformation techniques for inserting new genetic material into eukaryotic cells, including animal and plant cells, are well known. Viral vectors may be used for inserting expression cassettes into host cell genomes. Alternatively, the vectors may be transfected into the host cells. Transfection may be accomplished by methods as described in the art such as, but not limited to, calcium phosphate precipitation, electroporation, optical transfection, protoplast fusion, impalefection, and hydrodynamic delivery.
  • nucleic acid vaccines of the present disclosure provides methods of using nucleic acid vaccines of the present disclosure and pharmaceutical compositions and formulations comprising the nucleic acid vaccines and at least one pharmaceutically acceptable carrier.
  • compositions, methods, kits, and reagents for diagnosis, treatment, alleviation or prevention of a disease or condition in humans or other mammals where the active agent is the nucleic acid vaccine, cells containing the nucleic acid vaccine or polypeptides translated from nucleic acid vaccine polynucleotides.
  • the methods of use can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of disease progression, including stabilization, slowing down and complete arrest;
  • the nucleic acid vaccines described herein may be used to protect, treat or cure infection arising from contact with an infectious agent such as, but not limited to, viruses, bacteria, fungi, parasites and protozoa.
  • an infectious agent such as, but not limited to, viruses, bacteria, fungi, parasites and protozoa.
  • the infectious agent is a virus and the virus is SARS-CoV-2 and/or a variant thereof.
  • the variants of SARS-CoV-2 are VOI, VOC and VOHC variants.
  • nucleic acid vaccines described herein may be used as prophylactic agents where the nucleic acid vaccines are administered to a subject, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention of COVID-19.
  • nucleic acid vaccines described herein may be used as therapeutic agents where the nucleic acid vaccines are administered to a subject, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the alleviation of one or more symptoms of COVID-19.
  • kits for treating or preventing a viral infection and/or a disease, disorder, or condition associate with a viral infection or a symptom thereof, in a subject by administering a nucleic acid vaccine comprising one or more polynucleotides encoding a viral polypeptide.
  • the administration may be in combination with an anti-viral or anti-bacterial agent or a small molecule compound described herein or known in the art.
  • nucleic acid vaccines described herein may be used to protect against and/or prevent the transmission of an emerging or engineered threat which may be known or unknown.
  • a polypeptide e.g., one or more proteins, peptides, fragments or variants thereof of SARS- CoV-2 in a cell, tissue or organism using the nucleic acid polynucleotides described herein.
  • the translated polypeptide may be used for the prevention, alleviation and/or treatment of COVID-19.
  • Such translation can be in vitro, in vivo, ex vivo, or in culture.
  • the cell, tissue or organism may be contacted with an effective amount of a composition or pharmaceutical composition containing the nucleic acid vaccine which includes a polynucleotide with at least one region encoding the polypeptide of interest (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or prevention of CO VID-19.
  • a composition or pharmaceutical composition containing the nucleic acid vaccine which includes a polynucleotide with at least one region encoding the polypeptide of interest (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or prevention of CO VID-19.
  • the effective amount of the nucleic acid vaccine described herein provided to a cell, a tissue or a subject may be enough for immune prophylaxis.
  • an “effective amount” of the composition of the nucleic acid vaccine is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and the number of unmodified and modified nucleosides) and other components of the nucleic acid vaccine.
  • An effective amount of the composition containing the nucleic acid vaccine described herein is one that provides an induced or boosted immune response as a function of production in the cell of one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 as compared to an untreated cell. Increased production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid vaccine), increased protein translation from the polynucleotide or altered innate immune response of the host cell.
  • nucleic acid vaccine composition containing a polynucleotide that has at least one translatable region encoding the polypeptide (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2) is administered to the subject using the delivery methods described herein.
  • the polynucleotide is provided in an amount and under other conditions such that the polynucleotide is translated in the cell.
  • the cell in which the polynucleotide is localized, or the tissue in which the cell is present, may be targeted with one or more rounds of nucleic acid vaccine administration.
  • the administered nucleic acid vaccine comprising polynucleotides directs production of one or more polypeptides that provide a functional immune system-related activity which is substantially absent in the cell, tissue or organism n in which the polypeptide is translated.
  • the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
  • the administered polynucleotides direct production of one or more polypeptides that increases a functional activity related to the immune system which is present but substantially deficient in the cell in which the polypeptide is translated.
  • the polypeptide translated from the nucleic acid vaccine may antagonize, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell.
  • biological moi eties that may be antagonized include a nucleic acid, a carbohydrate, a protein toxin such as shiga and tetanus toxins, lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), or a small molecule toxin (e.g., cholera, botulinum, and diphtheria toxins).
  • the biological molecule which may be antagonized may be an endogenous protein that may have an undesirable activity such as, but not limited to, cytotoxic or cytostatic activity.
  • the proteins described herein may be engineered for localization within the cell, potentially within a specific compartment such as the cytoplasm or nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
  • the polynucleotides of the nucleic acid vaccines and their encoded polypeptides may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to viral infections (e.g., infections caused by SARS-CoV-2 and/or a variant thereof).
  • viral infections e.g., infections caused by SARS-CoV-2 and/or a variant thereof.
  • the subject to whom the nucleic acid vaccine may be administered suffers from or may be at risk of developing a disease, disorder, or deleterious condition.
  • GWAS genome-wide association studies
  • the agents can be administered simultaneously, for example in a combined unit dose (e.g., providing simultaneous delivery of both agents).
  • the agents can also be administered at a specified time interval, such as, but not limited to, an interval of minutes, hours, days or weeks.
  • the agents may be concurrently bioavailable, e.g., detectable, in the subject.
  • the agents may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents.
  • the agents may be delivered in separate unit dosages.
  • the agents may be administered in any order, or as one or more preparations that includes two or more agents.
  • At least one administration of one of the agents may be made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent.
  • combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than additive results.
  • the nucleic acid vaccine described herein may be administrated with other prophylactic or therapeutic compounds.
  • the prophylactic or therapeutic compound may be an adjuvant or a booster.
  • booster refers to an extra administration of the prophylactic composition.
  • a booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition.
  • the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 21 days, 28 days , 1 month, 2 months
  • administration of a booster may result in an increase in antibody titer at least 3 weeks after the administration of the booster as compared to antibody titer levels prior to the administration of the booster.
  • the increase may be seen, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or greater than 8 weeks after administration of the booster.
  • the difference in titer level after administration of the booster as compared to before the booster may be, but is not limited to at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11- fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80- fold, 85-fold, 90-fold, 95-fold, or 100-fold, or about 1 to 5-fold, about 5 to 8-fold, about 5 to 10-fold, about 5 to 20-fold, about 5 to 30-fold, about 5 to 40-fold, about 5 to 50-fold, about 5 to 60-fold, about 10 to 20-fold, about 10 to 30-fold, about 10 to 40-fold, about 10 to 50-fold, about 10 to 60-fold,
  • 125-fold about 100 to 150-fold, about 100 to 175-fold, about 100 to 200-fold, about 100 to 225-fold, about 100 to 300-fold, about 100 to 375-fold, about 125 to 150-fold, about 125 to 175-fold, about 125 to 200-fold, about 125 to 225-fold, about 125 to 300-fold, about 125 to 375-fold, about 150 to 175-fold, about 150 to 200-fold, about 150 to 225- fold, about 150 to 300-fold, about 150 to 375-fold, about 175 to 200-fold, about 175 to 225-fold, about 175 to 300-fold, about 175 to 375-fold, about 200 to 225-fold, about 200 to 300-fold, about 200 to 375-fold, about 225 to 300-fold, about 225 to 375-fold, about 300 to 375-fold, about 100 to 350-fold, about 125 to 350-fold, about 150 to 350-fold, about 175 to 350-fold, about 200 to 350
  • the nucleic acid vaccines may be formulated by the methods described herein.
  • the formulation may comprise a nucleic acid vaccine or polynucleotide which can have a therapeutic and/or prophylactic effect on more than one disease, disorder or condition.
  • the formulation may comprise polynucleotides encoding one or more proteins, polypeptide, peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-19.
  • nucleic acid vaccines described herein may be used for research in many applications, such as, but not limited to, identifying and locating intracellular and extracellular proteins, protein interaction, signal pathways and cell biology.
  • the nucleic acid vaccines comprising the polynucleotides described herein may act as a single composition as a vaccine.
  • a “vaccine” refers to a composition, a substance or preparation that stimulates, induces, causes or improves immunity in an organism, e.g., an animal organism, for example, a mammalian organism (e.g., a human).
  • a vaccine provides immunity against one or more diseases or disorders in the organism, including prophylactic and/or therapeutic immunity.
  • Exemplary vaccines include one or more agents that resembles an infectious agent, e.g., a disease-causing microorganism, and can be made, for example, from live, attenuated, modified, weakened or killed forms of disease-causing microorganisms, or antigens derived therefrom, including combinations of antigenic components.
  • a vaccine stimulates, induces causes or improves immunity in an organism or causes or mimics infection in the organism without inducing any disease or disorder.
  • a vaccine introduces an antigen into the tissues, extracellular space or cells of a subject and elicits an immune response, thereby protecting the subject from a particular disease or pathogen infection.
  • the nucleic acid vaccines described herein may encode an antigen and when the polynucleotides are expressed in cells, a desired immune response is achieved.
  • the nucleic acid vaccines described herein may encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 and when the polynucleotides are expressed in cells, a desired immune response against SARS-CoV-2 is achieved to treat and/or prevent COVID-19.
  • Nucleic acid vaccines may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms.
  • nucleic acid vaccines described herein may also be administered as a second line treatment after the standard first line treatments such as antibiotics and antivirals have failed to induce passive immunity.
  • the nucleic acid vaccines described herein are useful in settings where resistance to first line treatments has developed and disease persists and induces chronic disease.
  • Nucleic acid vaccines may be administered as part of a treatment regimen for latent viral infections, such as SARS-CoV-2 infections.
  • one or more polynucleotides are administered which ultimately produce proteins which result a desired immune response against SARS-CoV-2 is achieved to treat and/or prevent COVID-19.
  • the use of RNA in or as a vaccine overcomes the disadvantages of conventional genetic vaccination involving incorporating DNA into cells in terms of safeness, feasibility, applicability, and effectiveness to generate immune responses.
  • RNA molecules are considered to be significantly safer than DNA vaccines, as RNAs are more easily degraded. They are cleared quickly out of the organism and cannot integrate into the genome and influence the cell’s gene expression in an uncontrollable manner.
  • RNA vaccines it is also less likely for RNA vaccines to cause severe side effects like the generation of autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), vol. 2010, article ID623687). Transfection with RNA requires only insertion into the cell’s cytoplasm, which is easier to achieve than into the nucleus. However, RNA is susceptible to RNase degradation and other natural decomposition in the cytoplasm of cells. [0586] Various attempts to increase the stability and shelf life of RNA vaccines. US Pub. No. US 20050032730 to Von Der Mulbe et al. discloses improving the stability of mRNA vaccine compositions by increasing G(guanosine)/C(cytosine) content of the mRNA molecules. U.S.
  • Pat. No. 5,580,859 to Feigner et al. teaches incorporating polynucleotide sequences coding for regulatory proteins that binds to and regulates the stabilities of mRNA. While not wishing to be bound by theory, it is believed that the nucleic acid vaccines described herein may result in improved stability and therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs. Additionally, modified nucleosides, or combinations thereof, may be introduced into the nucleic acid vaccines described herein to activate the innate immune response. Such activating molecules are useful as adjuvants when combined with polypeptides and/or other vaccines. In certain embodiments, the activating molecules contain a translatable region which encodes for a polypeptide sequence useful as a vaccine, thus providing the ability to be a self-adjuvant.
  • the nucleic acid vaccines described herein may be used in the prevention, treatment and diagnosis of diseases and physical disturbances caused by infectious agents such as, but not limited to, SARS-CoV-2, or a VOC, VOI or VOHC of SARS-CovV-2.
  • the nucleic acid vaccines described herein may encode at least one polypeptide of interest (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2) and may be provided to an individual in order to stimulate the immune system to protect against the disease-causing agents.
  • the biological activity and/or effect from an infectious agent may be inhibited and/or abolished by providing neutralizing antibodies which have the ability to bind and neutralize the infectious agent; the neutralizing antibodies produced from the immune system stimulated by the polypeptides translated from the nucleic acid vaccine.
  • the polynucleotides encoding an immunogen may be delivered to cells to trigger multiple innate response pathways (see PCT Patent Application Publication Nos. W02012006377 and US Patent Publication No. US20130177639; the contents of each of which are herein incorporated by reference in their entirety).
  • the nucleic acid vaccines described herein may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see PCT Patent Application Publication Nos. W02012006372 and WO20 12006369 and US Publication Nos. US20130149375 and US20130177640; the contents of each of which are herein incorporated by reference in their entirety).
  • the nucleic acid vaccines described herein may be delivered to a mammal (e.g., human) in a dose amount large enough to be immunogenic for stimulating an immune response in the mammal.
  • the immune response can defend a viral infection, thereby, prevent and/or treat a disease.
  • the nucleic acid vaccines described herein may treat and/or prevent infectious diseases including viral infectious diseases such as COVID-19 caused by SARS-CoV-2, or a VOC, VOI or VOHC of SARS-CoV-2.
  • Nucleic acid vaccines described herein may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the nucleic acid vaccines described herein may be utilized to treat and/or prevent COVID-19 infection, including the diseases and conditions related to COVID-19 infection (including infection by the original and mutated versions of SARS- CoV-2).
  • Symptoms of COVID-19 infection are changing as more is learned about the disease but the current symptoms include fever or chills, cough, shortness of breath or difficulty breathing, fatigue, body aches, muscle aches, headaches, sore throat, congestion or runny nose, nausea and/or vomiting, diarrhea, and a new loss of taste or smell.
  • the nucleic acid vaccines described herein may be better designed, as compared to current anti-viral treatments, to produce the appropriate protein conformation on translation as the nucleic acid vaccines co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the nucleic acid vaccines are presented to the cellular system in a more native fashion. In some embodiments, the nucleic acid vaccines described herein are a tailored active vaccine for COVID-19 that not only can prevent infection by SARS-CoV-2 but can limit transmission of SARS-CoV-2.
  • the nucleic acid vaccines described herein may be used to prevent pandemic COVID-19 by reacting to emerging new strains with the very rapid nucleic acid based vaccine production process.
  • a single injection of a nucleic acid vaccine may provide protection for an entire season.
  • the nucleic acid vaccines described herein may be immunostimulatory.
  • the polynucleotide sequence of the nucleic acid vaccine may further comprise a sequence region encoding a cytokine that promotes the immune response, such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-y, GM-CFS, LT-a, or growth factors such as hGH.
  • a cytokine that promotes the immune response
  • a monokine such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-y
  • nucleic acid vaccines described herein encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 and may be used for the treatment and/or prevention of COVID-19
  • the nucleic acid vaccines described herein can produce much higher neutralizing antibody titers and they may produce responses early than commercially available anti-virals.
  • the nucleic acid vaccines described herein can produce 10 times, or 9X, or 8X, or 7X, or 6X, or 5X, or 4X, or 3X more neutralizing antibody titers than other vaccines.
  • administration of the nucleic acid vaccine may result in an increase in neutralizing antibody titer above a previous level (e.g., a pre-administration baseline level).
  • a previous level e.g., a pre-administration baseline level.
  • the difference in titer level between the post nucleic acid administration and the pre-administration levels may be, but is not limited to, at least 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14- fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95- fold, or 100-fold, or about 1 to 5-fold, about 5 to 10-fold, about 5 to 20-fold, about 5 to
  • administration of the nucleic acid vaccine results in neutralizing antibodies being present in a subject for at least 1 day post administration.
  • Antibodies may be present in a subject for, but not limited to, about 1 day, about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days, about 1 to 5 weeks, about 1 to 10 weeks, about 1 to 15 weeks, about 1 to 20 weeks, about 1 to 25 weeks, about 1 to 30 weeks, about 5 to 10 weeks, about 5 to 15 weeks, about 5 to 20 weeks, about 5 to 25 weeks, about 5 to 30 weeks, about 10 to 15 weeks,
  • administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at least 1 day post administration.
  • Antibodies may be detectable in a subject for, but not limited to, about 1 day, about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days, about 1 to 5 weeks, about 1 to 10 weeks, about 1 to 15 weeks, about 1 to 20 weeks, about 1 to 25 weeks, about 1 to 30 weeks, about 5 to 10 weeks, about 5 to 15 weeks, about 5 to 20 weeks, about 5 to 25 weeks, about 5 to 30 weeks, about 10 to 15
  • administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 1 day post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 14 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 21 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 28 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 35 days post administration.
  • administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 42 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 6 months (180 days) post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about one year post administration.
  • administration of the nucleic acid vaccine results in a subject producing neutralizing for at least 1 day post administration.
  • Antibodies may be produced by a subject for, but not limited to, about 1 day, about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days, about 1 to 5 weeks, about 1 to 10 weeks, about 1 to 15 weeks, about 1 to 20 weeks, about 1 to 25 weeks, about 1 to 30 weeks, about 5 to 10 weeks, about 5 to 15 weeks, about 5 to 20 weeks, about 5 to 25 weeks, about 5 to 30 weeks, about 10 to 15 weeks, about 10 to
  • administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at least about 1 day post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 14 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 21 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 28 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 35 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 42 days post administration.
  • administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 6 months (180 days or 26 weeks) post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 1 year post administration.
  • the nucleic acid vaccines described herein co-opt the natural cellular machinery to produce the appropriate protein conformation on translation. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the nucleic acid vaccines described herein are introduced to the cellular system in a way that is closer to the native way or the way normal cellular processing occurs. Additionally, formulations may be used to shield or target delivery of the nucleic acid vaccines to specific cells or tissues in the subject.
  • nucleic acid vaccines described herein represent a targeted active vaccine that not only can prevent infection but can limit transmission of COVID-19.
  • the nucleic acid vaccines may be used to prevent pandemic SARS-CoV-2 infection or COVID-19 by reacting to emerging new strains with the very rapid vaccine production process.
  • a single injection of nucleic acid vaccines encoding one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 may provide protection for at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, at least 13 years, at least 15 years or more than 15 years.
  • nucleic acid vaccines described herein may also be used to maintain or restore antigenic memory in a subject or population as part of a vaccination plan for COVID-19 or other diseases caused by SARS-CoV-2.
  • nucleic acid vaccines compositions may be created which include polynucleotides that encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 which are showing prevalence increased infection rates for the year.
  • the protein sequences of SARS-CoV-2 have been shown to change or mutate over time, wherein some of the mutations have shown increased infection rates.
  • nucleic acid vaccines compositions may be created which include polynucleotides that encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 which are showing prevalence increased infection rates for the year such as, but not limited to the D614G mutation in the spike protein.
  • the nucleic acid vaccines may be used to induce neutralizing antibodies in a subject.
  • the neutralization activity of the neutralizing antibodies induced by the present nucleic acid vaccines may correlate to the resulting effectiveness (e.g., immune protection) of the vaccines described herein.
  • the immune protection provided by the present nucleic acid vaccines may be greater than the immune protection provided in convalescent subjects.
  • the induced neutralization activity from the present nucleic acid vaccines may increase the recovery rate of those exposed to SARS-CoV-2 or whom are in convalescence.
  • the present nucleic acid vaccines induce higher neutralizing antibody titers in treated subjects.
  • the nucleic acid vaccines include at least 10 times, 9 times, 8 times, 7 times, 6 times, 5 times or 4 times more neutralizing antibody titers than other vaccines for COVID-19.
  • a vaccination scheme or plan is developed which allows for not only ongoing vaccination in the current year but memory booster vaccinations across years, strains, or groups thereof to establish and maintain memory in a population. In this manner, a population is less likely to succumb to any pandemic or outbreak involving recurrence of older strains. Any combination of a prior vaccine component strain can be utilized to create or design a memory booster vaccine.
  • nucleic acid vaccines which are memory booster vaccines are administered to boost memory across a time period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 years.
  • nucleic acid vaccines which are memory booster vaccines are administered to boost memory for alternating historic years including every other year from the past vaccine component strains relative to a current year.
  • selection of the vaccine components can be from every 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th or more years.
  • nucleic acid vaccines which are memory booster vaccines are administered to boost memory over ten-year periods.
  • the nucleic acid booster vaccine may be used in a population either once or periodically to create herd immunity which means greater than 30% of a population is protected.
  • the nucleic acid booster vaccine may be used in a population either once or periodically to create herd immunity against COVID-19 which means greater than 30% of a population is protected.
  • the nucleic acid vaccines are used to target at risk populations for COVID-19 such as those having pre-existing conditions including, but not limited to, cancer, chronic kidney disease, chronic obstructive pulmonary disease (COPD), immunocompromised state (weakened immune system) from solid organ transplant, blood or bone marrow transplant, immune deficiencies, HIV, and use of corticosteroids or other immune weaking medicines, obesity (body mass index (BMI) of 30 or higher), heart conditions such as heart failure, coronary artery disease, or cardiomyopathies, sickle cell disease, type 1 or type 2 diabetes mellitus, asthma (moderate-to-severe), cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, neurological conditions such as dementia, liver disease, pregnancy, pulmonary fibrosis, smoking, and thalassemia.
  • COVID-19 those having pre-existing conditions including, but not limited to, cancer, chronic kidney disease, chronic obstructive pulmonary disease (COPD), immunocompromised state (weakened immune system) from solid
  • the nucleic acid vaccines are used to protect healthcare workers who are at risk of being exposed to SARS-CoV-2.
  • the nucleic acid vaccine of the present disclosure comprises a LNP formulated polynucleotide encoding the full-length S protein with D614G (SEQ ID NO: 27) (as referred to as “PTX-B”).
  • PTX-B LNP formulated polynucleotide encoding the full-length S protein with D614G (SEQ ID NO: 27)
  • Methods for use of PTX-B vaccine to induce a protective immune response in a subject is provided.
  • the protective immune response can protect a subject against a viral infection, such as infection by SAR-CoV-2 original strain and its variant thereof.
  • the SARS-CoV-2 variant can be any VOC, VOI and/or VOHC strain.
  • the variant is an Alpha variant, a Beta variant, or a Delta variant.
  • the nucleic acid vaccine PTX-B is used to protect against an Alpha variant.
  • the Alpha variant is Alpha (B.1.1.7).
  • the nucleic acid vaccine PTX-B is used to protect against a Beta variant.
  • the Beta variant is beta (B.1.351).
  • the nucleic acid vaccine PTX-B is used to protect against a Delta variant.
  • the Delta variant is delta (B.1.617.2).
  • the nucleic acid vaccine PTX-B is used to induce neutralizing antibodies in a subject.
  • the nucleic acid vaccine PTX-B induces antibodies with high neutralization titers.
  • the neutralization titers of induced antibodies are as least 100 fold, 90 fold, 80 fold, 70 fold, 60 fold, 50 fold, 40 fold, 30 fold, 25 fold, 20 fold, 15 fold, 10 fold or 5 fold of the titers in sera from infected subjects or convalescent subjects.
  • a dosing regimen of the nucleic acid vaccine PTX-B is provided.
  • the dose of PTX-B ranges from 1 pg to 500 pg, from 1 pg to Img, from Img to lOmg, from Img to lOOmg, or from lOmg to lOOmg/kg of a subject body weight.
  • a dose of PTX-B can achieve a dose level of about 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 60pg, 70 pg, 80 pg, 90 pg, or 100 pg of the nucleic acid vaccine.
  • At least one dose of the nucleic acid vaccine PTX-B is administered.
  • the at least one dose administered is 16 ug.
  • the at least one dose administered is 40 ug.
  • the at least one dose administered is 100 ug.
  • at least one booster dose of PTX-B is administered.
  • the booster dose may be administered to the subject, one month, two months, three months, four months, six months, or one year or greater after the subj ect receives the first dose of PTX-B.
  • more than one booster dose e.g., two, three, four or more, is administered to the subject.
  • the booster dose administered is 16 ug.
  • the booster dose administered is 40 ug.
  • the booster dose administered is 100 ug.
  • the nucleic acid vaccine of the present disclosure comprises a LNP formulated polynucleotide encoding the full-length S protein of SARS- CoV-2 variant B.1.351 (South African variant (SEQ ID NO: 43).
  • the nucleic acid vaccine may be administered to a black/ African American population.
  • the nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) black/ African American subjects.
  • the nucleic acid vaccine may be administered to a population comprised of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% black/ African American subjects.
  • the nucleic acid vaccine may be administered to a population comprised about 88% black/ African American subjects.
  • the nucleic acid vaccine may be administered to subjects from any country in the world.
  • the nucleic acid vaccine may be administered to subjects from Afghanistan, Bulgaria, Norway, Andorra, Angola, Antigua and Barbuda, Argentina, Armenia, Australia, Austria, Azerbaijan, Bahamas,oane, Bangladesh, Barbados, Finland, Belgium, Caribbean, Benin, Bhutan, Cambodia, Laubo and Herzegovina, Botswana, Brazil, Brunei, Bulgaria, Burkina Faso, Burundi, Cabo Verde, Cambodia, Cameroon, Canada, Central African Republic (CAR), Chad, Chile, China, Colombia, Comoros, Congo, Democratic Republic of the, Congo, Republic of the, Costa Rica, Cote d'Ilude, Croatia, Cuba, Cyprus, Czechia, Denmark, Djibouti, Dominica, Dominican Republic, Ecuador, Egypt, El Salvador, Equatorial Guinea, Eritrea, Estonia, Eswatini, Ethiopia, Fiji, Finland, France, Gabon, Gamb
  • the nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of subjects from one country. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of subjects from South Africa.
  • the nucleic acid vaccine may be administered to a population comprised of both males and females. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of males or a majority (i.e., at least about 50.1%) of females. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of males. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of females. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of about 52% of males. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of about 48% of females.
  • the nucleic acid vaccine may be administered to a population comprised of subjects of any age. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages from about 1 year old to about 120 years old or more. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages from about birth to about 18 years old inclusive (i.e., minors). In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages from greater than about 18 years old to about 64 years old inclusive (i.e., adults). In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages greater than 64 years old to death (i.e., seniors or the elderly).
  • the nucleic acid vaccine may be administered to a population comprised of subjects whose mean age is about 5, 10, 15, 20, 25, 30, 35, 40,
  • the nucleic acid vaccine may be administered to a population comprised of subjects whose mean age is about 31 years old. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects whose mean age is about 32 years old. [0631] In some embodiments, subjects may be administered more than one dose of the nucleic acid vaccine. In certain embodiments, subjects may be administered at least 2 doses of the nucleic acid vaccine. In some embodiments, subjects may be administered at least one first dose and at least one second dose of the nucleic acid vaccine.
  • the at least one second dose may be administered between about 1 and 30 days after the at least one first dose. In some embodiments, the at least one second dose may be administered between about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days after the at least one first dose. In some embodiments, the at least one second dose may be administered between about 20 after the at least one first dose. In some embodiments, the at least one second dose may be administered between about 27 after the at least one first dose.
  • the safety and/or tolerability of the nucleic acid vaccine may be assayed.
  • Various methods of assaying unintended or unanticipated effects also known as “side-effects,” of a vaccine, including nucleic acid vaccines, are known in the art and may be suitable for use in assaying safety and/or tolerability.
  • Example methods for assaying safety and/or tolerability include but are not limited to monitoring for and recording incidences of: injection site reactions (e.g., arm check evaluations including pain, tenderness, erythema/redness, induration/swelling), solicited adverse events (SAEs; e.g., fever, chills, nausea/vomiting, diarrhea, headache, fatigue, myalgia), unsolicited adverse events (AEs), treatment emergent adverse events (TEAEs) including medically attendant adverse events (MAAEs new onset chronic diseases (NOCDs), adverse event of special interests (AESIs), potentially immune-mediated medical conditions (PIMMCs) and any combinations thereof.
  • injection site reactions e.g., arm check evaluations including pain, tenderness, erythema/redness, induration/swelling
  • SAEs solicited adverse events
  • AEs unsolicited adverse events
  • TEAEs treatment emergent adverse events
  • the safety and/or tolerability of the nucleic acid vaccine may be assayed for a period of time after administration of at least one dose of the nucleic acid vaccine.
  • safety and/or tolerability may be assayed for about 1 week, about 1 to 4 weeks, about 2 to 6 weeks, about 1 to 8 weeks, about 1 to 12 weeks, about 1 to 24 weeks, about 1 to 36 weeks, about 1 to 54 weeks, more than 54 weeks, or any combination thereof post administration of the first dose of the nucleic acid vaccine.
  • kits for conveniently and/or effectively carrying out methods of the present disclosure.
  • kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
  • kits for modulating the expression of genes in vitro or in vivo comprising nucleic acid vaccine compositions of the present disclosure or a combination of nucleic acid vaccine compositions of the present disclosure, nucleic acid vaccine compositions modulating other genes, siRNAs, miRNAs or other oligonucleotide molecules.
  • the kit may further comprise packaging and instructions and/or a delivery agent to form a formulation, e.g., for administration to a subject in need of treatment using the nucleic acid vaccine compositions described herein.
  • the delivery agent may comprise a saline, a buffered solution, a lipidoid, a dendrimer or any suitable delivery agent.
  • the buffer solution may include sodium chloride, calcium chloride, phosphate and/or EDTA.
  • the buffer solution may include, but is not limited to, saline, saline with 2mM calcium, 5% sucrose, 5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium, Ringer’s lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See U.S. Pub. No. 20120258046; herein incorporated by reference in its entirety).
  • the buffer solutions may be precipitated or it may be lyophilized.
  • the amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations.
  • the components may also be varied in order to increase the stability of nucleic acid vaccine compositions in the buffer solution over a period of time and/or under a variety of conditions.
  • the present disclosure provides for devices which may incorporate nucleic acid vaccine compositions of the present disclosure. These devices can contain a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient.
  • Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, electroporation devices, iontophoresis devices, multi-layered microfluidic devices.
  • the devices may be employed to deliver nucleic acid vaccine compositions of the present disclosure according to single, multi- or split-dosing regiments.
  • the devices may be employed to deliver nucleic acid vaccine compositions of the present disclosure across biological tissue, intradermal, subcutaneously, or intramuscularly. More examples of devices suitable for delivering oligonucleotides are disclosed in International Publication WO 2013/090648, the contents of which are incorporated herein by reference in their entirety.
  • Administered in combination means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
  • Adjuvant means a substance that enhances a subject’s immune response to an antigen.
  • the nucleic acid vaccines described herein may optionally comprise one or more adjuvants.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
  • mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig.
  • animals include, but are not limited to, mammals,
  • Antigen refers to a composition, for example, a substance or agent which causes an immune response in an organism, e.g., causes the immune response of the organism to produce antibodies against the substance or agent in particular, which provokes an adaptive immune response in an organism.
  • Antigens can be any immunogenic substance including, in particular, proteins, polypeptides, polysaccharides, nucleic acids, lipids and the like. Exemplary antigens are derived from infectious agents.
  • Such agents can include parts or subunits of infectious agents, for example, coats, coat components, e.g., coat protein or polypeptides, surface components, e.g., surface proteins or polypeptides, capsule components, cell wall components, flagella, fimbrae, and/or toxins or toxoids) of infectious agents, for example, bacteria, viruses, and other microorganisms.
  • infectious agents for example, coats, coat components, e.g., coat protein or polypeptides, surface components, e.g., surface proteins or polypeptides, capsule components, cell wall components, flagella, fimbrae, and/or toxins or toxoids
  • infectious agents for example, bacteria, viruses, and other microorganisms.
  • antigens for example, lipids and/or nucleic acids are antigenic, preferably, when combined with proteins and/or polysaccharides.
  • association means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
  • An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.
  • Bifunctional refers to any substance, molecule or moiety which is capable of or maintains at least two functions. The functions may affect the same outcome or a different outcome. The structure that produces the function may be the same or different.
  • Biocompatible As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.
  • Biodegradable As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.
  • biologically active refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • a polynucleotide described herein may be considered biologically active if even a portion of the polynucleotides is biologically active or mimics an activity considered biologically relevant.
  • Chimera As used herein, “chimera” is an entity having two or more incongruous or heterogeneous parts or regions.
  • Compound As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. [0654]
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. [0657]
  • the compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • conserved refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
  • two or more sequences are said to be “completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another.
  • two or more sequences are said to be “conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an polynucleotide or polypeptide or may apply to a portion, region or feature thereof.
  • Controlled Release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • Cytostatic refers to inhibiting, reducing, suppressing the growth, division, or multiplication of a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
  • Cytotoxic refers to killing or causing injurious, toxic, or deadly effect on a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
  • Delivery refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.
  • Delivery agent refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide to targeted cells.
  • Destabilized As used herein, the term “destable,” “destabilize,” or “destabilizing region” means a region or molecule that is less stable than a starting, wildtype or native form of the same region or molecule.
  • Detectable label refers to one or more markers, signals, or moieties which are attached, incorporated or associated with another entity that is readily detected by methods known in the art including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable labels may be located at any position in the peptides or proteins disclosed herein. They may be within the amino acids, the peptides, or proteins, or located at the N- or C-termini.
  • Digest As used herein, the term “digest” means to break apart into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.
  • Dosing regimen is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.
  • Encapsulate As used herein, the term “encapsulate” means to enclose, surround or encase.
  • Encoded protein cleavage signal refers to the nucleotide sequence which encodes a protein cleavage signal.
  • nucleic acid vaccines are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
  • Effective Amount As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.
  • Exosome is a vesicle secreted by mammalian cells or a complex involved in RNA degradation.
  • expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Feature refers to a characteristic, a property, or a distinctive element.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present disclosure relates to pharmaceutical compositions and methods for the preparation, manufacture and therapeutic use of nucleic acid vaccines comprising lipid nanoparticles (LNP) wherein the LNPs comprise cationic lipids, non-cationic lipids, sterols and conjugated lipids, wherein said LNPs comprise polynucleotide sequences set forth in SEQ ID NOS:7, 20, 26, 27 or 32 encoding one or more structural proteins of SARS-CoV-2 set forth in SEQ ID NO:2 and variants for the treatment, mitigation, amelioration and/or prevention of COVID-19.

Description

COMPOSITIONS AND METHODS FOR THE PREVENTION AND/OR TREATMENT OF COVID-19
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to US Provisional Application No. 63/378,263 filed October 4, 2022 entitled Compositions and Methods for the Prevention and/or Treatment of COVID-19, US Provisional Application No. 63/378,264 filed October 4, 2022 entitled Compositions and Methods for the Prevention and/or Treatment of COVID- 19, US Provisional Application No. 63/384,902 filed November 23, 2022 entitled Compositions and Methods for the Prevention and/or Treatment of COVID-19, and US Provisional Application No. 63/514,636 filed July 20, 2023 entitled Compositions and Methods for the Prevention and/or Treatment of CO VID-19; the contents of each of which are herein incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 2023-10-03-PCTSEQLST.xml, was created on October 3, 2023 and is 130,133 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
FIELD
[0003] The present disclosure generally relates to compositions, formulations, methods, and/or uses of nucleic acid vaccines, specifically nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment and/or prevention of COVID-19, including mitigation of physiologic effects of infection and/or symptoms.
BACKGROUND
[0004] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus which began infecting mammals in 2019 in China and has spread to a pandemic. SARS-CoV-2 infection causes coronavirus disease 2019 (termed “COVID- 19”), which affects mammals in different ways including individuals who are asymptomatic to individuals who have a wide range of symptoms that range from mild symptoms to severe illness or death.
[0005] Vaccines are an effective way to provide prophylactic protection against infective diseases. Currently, there are limited vaccines available to prevent, alleviate and/or treat COVID-19. Treatment of COVID-19 has been limited to the management of symptoms and/or the side effects of the disease. Thus, there remains a strong need for COVID-19 vaccines including formulations for delivering the vaccines to a range of different target T-cells.
SUMMARY
[0006] The present disclosure provides nucleic acid vaccines, compositions and formulations comprising nucleic acid vaccines, and methods of using same for preventing infection of coronavirus, and for the prevention, alleviation and treatment of COVID-19. The nucleic acid vaccines may include polynucleotides which encode at least one antigen protein, fragment or variant thereof of SARS-CoV-2. The SARS-CoV-2 antigen protein is a structural protein of SARS-CoV-2. The structural protein may be the spike protein, the membrane protein, the nucleocapsid phosphoprotein or the envelope protein. Non-limiting examples of the amino acid sequences of these structural proteins are shown in Table 1 (SEQ ID Nos: 1-6 and 15-19).
[0007] Provided herein are nucleic acid vaccines for COVID-19 for use in a method of vaccinating a subject for COVID-19, wherein the nucleic acid vaccine may include at least one polynucleotide encoding at least one structural protein or a fragment thereof of SARS- CoV-2.
[0008] Provided herein are methods of inducing an immune response in a subject by administering the nucleic acid vaccines described herein in an effective amount to produce an immune response. The immune response may be, but is not limited to, a T-cell response or a B cell response. As a non-limiting example, the immune response may be produced by a single administration of the nucleic acid vaccines described herein. As another non- limiting example, the immune response may be produced by a booster administration of the nucleic acid vaccines described herein. The administering the pharmaceutical composition may produce a dose-responsive immune response in the subject. As a nonlimiting example, the dose-responsive immune response may comprise induction of one or more of SARS-CoV-2 spike protein specific IgG, IgGl, IgG2a, IgG2b, IgM and IgA antibodies in the subject. As another non-limiting example, the dose-responsive immune response may comprise induction of one or more of IL-2+ T-cells, IL-4+ T-cells, and IFN- gamma+ T-cells. In some embodiments, the administering the pharmaceutical composition does not induce significant adverse reactions in the subject.
[0009] Provided herein are methods of treating and/or preventing CO VID-19 in a subject by administering the nucleic acid vaccines described herein.
[0010] Provided herein are pharmaceutical compositions and formulations of the nucleic acid vaccines for the treatment and prevention of COVID-19.
[0011] The nucleic acid vaccines described herein may be formulated in one or more lipid nanoparticles (LNPs).
[0012] In some embodiments the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
[0013] In some embodiments the LNP comprises (a) 50-65 mol% of a cationic lipid, (b) a non-cationic lipid comprising 4-10 mol% of phospholipids and 30-40 mol% of cholesterol, and (c) 0.5-2 mol% of a conjugated lipid.
[0014] In some embodiments the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid.
[0015] In some embodiments the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid.
[0016] In some embodiments the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid. [0017] In some embodiments the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid.
[0018] In some embodiments the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid.
[0019] In some embodiments the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid.
[0020] In some embodiments the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid.
[0021] In some embodiments the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid.
[0022] In some embodiments the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid.
[0023] In some embodiments the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5-50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid.
[0024] In some embodiments the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid.
[0025] In some embodiments the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid.
[0026] In some embodiments the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA. [0027] In some embodiments the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA.
[0028] Also provided herein are nucleic acid vaccines for COVID-19, comprising about 0.2 mg/mL mRNA, wherein the mRNA comprises a coding region with a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region with a nucleic acid sequence as set forth in SEQ ID NO: 7. The nucleic acid vaccines may be formulated as a 2 mL fill in a 3 mL glass vial. The nucleic acid vaccines may be formulated as a 2.4 mL fill in a 6 ml glass vial.
[0029] In some embodiments, administering the nucleic acid vaccines to a subject comprises administering about 5 pg to about 100 pg of the mRNA to the subject. For instance, the methods may comprise administering about 16 pg of the mRNA to the subject. Alternatively, the methods may comprise administering about 40 pg of the mRNA to the subject. Or the methods may comprise administering about 100 pg of the mRNA to the subject.
[0030] In some embodiments, administering the nucleic acid vaccines to a subject comprises administering about 0.025 mL to about 0.5 mL of the nucleic acid vaccine to the subject. For instance, the methods may comprise administering about 0.025 mL of the nucleic acid vaccine to the subject, about 0.05 mL of the nucleic acid vaccine to the subject, about 0.08 mL of the nucleic acid vaccine to the subject, about 0.2 mL of the nucleic acid vaccine to the subject, or about 0.5 mL of the nucleic acid vaccine to the subject.
[0031] In some embodiments of the methods provided, the administering comprises an intramuscular (IM) injection of the nucleic acid vaccine to the subject.
[0032] The nucleic acid vaccines may be administered to a subject in a first dose of the nucleic acid vaccine followed by a second dose of the nucleic acid vaccine after between about 1 and about 5 weeks. In some embodiments, the second dose of the nucleic acid vaccine is administered about 4 weeks after the first dose. [0033] In some embodiments, anti-Spike protein IgG antibodies are detected in the subject by day 28 after receiving a first dose of the nucleic acid vaccine.
[0034] In some embodiments, anti-Spike protein IgG antibodies are detected in the subject by day 28 after receiving a first dose of the nucleic acid vaccine and are enhanced after receiving a second dose of the nucleic acid vaccine in the subject by day 42.
[0035] In some embodiments, anti-Spike protein IgG antibodies in the subject are increased to 10-fold higher than the average values of anti-Spike protein IgG antibodies from serum samples from SARS-CoV-2 convalescent patients.
[0036] In some embodiments, the SARS-CoV-2 neutralizing antibodies are detected in the subject by day 28 after a first dose of the nucleic acid vaccine.
[0037] In some embodiments, the SARS-CoV-2 neutralizing antibodies are enhanced after a second dose of the nucleic acid vaccine in the subject by day 42.
[0038] In some embodiments, the subject produces neutralizing antibodies to SARS- CoV-2 for at least about 1 days post initial administration.
[0039] In some embodiments, the subject produces neutralizing antibodies to SARS- CoV-2 for about 21 days post initial administration.
[0040] In some embodiments, the subject produces neutralizing antibodies to SARS- CoV-2 for about 28 days post initial administration.
[0041] In some embodiments, the subject produces neutralizing antibodies to SARS- CoV-2 for about 42 days post initial administration.
[0042] In some embodiments, the subject produces neutralizing antibodies to SARS- CoV-2 for at least about 42 days post initial administration.
[0043] In some embodiments, the subject produces neutralizing antibodies to SARS- CoV-2 for about 1 year post initial administration.
[0044] In some embodiments, the amount of neutralizing antibody in a subject increased by at least about 4-fold relative to levels prior to administration of the nucleic acid vaccine. [0045] In some embodiments, the amount of neutralizing antibody in a subject increased by at least about 5-fold relative to levels prior to administration of the nucleic acid vaccine.
[0046] In some embodiments, the amount of neutralizing antibody in a subject increased by at least about 6-fold relative to levels prior to administration of the nucleic acid vaccine.
[0047] In some embodiments, the amount of neutralizing antibody in a subject increased by at least about 7-fold relative to levels prior to administration of the nucleic acid vaccine.
[0048] In some embodiments, the amount of neutralizing antibody in a subject increased by more than 4-fold relative to levels prior to administration of the nucleic acid vaccine.
[0049] In some embodiments, the amount of neutralizing antibody in a subject increased by more than 5-fold relative to levels prior to administration of the nucleic acid vaccine.
[0050] In some embodiments, the amount of neutralizing antibody in a subject increased by more than 6-fold relative to levels prior to administration of the nucleic acid vaccine.
[0051] In some embodiments, the amount of neutralizing antibody in a subject increased by more than 7-fold relative to levels prior to administration of the nucleic acid vaccine.
[0052] In some embodiments, the amount of neutralizing antibody in a subject increased by about 100-fold relative to levels prior to administration of the nucleic acid vaccine.
[0053] In some embodiments, the amount of neutralizing antibody in a subject increased by about 200-fold relative to levels prior to administration of the nucleic acid vaccine. [0054] In some embodiments, the nucleic acid vaccine is administered as a booster vaccine. The nucleic acid vaccine may be administered as the original vaccination or another vaccine may be administered as the original vaccination to the subject.
[0055] Thus, the disclosure provides nucleic acid vaccines for COVID-19 for use in a method of vaccinating a subject for COVID-19, wherein the nucleic acid vaccine comprises about 0.2 mg/mL mRNA, wherein the mRNA comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7, and wherein the nucleic acid vaccine is formulated for intramuscular (IM) injection and formulated in a lipid nanoparticle (LNP).
[0056] The details of various embodiments are set forth in the description below. Other features, objects and advantages will be apparent from the description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 A shows a histogram of solicited TEAEs of systemic events and local reactions reported in the Safety Population.
[0058] FIG. IB shows a histogram of solicited TEAEs (systemic events and local reactions) reported in the Safety Population (left graph) and evaluable immunogenicity population (right graph) At each time point (Dose 1 or Dose 2) the left histogram bar corresponds to subjects administered PTX-B vaccine, the right histogram bar corresponds to subject administered COMIRNATY®.
[0059] FIG. 1C shows a histogram of solicited systemic events in the Safety Population within 7 days post each dose.
[0060] FIG. ID shows a histogram of solicited local events in the Safety Population within 7 days post each dose.
[0061] FIG. 2 shows results from Nab titer assays in the EIP group. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
[0062] FIG. 3 shows results from Nab titer assays in the post-hoc EIP group with high baseline Nab titers removed. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
[0063] FIG. 4 shows the results of administering either PTX-B (grey bar) or COMIRNATY® (black bar) on Nab levels (as fold increase).
[0064] FIG. 5 shows results from Nab titer assays in the mITT group with high baseline Nab titers removed. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
[0065] FIG. 6A shows results from Nab titer assays in the full mITT. At each timepoint the left box-plot corresponds to subjects administered PTX-B vaccine, the right box plot corresponds to subject administered COMIRNATY®.
[0066] Fig. 6B shows a histogram where PTX-B was non-inferior versus COMIRNATY®.
[0067] Fig. 6C shows a histogram where PTX-B was non-inferior and numerically higher versus COMIRNATY®.
[0068] Fig. 6D shows histograms for the mITT population and the seronegative mITT population where was non-inferior and numerically higher versus COMIRNATY®.
[0069] Fig. 6E shows a histogram for Interferon-y (left graph) and Interleukin-5 (right graph) for PTX-B and COMIRNATY®.
[0070] Fig. 6F shows the saliva antibody response for PTX-B and COMIRNATY®. [0071] Fig. 6G shows a histogram for Cellular Immunity (Interferon-y) during the 6 months after primary vaccination.
[0072] FIG. 7 shows the results from Nab titer assays in subjects with a baseline titer greater than 100 (> 100) after PTX-B administration.
[0073] FIG. 8 shows the anti-RBD and anti-spike IgG at day 42 as determined by MSD.
[0074] FIG. 9 shows the anti-RBD and anti-spike IgG up to six months post vaccination by ELISA. [0075] FIG. 10 shows the anti-spike IgG up to 42 post vaccination determined by ELISA.
[0076] FIG. 11 shows the anti-RBD and anti-spike IgA up to six months post vaccination by ELISA.
[0077] FIG. 12 shows the pseudovirus neutralization titers for PTX-B vaccinated cohorts.
[0078] FIG. 13 shows the correlation of the Lentivirus and rVSV Neutralization Assay (Day 28 and Day 42).
[0079] FIG. 14 shows that PTX-B induces neutralizing antibodies against variants of concern (VOCs).
DETAILED DESCRIPTION
I. INTRODUCTION
[0080] The following description sets forth exemplary compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.
[0081] Described herein are polynucleotides (e.g., mRNAs), compositions, formulations, methods, and/or use of nucleic acid vaccines, specifically nucleic acid vaccines comprising polynucleotides encoding one or more antigen proteins, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19. The antigen protein may be a structural protein of SARS-CoV-2. The structural protein may be the spike(S) protein, the membrane(M) protein, the nucleocapsid(N) phosphoprotein or the envelope(E) protein.
[0082] In some embodiments, at least one component of the nucleic acid vaccine is a polynucleotide encoding at least one of the antigen proteins or the fragments or variants of the antigen proteins of SARS-CoV-2. The antigen protein may be a structural protein of SARS-CoV-2. The polynucleotide may be a RNA polynucleotide such as an mRNA polynucleotide. [0083] In some embodiments, the nucleic acid vaccine includes at least one mRNA polynucleotide encoding at least one of the structural proteins or the fragments or variants of the structural proteins of SARS-CoV-2.
[0084] 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 polypeptide of interest of SARS-CoV-2 may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides or variants of polypeptides, which independently may be encoded by one or more regions or parts or the whole of a polynucleotide from SARS-CoV-2. As used herein, the term “polypeptides of interest” refer to any polypeptide which is selected to be encoded within, or whose function is affected by, the polynucleotides described herein. Any of the peptides or polypeptides described herein may be antigenic (also referred to as immunogenic).
[0085] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function, or origin. In some embodiments, the polypeptides of interest are antigens encoded by the polynucleotides as described herein.
[0086] In some embodiments, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. [0087] The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, or at least about 85%, more preferably at least about 90%, even more preferably at least about 95% identical (homologous) to a native or reference sequence.
[0088] In some embodiments “variant mimics” are provided. As used herein, the term “variant mimic” is one which contains one or more amino acids which would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.
[0089] “Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gap and penalties introduced in the calculation.
[0090] By “homologs” as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
[0091] “Analogs”, as used herein, is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide. [0092] In some embodiments, the present disclosure contemplates several types of compositions which are polypeptide based 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 has been modified and/or changed in any way relative to a reference molecule or starting molecule.
[0093] For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences described herein (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support.
[0094] “ Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
[0095] As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of nonconservative substitutions include the substitution of a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
[0096] “ Insertional variants” when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. “Immediately adjacent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.
[0097] “Deletional variants” when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.
[0098] “ Covalent derivatives” when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant hosT-cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.
[0099] “Features” when referring to polypeptides are defined as distinct amino acid sequence-based components of a molecule. Features of the polypeptides encoded by the polynucleotides described herein include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof. [0100] As used herein when referring to polypeptides the term “surface manifestation” refers to a polypeptide-based component of a protein appearing on an outermost surface. [0101] As used herein when referring to polypeptides the term “local conformational shape” means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.
[0102] As used herein when referring to polypeptides the term “fold” refers to the resultant conformation of an amino acid sequence upon energy minimization. A fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces. Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.
[0103] As used herein the term “turn” as it relates to polypeptide conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
[0104] As used herein when referring to polypeptides the term “loop” refers to a structural feature of a polypeptide which may serve to reverse the direction of the backbone of a peptide or polypeptide. Where the loop is found in a polypeptide and only alters the direction of the backbone, it may comprise four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Bio., 1266 (4): 814- 830; 1997). Loops may be open or closed. Closed loops or “cyclic” loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties. Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having disulfide bridges or alternatively bridging moieties may be non-protein based such as the dibromozylyl agents used herein.
[0105] As used herein when referring to polypeptides the term “half-loop” refers to a portion of an identified loop having at least half the number of amino acid resides as the loop from which it is derived. It is understood that loops may not always contain an even number of amino acid residues. Therefore, in those cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of amino acids of the loop/2+/-0.5 amino acids).
[0106] As used herein when referring to polypeptides the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
[0107] As used herein when referring to polypeptides the term “half-domain” means a portion of an identified domain having at least half the number of amino acid resides as the domain from which it is derived. It is understood that domains may not always contain an even number of amino acid residues. Therefore, in those cases where a domain contains or is identified to comprise an odd number of amino acids, a half-domain of the odd- numbered domain will comprise the whole number portion or next whole number portion of the domain (number of amino acids of the domain/2+/-0.5 amino acids). For example, a domain identified as a 7 amino acid domain could produce half-domains of 3 amino acids or 4 amino acids (7/2=3.5+1-0.5 being 3 or 4). It is also understood that sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
[0108] As used herein, when referring to polypeptides the term “site” as it pertains to amino acid-based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.” A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide-based molecules described herein.
[0109] As used herein the terms “termini” or “terminus” when referring to polypeptides refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide-based molecules described herein may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins described herein are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide-based moiety such as an organic conjugate. [0110] Once any of the features have been identified or defined as a desired component of a polypeptide to be encoded by a polynucleotide described herein, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules described herein. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full-length molecule would.
[OHl] In a polypeptide, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids. The modifications may be various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) modifications.
[0112] Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis or a priori incorporation during chemical synthesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
[0113] In some embodiments, the polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation. As used herein a “consensus” sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites. [0114] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical to a reference protein. The protein fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids in length. In another example, any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids, or more, which are about 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 can be utilized in accordance with the nucleic acid vaccines described herein. In certain embodiments, a polypeptide to be utilized in accordance with the nucleic acid vaccines 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. [0115] As such, polynucleotides of the present disclosure encode peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the peptide or polypeptide sequences disclosed herein. The polynucleotides may also contain substitutions, insertions and/or additions, deletions and covalent modifications with respect to the polynucleotide reference sequences.
[0116] Reference molecules (polypeptides or polynucleotides) may share a certain identity with the designed molecules (polypeptides or polynucleotides). The term “identity” as known in the art, refers to a relationship between the sequences of two or more peptides, polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleosides. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, N.Y., 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, N.Y, 1991; and Carillo et al., SIAM J. Applied Math. 48: 1073; 1988).
[0117] In some embodiments, the encoded polypeptide variant may have the same or a similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide. Generally, variants of a particular polynucleotide or polypeptide described herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul et al.„ Gapped BLAST and PSLBLAST: a new generation of protein database search programs, Nucleic Acids Res. 1997, 25:3389-3402.) Other tools are described herein, specifically in the definition of “Identity.”
II. COMPOSITIONS OF THE PRESENT DISCLSOURE
SARS-CoV-2
[0118] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus which causes coronavirus disease 2019 termed “COVID-19.” COVID-19 affects mammals in different ways including individuals who are asymptomatic to individuals who have a wide span of symptoms that range from mild symptoms to severe illness or death. To date, about 80% of COVID-19 patients have mild to moderate symptoms whereas about 20% may develop complications such as sever 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 difficulty breathing, fatigue, body aches, muscle aches, headaches, sore throat, congestion or runny nose, nausea and/or vomiting, diarrhea, and a new loss of taste or smell.
[0119] The genome of SARS-CoV-2 encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and nonstructural proteins (named nspl to nspl6) and accessory proteins. The viral surface spike protein is generally the leading mediator for viral entry into cells. SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Another feature of the spike protein of SARS-CoV-2 is that the protein has a functional furin cleavage site at the S1-S2 boundary (SI is the receptor binding unit and S2 is the membrane fusion unit), which can preactivate the entry of many viruses including SARS-CoV-2. SARS-CoV-2 spike has been used as a protective antigen that elicits neutralizing antibodies in various vaccine developing strategies. The membrane protein and the envelope protein are for viral assembly. The envelop protein (E) can forms a homopentameric cation channel that is important for virus 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 has 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 herein incorporated by reference in their entirety). Additionally, the nucleocapsid protein in SARS-CoV-2 can modulate the host cell machinery and may be included in regulatory roles in the viral life cycle.
[0120] 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. There are six RBD amino acids have been shown to be critical for binding to ACE2 receptors and the SARS- CoV-2 genome appears to have a RBD that has a high affinity binding to ACE2 for 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 herein incorporated by reference in their entirety).
[0121] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein encode the full-length polypeptide of a structural protein, or a fragment or variant of the structural protein of SARS-CoV-2, such as the spike protein, the nucleocapsid protein, the envelop protein or the membrane protein.
[0122] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein encode more than one fragment or variant of a structural protein of SARS-CoV-2, such as the spike protein, the nucleocapsid protein, the envelop membrane and/or the membrane protein.
[0123] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein encode a mutated variant of one of the structural proteins, or a fragment of the mutated variant of the structural proteins of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change from Aspartic Acid to Glycine in one of the structural proteins of SARS-CoV-2.
[0124] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein encode a full-length polypeptide of the spike protein, or a fragment, or a variant of the spike protein of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change from Aspartic Acid to Glycine in the spike protein of SARS-CoV-2. As a non-limiting example, the variant may be a single amino acid change from Aspartic Acid to Glycine at position 614 (D614G) in the spike protein of SARS-CoV-2 (Korber et al.;
Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus; Cell,' 2020, 182(4): 812-827; the contents of which is herein incorporated by reference in its entirety).
[0125] In some embodiments, the nucleic acid vaccine described herein may encode one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2. Non-limiting examples of proteins, peptides, fragments or variants of the structural proteins of SARS-CoV-2 are provided in Table 1. In the table, the NCBI reference number is also provided if known. Table 1. Structural Protein Sequences of SARS-CoV-2
Figure imgf000024_0001
[0126] In some embodiments, the nucleic acid vaccine described herein may encode at least one structural protein with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1.
[0127] In some embodiments, the nucleic acid vaccine may be an mRNA vaccine that, when translated, produces one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2. Accordingly, the polynucleotides of the mRNA vaccine are mRNA polynucleotides encoding one or more proteins, peptides, fragments or variants thereof of the structural proteins of SARS-CoV-2.
[0128] In one embodiment, the coding sequences of mRNA vaccines described herein may be based on the coding sequence of the spike(S) protein from the genome of SARS- CoV-2 Wuhan-Hu-1 isolate (GenBank: NM908947.3, complete genome sequence). In some embodiments, a change of the code for a single amino acid change from D614 to G614 is introduced to match the amino acid of the current dominant circulating strains. [0129] Non-limiting examples of a RNA sequence encoding proteins, peptides, fragments or variants of the structural proteins of SARS-CoV-2 are provided in Table 2.
Table 2. Sequences of Spike protein of SARS-CoV-2
Figure imgf000025_0001
[0130] In some embodiments, the mRNA sequence encoding the spike protein with D614G mutation of SARS-CoV-2 comprises the coding region of SEQ ID NO: 7, or a fragment or variant thereof.
[0131] In some embodiments, the mRNA sequence encoding the spike protein with D614G mutation of SARS-CoV-2 comprises SEQ ID NO: 27, or a fragment or variant thereof.
[0132] In some embodiments, the nucleic acid vaccines may comprise a region encoding any of the sequences listed in Table 1 or a fragment or variant thereof. The nucleic acid vaccines may comprise hybrid or chimeric regions, or mimics or variants. In some embodiments, the nucleic acid vaccines may comprise any of the polynucleotide sequences listed in Table 3.
Table 3. Exemplary Sequences to be used in the Nucleic Acid Vaccines for treating or preventing COVID-19
Figure imgf000026_0001
[0133] Any of the sequences referred to in Tables 1-3 or variants thereof may also be used in a memory booster vaccine described herein.
[0134] In some embodiments, the nucleic acid vaccine described herein encodes a protein or fragment 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 a protein provided by an amino acid sequence in Table 1. The terms “identical” or percent “identity” in the context of two or more polypeptide sequences refer to two or more sequences that are the same. The percent identity between polypeptide sequences may be performed using algorithms known in the art, such as BLAST and CLUSTAL.
[0135] The sequence of the SARS-CoV-2 protein or fragment or variant thereof may be obtained from any source. In some embodiments, the sequence of the SARS-CoV-2 protein or fragment or variant thereof is from a strain that is capable of or at risk of infecting human subjects.
[0136] In some embodiments, the sequence of the SARS-CoV-2 protein or fragment or variant thereof may be modified or optimized (such as codon optimized) for expression in a particular cell or host organism.
[0137] In some embodiments, the nucleic acid vaccine described herein may be a multivalent vaccine. The multivalent vaccine may include polynucleotides that encodes at least two different one or more proteins, peptides, fragments or variants thereof of SARS- CoV-2. As a non-limiting example, the polynucleotides may encode the same or a different structural protein. As a non-limiting example, the polynucleotides may encode the same structural protein but different variants of the structural protein.
[0138] 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 a fragment of the S protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the 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 a S protein sequence of SARS-CoV-2 (e.g., full-length, fragment or variant) where the S protein has a mutated furin cleavage site. The S protein furin cleavage site mutant will remove or disable the furin cleavage site(s) in S protein (e.g., between the SI and S2 boundary). In some viral envelope proteins, disruption of a furin cleavage site was found to enhance expression and stability. In some embodiments, the nucleic acid vaccine encodes a S protein sequence of SARS-CoV-2 (e.g., full-length, fragment or variant) where the S protein includes the D614G mutation. The nucleic acid vaccine encoding the S protein of SARS-CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
[0139] 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 a fragment of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the topological domain (e.g., virion surface or intravirion region) of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the M protein (e.g., full-length protein or fragment) of SARS-CoV-2. The nucleic acid vaccine encoding the M protein of SARS- CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
[0140] 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 the dimerization domain of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the N protein (e.g., full-length protein or fragment) of SARS-CoV-2. The nucleic acid vaccine encoding the N protein of SARS-CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
[0141] 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 E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the topological domain (e.g., virion surface or intravirion region) of the E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes the transmembrane domain of the E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a variant of the E protein (e.g., full-length protein or fragment) of SARS-CoV-2. The nucleic acid vaccine encoding the E protein of SARS- CoV-2, a fragment or variant thereof may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
[0142] In some embodiments, the nucleic acid vaccine encodes two different structural proteins of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and a M protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and a N protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and an E protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a M protein, fragment or variant thereof of SARS-CoV-2 and a N protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a M protein, fragment or variant thereof of SARS-CoV-2 and an E protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a N protein, fragment or variant thereof of SARS-CoV-2 and an E protein, fragment or variant thereof of SARS-CoV-2. The nucleic acid vaccine encoding two different structural proteins, fragment or variant thereof of SARS-CoV-2, may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized. In some embodiments, the sequences encoding the two different structural proteins or fragments or variants thereof of SARS- Cov-2 of the nucleic acid vaccine are constructed as a single polynucleotide. [0143] In some embodiments, the nucleic acid vaccine encodes at least three different sequences of the structural proteins fragment or variant thereof for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S proteins, fragments or variants sequences for SARS-CoV-2 and a M protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S proteins, fragments or variants sequences for SARS-CoV-2 and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different S proteins, fragments or variants sequences for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M proteins, fragments or variants sequences for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N proteins, fragments or variants sequences for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E proteins, fragments or variants sequences for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M proteins, fragments or variants sequences for SARS-CoV-2 and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different M proteins, fragments or variants sequences for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N proteins, fragments or variants sequences for SARS-CoV-2 and a M protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different N proteins, fragments or variants sequences for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two different E proteins, fragments or variants sequences for SARS-CoV-2 and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant sequence for SARS-CoV-2, a M protein, fragment or variant sequence for SARS-CoV-2, and a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant sequence for SARS-CoV-2, a M protein, fragment or variant sequence for SARS-CoV-2, and an E protein, fragment or variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein, fragment or variant sequence for SARS-CoV-2, a N protein, fragment or variant sequence for SARS-CoV-2, and an E protein, fragment or variant sequence for SARS- CoV-2. In some embodiments, the nucleic acid vaccine encodes a M protein, fragment or variant sequence for SARS-CoV-2, a N protein, fragment or variant sequence for SARS- CoV-2, and an E protein, fragment or variant sequence for SARS-CoV-2. The nucleic acid vaccine encoding at least three different sequences of the structural proteins fragment or variant thereof for SARS-CoV-2, may also include a signal peptide and/or at least one linker (e.g., GSG linker) sequence and one or more sequences in the nucleic acid vaccine may be codon optimized.
SARS-CoV-2 Variants
[0144] SARS-CoV-2 is a member of the large coronavirus family of viruses. Multiple variants (sometimes referred to as “strains” or “lineages”) of SARS-CoV-2 have been identified globally. The nomenclature for SARS-CoV-2 variants used in this description is consistent with the PANGO nomenclature for new virus lineages (Rambaut, Andrew, et al., A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nature microbiology, 2020, 5:: 1403-1407, the contents of which are incorporated herein by reference in their entirety). Near real-time data relating to PANGO SARS-CoV-2 lineages or variants can be accessed online using Nextstrain’s SARs-CoV-2 analysis user interface (nextstrain.org/ncov/global).
[0145] As of this disclosure, numerous PANGO lineage variants of SARS-CoV-2 have been identified, including the following (number in parentheses represents number of cases per each submitted PANGO lineage: A (37); A.l (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.l (1); AE.2 (2); AE.4 (1); AE.5 (1); AE.7 (1); AE.8 (1); AG. l (1); AY. l; AY.2; AY.3; B (47); B.l (374); B.1.1 (237); B.1.1.1 (40); B.1.1.10 (2); B.1.1. I l l (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); B.1.1.214
(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. l.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. l (2);
C.1.1 (1); C. l l (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); C.36.3 (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. l (57); P.2 (47); P.6 (2); P.7 (3); Q.l (6), Q.3 (7); Q.4 (2); Q.5 (1); Q.6 (1). Q.8 (7), R.l (9); S. l (1); U.2 (1); U.3 (1); W. l (1); Y. l (2); and Z. l (1).
[0146] From an epidemiological perspective, variants are typically categorized as Variants of Interest (VOIs), Variants of Concern (VOCs), and Variants of High Consequence (VOHCs). For information relevant to categorizing specific variants as VOIs, VOCs, or VOHCs see, for example, cdc.gov/coronavirus/2019-ncov/cases- updates/variant-surveillance/vari ant-info.html.
[0147] VOIs may have certain genetic markers associated with changes to receptor binding, reduced neutralization by antibodies generated against previous infection or vaccination, reduced efficacy of treatments, potential diagnostic impact, or predicted increase in transmissibility or disease severity. In some instances, VOIs have specific genetic markers that are predicted to affect transmission, diagnostics, therapeutics, or immune escape, or cause an increased proportion of cases or unique outbreak clusters. SARS-CoV-2 VOIs include, for example, PANGO lineage B. 1.1.7 (Alpha), B.1.351 (Beta); B.1.427/429 (Epsilon); B.1.526 (Iota); B.1.525 (Eta); B.1.617.1 (Kappa);
B.1.617.2 (Delta); B.1.621 (Mu); C37 (Lamba); P. l (Gamma)and P.2.
[0148] VOCs may include variants for which there is evidence of an increase in transmissibility, more severe disease (increased hospitalizations or deaths), significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures. In some instances, VOCs have evidence of impact on diagnostics, treatments, and vaccines, widespread interference with diagnostic test targets, evidence of substantially increased resistance to one or more class of therapies, evidence of significant decreased neutralization by antibodies generated during previous infection or vaccination, evidence of reduced vaccine-induced protection from severe disease, evidence of increased transmissibility, or evidence of increased disease severity. SARS-CoV-2 VOCs may include, for example, PANGO lineage B.l.1.7 (Alpha), P.l (Gamma), B.1.351(Beta), B.1.427and B.1.429 ((Epsilon), B.1.526 (Iota), B.1.525 (Eta), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), and C37 (Lamba).
[0149] VOHCs may have clear evidence that prevention measures or medical countermeasures (MCMs) have significantly reduced effectiveness relative to previously circulating variants. In some instances, VOHCs have impact on Medical Countermeasures (MCM), demonstrated failure of diagnostics, evidence to suggest a significant reduction in vaccine effectiveness, a disproportionately high number of vaccine breakthrough cases, very low vaccine-induced protection against severe disease, significantly reduced susceptibility to multiple Emergency Use Authorization (EUA) or approved therapeutics, more severe clinical disease and increased hospitalizations.
[0150] The nucleic acid vaccines disclosed herein may encode one or more polypeptides, e.g., one or more proteins, peptides, fragments or variants thereof, of any of the SARS-CoV-2 variants described herein. In some embodiments, the nucleic acid vaccines disclosed herein may encode one or more polypeptides, e.g., one or more proteins, peptides, fragments or variants thereof, of a SARS-CoV-2 VOI, VOC, and/or VOHC. In some embodiments, the nucleic acid vaccines encode a polypeptide comprising the specific mutation called D614G.
[0151] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.526 SARS-CoV-2 variant (i.e., Iota variant), such as one or more of Spike protein substitutions L5F, T95I, D253G, S477N, E484K, D614G, and/or A701 V; ORFla substitutions L3201P, T265I, and/or A3675/3677; ORFlb substitutions P314L and/or Q1011H; ORF3a substitutions P42L, Q57H; ORF8 substitution Ti ll; and/or 5’UTR substitution R81C.
[0152] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.525 SARS-CoV-2 variant (i.e., Eta variant), such as one or more of Spike protein substitutions A67V, A69/70, A144, E484K, D614G, Q677H and/or F888L; ORFlb substitution P314F; ORFla substitution T2007I; M protein substitution I82T; N protein substitutions A12G and/or T205I; and/or 5’UTR substitution R81C.
[0153] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the P.2 SARS- CoV-2 variant, such as one or more of: Spike protein substitutions E484K, D614G, and/or V1176F; ORFla substitutions L3468V and/or L3930F; ORFlb substitution P314L; N protein substitutions Al 19S, R203K, G204R, and/or M234I; 5’UTR substitution R81C.
[0154] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.l.1.7 SARS-CoV-2 variant (i.e., Alpha variant), such as one or more of: Spike protein substitutions A69/70, A144Y, E484K, S494P, N501Y, A570D, D614G, and/or P681H.
[0155] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the P.l SARS- CoV-2 variant (i.e., Gamma variant), such as one or more of: Spike protein substitutions K417N/T, E484K, N501Y, and/or D614G.
[0156] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.351 SARS-CoV-2 variant (i.e., Beta variant), such as one or more of: Spike protein substitutions K417N, E484K, N501Y, and/or D614G. The B.1.351 variant is also referred to as the South African variant, as it first originated in South Africa.
[0157] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.427 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions L452R and/or D614G.
[0158] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.429 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions SI 31, W152C, L452R, and/or D614G. [0159] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.617.1 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions G142D, E154K, L452R, E484Q, D614G, P681R, and/or Q1071H.
[0160] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant (i.e., Delta variant), such as one or more of: Spike protein substitutions T19R, T95I, G142D, A156/157, R158G, L452R, T478K, D614G, P681R, and/or D950N. In other embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more further mutations or substitutions present in the B.1.617.2 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions V70F, A222V, W258L, and/or K417N.
[0161] In some embodiments, the nucleic acid vaccines encode one or more polypeptide comprising one or more mutations or substitutions present in the B.1.617.3 SARS-CoV-2 variant, such as one or more of: Spike protein substitutions T19R, G142D, L452R, E484Q, D614G, P681R, and/or D950N.
[0162] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 Spike protein, e.g., protein, peptide, fragment, or variant, comprising one or more substitutions and/or deletions selected from: A570D, A67V, A701V, D253G, D614G, E484K, F888L, K417N/T, L452R, L5F, N501Y, P681H, Q677H, S13I, S477N, S494P, T95I, V1176F, W152C, A144, A144Y, and A69/70.
[0163] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 ORF la comprising one or more substitutions and/or deletions selected from: L3201P, T265I, T2007I, L3468V, A3675-3677, and L3930F.
[0164] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 ORF lb comprising one or more substitutions selected from: P314F, P314L, and Q1011H.
[0165] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 ORF3a comprising one or more substitutions selected from: P42L and Q57H. [0166] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 ORF8 comprising a Ti ll substitution.
[0167] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 5’UTR comprising a R81C substitution.
[0168] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 M protein, e.g., protein, peptide, fragment, or variant, comprising I82T substitution.
[0169] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 N protein, e.g., protein, peptide, fragment, or variant, comprising one or more substitutions selected from: A12G, A119S, R203K, G204R, T205I, and M234I.
Components of Nucleic Acid Vaccines
[0170] In some embodiments, the polynucleotides described herein encode at least one polypeptide of interest, e.g., one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2. The proteins, peptides, fragments or variants thereof of SARS-CoV-2 of the present disclosure may be wild type where they are derived from the infectious agent, or modified (e.g., the structural proteins or fragments and variants thereof are engineered, designed or artificial). They may have any combination of the features described herein. [0171] 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, or fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19. As a non-limiting example, these peptides or polypeptides may serve as an antigen or antigenic molecule (also preferred to as immunogenic molecule). The term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.
[0172] Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'- amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
[0173] In some embodiments, in vitro transcription (IVT) enzymatic synthesis methods may be used to make linear polynucleotides (referred to as “IVT polynucleotides”) encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 of the present disclosure.
[0174] In some embodiment, the nucleic acid vaccines may include “chimeric polynucleotides” which have portions or regions which differ in size and/or encoded protein (e.g., structural protein of SARS-CoV-2). A “chimera” is an entity having two or more incongruous or heterogeneous parts or regions. As used herein a “part” or “region” of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide.
[0175] In some embodiments, the nucleic acid vaccine includes polynucleotides from about 30 to about 100,000 nucleotides in length(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). [0176] 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 polynucleotides may be non-coding.
[0177] In some embodiments, the length of a region encoding at least one peptide or polypeptide of interest of the polynucleotides of the nucleic acid vaccine is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 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 a region may be referred to as a “coding region” or “region encoding.” [0178] In some embodiments, the polynucleotides of the nucleic acid vaccine is or functions as a messenger RNA (mRNA). As used herein, the term “messenger RNA (mRNA)” refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide or polypeptide of interest in vitro, in vivo, in situ or ex vivo.
[0179] The shortest length of a region of the polynucleotide of the nucleic acid vaccine can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g., no longer than35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids. Examples of dipeptides that the polynucleotide sequences can encode or include, but are not limited to, carnosine and anserine.
[0180] The region of the polynucleotide of the nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19 may be greater than about 30 nucleotides in length. The length may be, but is not limited to, at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,
2.500, and 3,000, 4,000, 5,000, 6,000, 7,000, 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 from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to
1.500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). mRNA Components
[0181] The nucleic acid vaccines described herein may be an mRNA vaccine. The mRNA vaccine includes at least one mRNA molecule which, when translated, produce at least one peptide or polypeptide of interest for the prevention, alleviation and/or treatment of COVID-19. In general, 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
[0182] In some embodiments, the mRNA includes a region to initiate 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”.
[0183] In some embodiments, the mRNA includes a region to stop 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.”
[0184] In some embodiments, the regions to initiate or terminate translation may independently range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. Additionally, these regions may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
[0185] In some embodiments, a masking agent may be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.
[0186] In some embodiments, the start codon may be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon which is not the start codon. Translation of the polynucleotide may begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon. The polynucleotide sequence where the start codon is removed may further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide. mRNA Components: Coding Region
[0187] In some embodiments, the coding region of the polynucleotide of the nucleic acid vaccine may encode at least one peptide or polypeptide of interest. Non-limiting examples of peptides or polypeptides of interest include one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19. mRNA Components: Untranslated Region
[0188] The polynucleotides of the nucleic acid vaccines described herein may comprise one or more regions or parts which act or function as an untranslated region (UTR). Wild type UTRs of a gene are transcribed but not translated. In mRNA, the 5 'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, UTRs may have a role in terms of stability and translation of the nucleic acid molecule and translation. Variants of UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
[0189] In some embodiments, the UTRs of the polynucleotide of the nucleic acid vaccine may range independently from 15-1,000 nucleotides in length (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).
[0190] Wild type 5' UTRs include features which play roles in translation initiation as these 5’ UTRs include sequences such as Kozak sequences which are known to be involved in how the ribosome initiates translation of many genes. 5' UTRs also have been known to form secondary structures which are involved in elongation factor binding. Other non-UTR sequences (e.g., introns or portions of intron sequences) may also be used as regions or subregions which may increase protein production as well as polynucleotide levels. [0191] Natural or wild type 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides of the nucleic acid vaccines.
[0192] The UTR from any gene may be incorporated into the regions of the polynucleotides of the nucleic acid vaccines. Alternatively, artificial UTRs, which are not variants of wild type regions, may also be used in the polynucleotides of the nucleic acid vaccines. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. As a non-limiting example, a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs from a different parental sequence.
[0193] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
[0194] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). In some embodiments, the 5’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). In some embodiments, the 5’UTR is directly 5’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine. In some embodiments, the 5’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 5’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine; e.g., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 5’UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO:
47 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 (DNA) or SEQ ID NO: 47 (RNA).
[0195] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). In some embodiments, the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). In some embodiments, the 3’UTR is directly 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine. In some embodiments, the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine; e.g., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 3’UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO:
48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 (DNA) or SEQ ID NO: 48 (RNA).
[0196] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). In some embodiments, the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). In some embodiments, the 3’UTR is directly 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine. In some embodiments, the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine; e.g., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides separates the 3’UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 (DNA) or SEQ ID NO: 53 (RNA). mRNA Components: Cap and IRES Sequences
[0197] In some embodiments, the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’ cap structure. The 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.
[0198] In some embodiments, the 5’ terminal capping region of the polynucleotide of the nucleic acid vaccine may comprise a single cap or a series of nucleotides forming the cap. The capping region may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some examples, the capping region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, the cap is absent.
[0199] In some embodiments, cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs may be used in the nucleic acid vaccines. Cap analogs, which may be chemically (e.g., non-enzymatically) or enzymatically synthesized, differ from natural (e.g., endogenous, wild-type or physiological) 5'-caps in their chemical structure, but they retain cap function. [0200] In some embodiments, the 5' terminal caps of the polynucleotides of the nucleic acid vaccines may include endogenous caps or cap analogs. As a non-limiting example, 5' terminal caps may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine (mlG), 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
[0201] The skilled artisan will appreciate that 5' capping can be generated via enzymatic or other synthetic processes. Endogenous mRNA molecules are 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. This 5 '-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or ante-terminal transcribed nucleotides of the 5' end of the mRNA can optionally also be 2'-O-methylated. 5 '-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
[0202] Polynucleotides, e.g., mRNAs, of the nucleic acid vaccine described herein may be modified to include a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp- 5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a vaccinia virus capping enzyme available from, e.g., New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 '-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.
[0203] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5 '-terminal and/or 5 '-ante-terminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule. [0204] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (e.g., non-enzymatically) or enzymatically synthesized and linked to a nucleic acid molecule, such as an mRNA molecule.
[0205] For example, the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5 '-triphosphate-5 '- guanosine (m7G-3'mppp-G; which may equivalently be designated 3' O-Me- m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA). The N7- and 3 '-O-methlyated guanine provide the terminal moiety of the capped nucleic acid molecule (e.g., mRNA).
[0206] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O- methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5 '-triphosphate-5 '- guanosine, m7Gm-ppp-G).
[0207] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of cap analogs from endogenous 5 '-cap structures may lead to reduced translational competency and reduced cellular stability.
[0208] In exemplary aspects of the present disclosure, polynucleotides, e.g., mRNAs, can be capped post-transcriptionally, using enzymes. For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'- terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is termed the Cap 1 structure. In some embodiments, the Cap 1 structure provides a higher translational- competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')NlmpNp (Cap 1), and 7mG(5')-ppp(5')NlmpN2mp (Cap 2).
[0209] In one embodiment, the polynucleotide of the nucleic acid vaccine described herein comprises a Cap 1 structure.
[0210] Because the polynucleotides, e.g., mRNA, may be capped post- transcriptionally, and because this process is more efficient, up to 100% of the polynucleotides, e.g., mRNA, may be capped. This is in contrast to -80% when a cap analog is linked to an mRNA in the course of an in vitro transcription reaction.
[0211] In some embodiments, the polynucleotides of the nucleic acid vaccines may contain an internal ribosome entry site (IRES) sequence. While not wishing to be bound by theory, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site or may serve as one of multiple ribosome binding sites of an mRNA. mRNA Components: Tailing Region
[0212] In some embodiments, the polynucleotide of the nucleic acid vaccine, e.g., the mRNA includes a tailing region. Non-liming examples of a tailing region include a poly-A sequence, a poly-C sequence, and/or a polyA-G quartet.
[0213] In some embodiments the mRNA includes a chain terminating nucleoside. Nonlimiting examples of chain terminating nucleosides include 2'-0 methyl, F and locked nucleic acids (LNA).
[0214] In some embodiments, the sequence of the tailing region of the polynucleotide of the nucleic acid vaccine may range from absent to 500 nucleotides in length (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 or as a function of poly-A Binding Protein binding.
[0215] In some embodiments, poly-A tails may also be added after the construct is exported from the nucleus. [0216] 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 in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
[0217] In some embodiments, the length of a poly-A tail, when present, is greater than 30 nucleotides in length (e.g., at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A tail region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000 nucleotides).
[0218] In some embodiments, the poly-A tail is approximately 99 nucleotides in length (SEQ ID NO: 44).
[0219] In some embodiments, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design may be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides. [0220] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail may also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly- A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein may enhance expression.
Signal Sequences
[0221] In some embodiments, the polynucleotides of the nucleic acid vaccines may also encode additional features which may facilitate the trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3- 60 amino acids) in length which is incorporated at the 5' terminus of the coding region or the N-terminus polypeptide encoded, respectively. In some embodiments, addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.
[0222] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein include a signal sequence comprising SEQ ID NO: 45 (DNA) or SEQ ID NO: 49 (RNA).
Codon Optimization
[0223] The polynucleotides of the nucleic acid vaccines, their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include, but are not limited to, match codon frequencies in target and host organisms to ensure proper folding, alter GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In some embodiments, the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 4.
Table 4. Codon Options
Figure imgf000055_0001
[0224] 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, cosmids, and artificial chromosomes.
Modifications
[0225] Nucleic acid vaccines of the present disclosure, including mRNA vaccines, may include one or more modifications. The terms “modification” or, as appropriate, “modified” refer to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties. In a polypeptide, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids.
[0226] As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group or other backbone linkage (intemucleoside linkage).
[0227] The modifications may be various distinct modifications. In some embodiments, the coding region(s), the untranslated region(s), the flanking region(s), and/or the terminal or tailing regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, nucleic acid vaccines of the present disclosure comprise one or more modifications which render the nucleic acid molecules, when introduced to a cell, more resistant to degradation in the cell and/or more stable in the cell as compared to unmodified polynucleotides.
[0228] The polynucleotides of the nucleic acid vaccines described herein can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted, for example, with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or groups. In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present disclosure may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional modifications are described herein.
[0229] In some embodiments, the modifications include 2’-O-Methyl-modified or 2’- O-Methoxyethyl-modified nucleotides (2’-0Me and 2’-M0E modifications, respectively). [0230] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may comprise at least one modification described herein.
[0231] The polynucleotides of the nucleic acid vaccines described herein can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage.
[0232] Modifications of polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that are useful in the vaccines of the present disclosure include, but are not limited to, any modifications as described in PCT Publication WO2017070626, the contents of which are incorporated herein by reference in their entirety, including, for example, modification or deletion of nucleotides (or codons) encoding one or more N- linked glycosylation site in a translated polypeptide. Modifications that are useful in the vaccines of the present disclosure may also comprise any modifications as described in PCT Publication WO2018200892, the contents of which are incorporated herein by reference in their entirety. The vaccines of the present disclosure may further comprise features or modifications as described in PCT patent application publications W02020255063, WO2020182869, W02016011222, W02016011226, W02016005004, W02016000792, WO2015176737, WO2015085318, WO2015048744, and WO2015034925, and United States patent application publications US20200254086, US20200206362, US20180311336 and US20180303929; the contents of each of which are incorporated herein by reference in their entireties. [0233] For example, the polynucleotides, including the mRNA molecules of the nucleic acid vaccines described herein, can include modifications as follows. The intemucleoside linkages of the polynucleotides may be partially or fully modified. The polynucleotides may comprise modifications to one or more nucleobases. The polynucleotides may comprise 5-methylcytosines in place of all cytosine nucleobases/cytidine nucleotides. Further the polynucleotides may have one or more modifications to one or more of the sugar subunits of a nucleoside. The sugar modification can be one or more locked nucleic acids (LNAs) or 2’-O-Methoxyethyl-modified (“2’-M0E”) modifications. The polynucleotides can be designed with a patterned array of sugar, nucleobase or linkage modifications. In some embodiments, the polynucleotides can comprise modifications to maximize stability. In some embodiments, the polynucleotides can be fully 2’-MOE-sugar modified.
Modified Nucleobases
[0234] The modified nucleosides and nucleotides can include a modified nucleobase. 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.
[0235] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (y), pyridin- 4-one ribonucleoside, 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), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5- carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2 -thio-uridine (mcm5s2U), 5-aminomethyl-2 -thiouridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio- uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5- carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5 -propynyl -uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4-thio-pseudouridine, 5-methyl- uridine (m5U, i.e., having the nucleobase deoxythymine), 1 -methylpseudouridine (m1!]/), 5- methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (m y), 4-thio-l-methyl- pseudouridine, 3-methyl-pseudouridine (m3y), 2-thio-l-methyl-pseudouridine, 1-methyl- 1-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-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, Nl-methyl- pseudouridine (also known as 1 -methylpseudouridine (m1!]/)), 3-(3-amino-3- carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 y), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2- thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ym), 2-thio-2'-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O- methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl- uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH- ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine. [0236] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f’C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5- halo-cytidine (e.g., 5 -iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2- thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4- thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio- zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, lysidine (I C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl- cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (CCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F- cytidine, and 2'-OH-ara-cytidine.
[0237] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6- halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza- adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyl -ad enosine (nfA), 2-methyl-adenine (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 (io6 A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6- threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl- adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl- adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m Am), l,2'-O-dimethyl-adenosine (m*Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F- adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.
[0238] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1- methyl-inosine (m1!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (02yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl- queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza- guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7- methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m'G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl- guanosine (m2’7G), N2, N2,7-dimethyl-guanosine (m2’2’7G), 8-oxo-guanosine, 7-methyl-8- oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2- dimethyl-6-thio-guanosine, a-thio-guanosine, 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 (m'Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), l,2'-O-dimethyl-inosine (m^m), and 2'-O-ribosylguanosine (phosphate) (Gr(p)).
[0239] The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluorom ethyl and other 5 -substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3- deazaguanine, deazaadenine, 7-deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a]l,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5- d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.
[0240] Different sugar modifications, nucleotide modifications, and/or intemucleoside linkages (e.g., backbone structures) may be introduced at various positions in a polynucleotide described herein. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. The polynucleotides of the present disclosure may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, T/U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
[0241] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may be modified to be a circular nucleic acid. The termini of the polynucleotides may be linked by chemical reagents or enzymes, producing circular polynucleotides that have no free ends. Circular polynucleotides are expected to be more stable than linear counterparts and to be resistant to digestion with exonucleases. Circular polynucleotides may further comprise other structural and/or chemical modifications with respect to A, G, T/U or C ribonucleotides/deoxyribonucleotides.
[0242] In some embodiments, the polynucleotides are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the polynucleotides are at least 75% modified, e.g., at least 75% of the nucleotides are modified. It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linkage) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification.
[0243] In some embodiments, the polynucleotides are at least 10% modified in only one component of the nucleotide, with such component being the nucleobase, sugar, or linkage between nucleosides. For example, modifications may be made to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleobases, sugars, or linkages of a polynucleotide described herein.
[0244] As non-limiting examples, the uracil nucleosides of the polynucleotide of the nucleic acid vaccine are all modified. The modifications may be the same or different. In some embodiments, the guanine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified. The modifications may be the same or different. In some embodiments, the guanine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified. The modifications may be the same or different. In some embodiments, the cytosine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified. The modifications may be the same or different. In some embodiments, the adenine nucleosides of the polynucleotide of the nucleic acid vaccine are all modified. The modifications may be the same or different.
[0245] In one embodiment of the disclosure, the polynucleotide of the nucleic acid vaccine is modified to comprise Nl-methyl-pseudouri dine nucleotides.
Sugar Modifications
[0246] The modified nucleosides and nucleotides which may be incorporated into polynucleotides (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted Cl -6 alkyl; optionally substituted Cl-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-Cl-6 alkoxy, optionally substituted Cl-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in which the 2'-hydroxyl is connected by a Cl -6 alkylene or Cl -6 heteroalkylene bridge to the 4’ -carbon of the same ribose sugar, where exemplary bridges include methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.
[0247] In some embodiments, the polynucleotide, such as the mRNA of the nucleic acid vaccine described herein comprises at least one sugar modification. Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multi cyclic forms (e.g., tri cyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3'^2')), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, polynucleotide molecules as described herein, including mRNAs, can include nucleotides containing, e.g., arabinose, as the sugar.
[0248] Nonlimiting examples of the sugar modification may include the modifications provided in Table 5. The polynucleotides of the present disclosure can have one or more nucleotides carrying a modification as provided in Table 5. In some embodiments, each of the nucleotides of a polynucleotide described herein carries any one of the modifications as provided in Table 5, or none of the modifications as provided in Table 5.
Table 5. Nucleotide Sugar Modifications
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
[0249] In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -OMe, referred to as 2’ -OMe. In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -F, referred to as 2’-F.
Internucleoside Linkages
[0250] The polynucleotides of the present disclosure can include any modification to the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). In the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonates phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both nonlinking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
[0251] The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
[0252] In specific embodiments, a modified nucleoside includes an alpha-thio- nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a- thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O- (l-thiophosphate)-pseudouridine).
[0253] In some embodiments, the polynucleotides comprise at least one phosphorothioate linkage or methylphosphonate linkage between nucleotides.
[0254] In some embodiments, the polynucleotides comprise at least one 5 ’-(E)- vinylphosphonate (5’- -VP), a phosphate mimic, as a modification.
[0255] In one embodiment of the present disclosure, the polynucleotide (e.g., mRNA) of the nucleic acid vaccine for CO VID-19 may be modified.
Valency
[0256] Nucleic acid vaccines of the present disclosure may vary in their valency. “Valency” refers to the number of antigenic components in the nucleic acid vaccine or the polynucleotide of the nucleic acid vaccines. The antigenic components of the nucleic acid vaccine may be on the same polynucleotide or they may be on different polynucleotides. In some embodiments, the nucleic acid vaccine may be monovalent. In some embodiments, the nucleic acid vaccine may be divalent. In some embodiments, the nucleic acid vaccine may be trivalent. In some embodiments, the nucleic acid vaccine may be multivalent which may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 antigens or antigenic moi eties such as, but not limited to, antigenic peptides. As a non-limiting example, antigenic peptides may be one or more fragments or variants of the structural proteins of SARS-CoV-2.
Synthesis
Enzymatic Methods
In Vitro Transcription-Enzymatic Synthesis
[0257] cDNA encoding the polynucleotides of the nucleic acid vaccines described herein may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and polymerase variants.
[0258] In some embodiments, the DNA template is removed from the IVT reaction, using a DNase I enzyme. The digested DNA and nucleotides are then removed during oligo dT purification of the mRNA. This purification method is based on affinity of the poly-A tail of the mRNA to the poly-dT column bed. Centrifugation may be used but may not be required to remove the digested DNA and nucleotides. After purification by a reverse phase column (e.g., SDVB) to remove double stranded RNA from the mRNA, ultrafiltration may be utilized, followed by one or more filtration steps. Following purification, residual DNA may be measured to confirm that the DNA has been removed by using PCR for a region of the plasmid outside of the region transcribed into mRNA. In some embodiments, where concentration of the product is desired, diafiltration methods may be used followed by one or more filtration steps to remove any bioburden (e.g., biomolecules, or other biomaterial). [0259] Any number of RNA polymerases or variants may be used in the synthesis of the polynucleotides of the nucleic acid vaccine described herein. RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence. [0260] Polynucleotide or nucleic acid synthesis reactions may be carried out by enzymatic methods utilizing polymerases. Polymerases catalyze the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain. Currently known DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis. DNA polymerase I (pol I) or A polymerase family, including the KI enow fragments of E. Coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among the best studied of these families. Another large family is DNA polymerase a (pol a) or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from phages T4 and RB69. Although they employ similar catalytic mechanism, these families of polymerases differ in substrate specificity, substrate analogincorporating efficiency, degree and rate for primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity against inhibitors.
Solid-Phase Chemical Synthesis
[0261] In some embodiments, polynucleotides of the nucleic acid vaccines described herein may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer-controlled solidphase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids.
[0262] In some embodiments, automated solid-phase synthesis is used where the chain is synthesized in 3' to 5' direction. The hydroxyl group in the 3' end of a nucleoside is tethered to a solid support via a chemically cleavable or light-cleavable linker. Activated nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, are added to the support-bound nucleoside sequentially. At the end of the synthesis, a cleaving agent such as ammonia or ammonium hydroxide is added to remove all the protecting groups and release the polynucleotide chains from the solid support. Light may also be applied to cleave the polynucleotide chain. The product can then be further purified with high pressure liquid chromatography (HPLC) or electrophoresis.
Liquid Phase Chemical Synthesis
[0263] The synthesis of polynucleotides of the nucleic acid vaccines described herein by the sequential addition of monomer building blocks may be carried out in a liquid phase. A covalent bond is formed between the monomers or between a terminal functional group of the growing chain and an incoming monomer. Functional groups not involved in the reaction must be temporarily protected. After the addition of each monomer building block, the reaction mixture has to be purified before adding the next monomer building block. The functional group at one terminal of the chain has to be deprotected to be able to react with the next monomer building blocks. A liquid phase synthesis is labor- and timeconsuming and cannot not be automated. Despite the limitations, liquid phase synthesis is still useful in preparing short polynucleotides in a large scale. Because the system is homogenous, it does not require a large excess of reagents and is cost- effective in this respect.
Quantification and Purification
[0264] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may be quantified in exosomes or when derived from one or more bodily fluid. As used herein “bodily fluids” include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alter natively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
[0265] In the exosome quantification method, a sample of not more than 2 mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof. In the analysis, the level or concentration of a polynucleotide may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker. The assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.
[0266] These methods afford the investigator the ability to monitor, in real time, the level of polynucleotides remaining or delivered. This is possible because the polynucleotides described herein differ from the endogenous forms due to the structural modifications.
[0267] In some embodiments, the polynucleotide may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). Anon-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). The quantified polynucleotide may be analyzed in order to determine if the polynucleotide may be of proper size, check that no degradation of the polynucleotide has occurred. Degradation of the polynucleotide may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
[0268] Purification of the polynucleotides of the nucleic acid vaccines described herein may include, but is not limited to, polynucleotide clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGEN- COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes (EX- IQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a polynucleotide such as a “purified polynucleotide” refers to one that is separated from at least one contaminant. As used herein, a “contaminant” is any substance which makes another unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.
[0269] A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
III. PHARMACEUTICAL COMPOSITIONS AND DELIVERY
[0270] The nucleic acid vaccines described herein may be used as therapeutic or prophylactic agents. In some embodiments, the present disclosure provides pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and a nucleic acid vaccine, i.e., a nucleic acid vaccine for COVID-19. In accordance, the pharmaceutical compositions comprising the nucleic acid vaccine described herein can be used for preventing, alleviating and/or treating COVID-19.
[0271] Provided herein are nucleic acid vaccines and pharmaceutical compositions thereof which may be used in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances. Pharmaceutical compositions of the nucleic acid vaccines described herein may be sterile and/or pyrogen-free.
[0272] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the nucleic acid vaccines or the polynucleotides contained therein, e.g., polynucleotides encoding one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention, alleviation and/or treatment of COVID-19, to be delivered as described herein.
[0273] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., nonhuman mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated 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 birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
Formulations [0274] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
[0275] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
[0276] A pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
[0277] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
[0278] In some embodiments, the formulations described herein may contain at least one nucleic acid vaccine composition, e.g., nucleic acid vaccine for COVID-19, e.g., one mRNA vaccine for CO VID-19. As a non-limiting example, the formulations may contain 1, 2, 3, 4 or 5 nucleic acid vaccine compositions with different sequences, e.g., 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.
[0279] The nucleic acid vaccine compositions of the present disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the nucleic acid vaccine composition); (4) alter the biodistribution (e.g., target the nucleic acid vaccine composition to specific tissues or cell types); (5) increase the translation of encoded protein in vivo, and/or (6) alter the release profile of encoded protein in vivo. [0280] In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with nucleic acid vaccine compositions (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the present disclosure can include one or more excipients, each in an amount that together increases the stability of the nucleic acid vaccine compositions and/or increases cell transfection by the nucleic acid vaccine compositions. Further, the nucleic acid vaccine compositions of the present disclosure may be formulated using selfassembled nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients, and delivery agents for nucleic acids that may be used in the formulation with the nucleic acid vaccine compositions of the present disclosure are disclosed in PCT Patent Application Publication WO 2013/090648, the contents of which are incorporated herein by reference in their entirety.
Lipidoids
[0281] The nucleic acid vaccine compositions of the disclosure can be formulated using one or more lipidoids.
[0282] The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of oligonucleotides or nucleic acids (see Mahon et al., Bioconjug Chem. 2010, 21 : 1448-1454; Schroeder et al., J InternMed. 2010, 267:9-21; Akinc et al., Nat Biotechnol. 200,8 26:561-569; Love et al., Proc Natl Acad Set USA. 2010, 107: 1864-1869; Siegwart et al., Proc Natl Acad Set U S A. 2011, 108: 12996-3001; the contents of all of which are incorporated herein by references in their entirety).
[0283] While these lipidoids have been used to effectively deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008, 105: 11915-11920; Akinc et al., Mol Ther. 2009, 17:872-879; Love et al., Proc Natl A cad Sci U SA. 2010, 107: 1864-1869; Leuschner et al., Nat Biotechnol. 2011, 29: 1005-1010; the contents of all of which is incorporated herein by reference in their entirety), the present disclosure contemplates their formulation and use in delivering at least one pharmaceutically acceptable carrier, including nucleic acid vaccines. Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the nucleic acid vaccine compositions following the injection of a lipidoid formulation via localized and/or systemic routes of administration. Lipidoid complexes containing nucleic acid vaccine compositions can be administered by various means including, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), or intracerebroventricular (ICV) administration.
[0284] In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are herein incorporated by reference in their entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 2010, 401 :61 ; the contents of which are herein incorporated by reference in their entirety), Cl 2-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
[0285] The lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009, 17:872-879 and the contents of which is incorporated herein by reference in their entirety.
[0286] The lipidoid referred to herein as “C 12-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010, 107: 1864-1869 and Liu and Huang, Molecular Therapy. 2010, 669-670; the contents of both of which are herein incorporated herein by reference in their entirety. The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to the nucleic acid vaccine compositions. As an example, formulations with certain lipidoids, include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 alkyl chain length). As another example, formulations with certain lipidoids, include, but are not limited to, Cl 2-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
[0287] In some embodiments, nucleic acid vaccine compositions formulated with a lipidoid for systemic intravenous administration. For example, a final optimized intravenous formulation using nucleic acid vaccine compositions and comprising a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid to nucleic acid vaccine compositions and a C14 alkyl chain length on the PEG lipid, with a mean particle size of roughly 50-60 nm, can result in the distribution of the formulation to be greater than 90% to the liver, (see, Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are herein incorporated by reference herein in their entirety). In another example, an intravenous formulation using a C12-200 lipidoid (see PCT Patent Application Publication W02010129709, the contents of which are herein incorporated by reference in their entirety) may have a molar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipid to nucleic acid and a mean particle size of 80 nm may be effective to deliver nucleic acid vaccine compositions (see, Love et al., Proc Natl Acad Sci USA. 2010, 107: 1864-1869, the contents of which are herein incorporated by reference herein in their entirety).
[0288] In some embodiments, an MD1 lipidoid-containing formulation may be used to effectively deliver nucleic acid vaccine compositions to hepatocytes in vivo. The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009,17:872-879, the contents of which are herein incorporated by reference in their entirety), use of a lipidoid-formulated nucleic acid vaccine compositions to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly sizelimited. [0289] Use of lipidoid formulations to deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and endothelium 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 each of which are herein incorporated by reference herein in their entirety). For effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of nucleic acid vaccine compositions for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. For example, the component molar ratio may include, but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011, 29: 1005-1010; the contents of which are herein incorporated by reference in their entirety). The use of lipidoid formulations for the localized delivery of nucleic acids to cells via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and nucleic acid vaccine compositions.
Liposomes
[0290] The nucleic acid vaccine compositions of the disclosure can be formulated using one or more liposomes.
[0291] In some embodiments, pharmaceutical compositions of nucleic acid vaccine compositions include liposomes. Liposomes are artificially prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
[0292] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, poly dispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
[0293] In some embodiments, pharmaceutical compositions comprising the nucleic acid vaccines described herein may include, without limitation, liposomes such as those formed from 1,2-di oleyloxy -A, A-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), SMARTICLES®/NOV340 (Marina Biotech, Bothell), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US Patent Application Publication US20100324120; the contents of which are herein incorporated by reference in their entirety), neutral DOPC (l,2-dioleoyl-sn-glycero-3- phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006, 5(12): 1708-1713); the contents of which is herein incorporated by reference in its entirety), hyaluronan-coated liposomes (Quiet Therapeutics, Israel), and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).
[0294] In some embodiments, pharmaceutical compositions comprising the nucleic acid vaccines described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (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 Ret. 2005, 107:276-287; Semple et al. Nature Biotech. 2010, 28: 172-176; Judge et al. J Clin Invest. 2009, 119:661-673; deFougerolles Hum Gene Ther. 2008, 19: 125-132; the contents of each of which are incorporated herein in their entireties). The original manufacturing method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations may be composed of 3 to 4 lipid components in addition to the nucleic acid vaccine compositions. As a non-limiting example, a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%
1.2-dioleyloxy-MMdimethylaminopropane (DODMA), as described by Jeffs et al. In another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-A,A-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or
1.2-dilinolenyloxy-3 -dimethylaminopropane (DLenDMA), as described by Heyes et al. In another example, the nucleic acid-lipid particle may comprise a cationic lipid comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; a noncationic lipid comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and a conjugated lipid that inhibits aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle as described in W02009127060 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 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 a cationic lipid of Formula I, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles. [0295] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
[0296] In some embodiments, the liposome may contain a sugar-modified lipid disclosed in US Pat. No.; US5595756 to Bally et al., the contents of which are incorporated herein by reference in their entirety. The lipid may be a ganglioside and cerebroside in an amount of about 10 mol percent.
[0297] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a liposome comprising a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phosphates in the nucleic acid vaccine compositions (N:P ratio) of between 1 : 1 and 20: 1 as described in PCT Patent Application Publication No. W02013006825, the contents of which are herein incorporated by reference in their entirety. In some embodiments, the liposome may have a N:P ratio of greater than 20: 1 or less than 1 : 1.
[0298] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid-polycation complex. The formation of the lipidpolycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, the contents of which are herein incorporated by reference in their entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in PCT Patent Application Publication No. WO2012013326; the contents of which are herein incorporated by reference in their entirety. In some embodiments, the nucleic acid vaccine compositions may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
[0299] The liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, 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 herein incorporated by reference in their entirety), the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA.
[0300] In some embodiments, the pharmaceutical compositions may be formulated with any amphoteric liposome disclosed in PCT Patent Application Publication No.: WO 2008043575 to Panzner and US Pat. No.: US 8,580,297 to Essler et al. (Marina Biotech), the contents of which are incorporated herein by reference in their entirety. The amphoteric liposome may comprise a mixture of lipids including a cationic amphiphile, an anionic amphiphile and optional one or more neutral amphiphiles. The amphoteric liposome may comprise amphoteric compounds based on amphiphilic molecules, the head groups of which being substituted with one or more amphoteric groups. In some embodiments, the pharmaceutical compositions may be formulated with an amphoteric lipid comprising one or more amphoteric groups having an isoelectric point between 4 and 9, as disclosed in US Patent Application Publication No.: US20140227345 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 US Pat. No.: US7312206 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 amphipathic cationic lipid, at least one amphipathic anionic lipid, and at least one neutral lipid, or liposomes comprise at least one amphipathic lipid with 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 US Pat. No. 7780983 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 liposomes comprising a serum-stable mixture of lipids taught in US Patent Application Publication No.: US 20110076322 to Panzner et al, the contents of which are incorporated herein by reference in their entirety, capable of encapsulating the nucleic acid vaccine compositions of the present disclosure. The lipid mixture comprises phosphatidylcholine and phosphatidylethanolamine in a ratio in the range of about 0.5 to about 8. The lipid mixture may also include pH sensitive anionic and cationic amphiphiles, such that the mixture is amphoteric, being negatively charged or neutral at pH 7.4 and positively charged at pH 4. The drug/lipid ratio may be adjusted to target the liposomes to particular organs or other sites in the body. In some embodiments, liposomes loaded with the nucleic acid vaccine compositions of the present disclosure as cargo, are prepared by the method disclosed in US Patent Application Publication No.: US 20120021042 to Panzner et al., the contents of which are incorporated herein by reference in their entirety. The method comprises steps of admixing an aqueous solution of a polyanionic active agent and an alcoholic solution of one or more amphiphiles and buffering said admixture to an acidic pH, wherein the one or more amphiphiles are susceptible of forming amphoteric liposomes at the acidic pH, thereby to form amphoteric liposomes in suspension encapsulating the active agent.
Lipoplexes
[0301] The nucleic acid vaccine compositions of the disclosure can be formulated using one or more lipoplexes.
[0302] In some embodiments, the nucleic acid vaccine compositions may be formulated as a lipoplex, such as, without limitation, the ATUPLEX™ system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and nontargeted delivery of nucleic acids (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- 293Weide et al. J Immunother ., 2009, 32:498-507; Weide et al. J Immunother., 2008, 31 : 180-188; Pascolo ., Expert Opin. Biol. Ther. 4: 1285-1294; Fotin-Mleczek et al., J.
Immunother., 2011, 34: 1-15; Song et al., Nature Biotechnol . 2005, 23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007, 6; 104:4095-4100; deFougerolles Hum Gene Ther. 2008, 19: 125-132; the contents of each of which are incorporated herein by reference in their entirety).
Lipid Nanoparticles (LNPs)
[0303] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid nanoparticle (LNP). In general, LNPs can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, LNPs may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.
[0304] LNPs useful herein are known in the art and generally comprise cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from nonspecific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP-forming composition.
[0305] The components of the LNP may be selected based on the desired target, tropism, cargo, size, or other desired feature or property.
[0306] The LNP may be the lipid nanoparticles described in PCT Patent Application Publication No. W02012170930, the contents of which are herein incorporated by reference in their entirety.
[0307] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a LNP that comprises at least one cationic lipid.
[0308] In some embodiments, the cationic lipid which may be used in formulations of the present disclosure may be selected from, but not limited to, a cationic lipid described in PCT Patent Application Publication Nos. W02012040184, WO2011153120, WO201 1149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865 and W02008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No. US20100036115 and US20120202871; the contents of each of which are herein incorporated by reference in their entirety. The cationic lipid may be also selected from, but not limited to, formula A described in PCT Patent Application Publication Nos. W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO201 1022460, WO2012061259, WO2012054365 and WO2012044638; the contents of each of which are herein incorporated by reference in their entirety. Alternatively, the cationic lipid may be selected from, but not limited to, formula CLLCLXXIX of PCT Patent Application No. W02008103276, formula CLLCLXXIX of US Patent No.
7,893,302, formula CLLCLXXXXII of US Patent No. 7,404,969 and formula I- VI of US Patent Publication No. US20100036115; the contents of each of which are herein incorporated by reference in their entirety. The cationic lipid may be a multivalent cationic lipid such as the cationic lipid disclosed in US 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 including two quaternary amine groups and a hydrophobic portion including four hydrocarbon chains as described in US Patent No. 7,223,887 to Gaucheron et al.. The cationic lipid may be biodegradable as the biodegradable lipids disclosed in US Patent Application Publication No.: US20130195920 to Maier et al., the contents of which are incorporated herein by reference in their entirety. The cationic lipid may have one or more biodegradable groups located in a lipidic moiety of the cationic lipid as described in formula I-IV in US20130195920 to Maier et al.. In some embodiments, the cationic lipid may also be the cationic lipids disclosed in US20130156845 to Manoharan et al. and US 20130129785 to Manoharan et al., WO 2012047656 to Wasan et al., WO 2010144740 to Chen et al., WO 2013086322 to Ansell et al., or WO 2012016184 to Manoharan et al., the contents of each of which are incorporated herein by reference in their entirety. [0309] As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)- N,N-dimethylnonacosa-20,23 -dien- 10-amine, ( 17Z,20Z)-N,N-dimemylhexacosa- 17,20- dien-9-amine, (lZ,19Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)-N,N- dimethyldocosa- 13,16-dien-5-amine, (12Z, 15Z)-N,N-dimethylhenicosa- 12, 15-dien-4- amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N- dimethyltetracosa- 15,18-dien-7-amine, ( 18Z,2 lZ)-N,N-dimethylheptacosa- 18,21 -dien- 10- amine, (15Z, 18Z)-N,N-dimethyltetracosa-l 5, 18-dien-5-amine, (14Z, 17Z)-N,N- dimethyltricosa- 14, 17-dien-4-amine, ( 19Z,22Z)-N,N-dimeihyloctacosa- 19,22-dien-9- amine, (18Z,21Z)-N,N-dimethylheptacosa- 18 ,21 -dien-8 -amine, (17Z,20Z)-N,N- dimethylhexacosa- 17,20-dien-7-amine, ( 16Z, 19Z)-N,N-dimethylpentacosa- 16,19-dien-6- amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z ,24Z)-N,N- dimethyltriaconta-21 ,24-dien-9-amine, ( 18Z)-N,N-dimetylheptacos- 18-en- 10-amine, ( 17Z)-N,N-dimethylhexacos- 17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa- 19,22- dien-7-amine, N,N-dimethylheptacosan-l 0-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa- 20,23 -dien-10-amine, 1-[(1 lZ,14Z)-l-nonylicosa-l 1 , 14-dien-l-yl] pyrrolidine, (20Z)-N,N- dimethylheptacos-20-en-l 0-amine, (15Z)-N,N-dimethyl eptacos-15-en-l 0-amine, (14Z)- N,N-dimethylnonacos- 14-en-10-amine, ( 17Z)-N,N-dimethylnonacos- 17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-l 0- amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, ( 16Z)-N,N-dimethylpentacos- 16- en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine, (13Z,16Z)- N,N-dimethyl-3-nonyldocosa-13,16-dien-l-amine, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl] eptadecan-8-amine, 1 -[( 1 S,2R)-2-hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine, N,N-dimethyl-l-[(l S ,2R)-2-octylcyclopropyl]nonadecan- 10-amine, N,N-dimethyl-21-[(lS,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N- dimethyl- 1 -[( 1 S,2S)-2- { [(lR,2R)-2-pentylcyc!opropyl]methyl } cyclopropyl]nonadecan- 10- amine,N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl- [(lR,2S)-2-undecy!cyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(l S,2R)-2- octylcyclopropyl]heptyl] dodecan-1 -amine, l-[(lR,2S)-2-hepty lcyclopropyl]-N,N- dimethyloctadecan-9-amine, l-[(lS,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6- amine, N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N- dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, S-N,N- dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, l-{2- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-l -[(octyloxy )methyl]ethyl}pyrrolidine, (2S)-N,N- dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l-yloxy]propan-2- amine, 1 -{2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]- 1 -[(octyloxy )m ethyl] ethyl } azetidine, (2S)-l-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2- amine, (2S)-l-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan- 2-amine, N,N-dimethyl-l-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2- amine, N,N-dimethyl-l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2S)- N,N-dimethyl-l-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)propan-2- amine, (2S)-1-[(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethyl-3-(pentyloxy)propan- 2-amine, (2S)-l-(hexyloxy)-3-[(l lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N- dimethylpropan-2-amine, 1-[(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, l-[(13Z,16Z)-docosa-13,16-dien-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2S)- 1 -[(13Z, 16Z)-docosa- 13,16-dien- 1 -yloxy]-3 -(hexyloxy)- N,N-dimethylpropan-2-amine, (2S)- 1 -[( 13Z)-docos- 13 -en- 1 -yloxy]-3 -(hexyloxy)-N,N- dimethylpropan-2-amine, l-[(13Z)-docos-13-en-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, l-[(9Z)-hexadec-9-en-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(l-metoylo ctyl)oxy]-3-[(9Z,12Z)- octadeca-9,12-dien- l-yloxy]propan-2-amine, (2R)-l-[(3,7-dimethyloctyl)oxy]-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l -yloxy ]propan-2-amine, N,N-dimethyl-l- (octyloxy)-3-({8-[(lS,2S)-2-{[(lR,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-l-{[8- (2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,2OZ,23Z)-N,N- dimethylnonacosa-ll,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.
Lipid Nanoparticle (LNP) compositions [0310] In some embodiments a lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, at least one additional LNP functional component, or any combination thereof. In some embodiments a lipid nanoparticle may be comprised of at least one cationic lipid, at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one cationic lipid, at least one noncationic lipid, and at least one sterol. In some embodiments, the LNP may be comprised of at least one cationic lipid, at least one non-cationic lipid, and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one non-cationic lipid. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one sterol. In some embodiments, the LNP may be comprised of at least one cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one non-cationic lipid and at least one sterol. In some embodiments, the LNP may be comprised of at least one non-cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one sterol and at least one additional LNP functional component. In some embodiments, the LNP may be comprised of at least one cationic lipid. In some embodiments, the LNP may be comprised of at least one noncationic lipid. In some embodiments, a LNP may be comprised of a sterol. In some embodiments, the LNP may be comprised of a additional LNP functional component.
[0311] In some embodiments, the at least one cationic lipid may comprise any of at least one ionizable cationic lipid, at least one amino lipid, at least one saturated cationic lipid, at least one unsaturated cationic lipid, at least one zwitterionic lipid, at least one multivalent cationic lipid, or any combination thereof. In some embodiments, the LNP may be essentially devoid of the at least one cationic lipid. In some embodiments, the LNP may contain no amount of the at least one cationic lipid. [0312] In some embodiments, at least one cationic lipid may be selected from, but not limited to, at least one of l,3-Bis-(l,2-bis-tetradecyloxy-propyl-3- dimethylethoxyammoniumbromide)-propan-2-ol ((R)-PLC-2), 2-(Dinonylamino)ethan-l- ol (17-10), 2-(Didodecylamino)ethan-l-ol (17-11), 3-(Didodecylamino)propan-l-ol (17- 12), 4-(Didodecylamino)butan-l-ol (17-13), 2-(Hexyl((9Z,12Z)-octadeca-9,l 2-dien- 1- yl)amino)ethan- 1 -ol (17-2), 2-(Nonyl((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)ethan- 1 -ol (17-3), 2-(Dodecyl((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethan-l-ol (17-4), 2- (((9Z, 12Z)-Octadeca-9, 12-dien- 1 -yl)(tetradecyl)amino)ethan- 1 -ol ( 17-5 ), 2-(((9Z, 12Z)- Octadeca-9, 12-dien- l-yl)(octadecyl)amino)ethan-l-ol (17-6), 2-(Ditetradecylamino)ethan- l-ol (17-7), 2-(Di((Z)-octadec-9-en-l-yl)amino)ethan-l-ol (17-8), (9Z,12Z)-N-(2- Methoxyethyl)-N-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)octadeca-9, 12-dien- 1 -amine ( 17-9), N-Nonyl-N-(2-(piperazin- 1 -yl)ethyl)nonan- 1 -amine (19-1), N-Dodecyl-N-(2-(piperazin- 1 - yl)ethyl)dodecan- 1 -amine (19-2), (9Z, 12Z)-N-((9Z, 12Z)-Octadeca-9, 12-dien- 1 -yl)-N-(2- (piperazin- 1 -yl)ethyl)octadeca-9, 12-dien- 1 -amine ( 19-3), N-Dodecyl-N-(2-(4- methylpiperazin- 1 -yl)ethyl)dodecan- 1 -amine 1 ntermediate 1 : 2-(Didodecylamino)ethan- 1 -ol ( 19-4), N-Dodecyl-N-(2-(4-(4-methoxybenzyl)piperazin- 1 -yl)ethyl)dodecan- 1 -amine (19- 5), (9Z,12Z)-N-(2-(4-Dodecylpiperazin-l-yl)ethyl)-N-((9Z,12Z)-octadeca-9,l 2-dien- 1- yl)octadeca-9, 12-dien- 1 -amine (19-6), (3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yloxy)-N,N-dimethylpropan-l-amine) (1 -Bl 1), N-(2-(Didodecylamino)ethyl)- N-dodecylglycine (20-1), Dinonyl8,8'-((2-(dodecyl(2- hydroxyethyl)amino)ethyl)azanediyl)dioctanoate (20-10), 3-((2- (Ditetradecylamino)ethyl)(dodecyl)amino)propan-l-ol (20-11), 2-((2- (Ditetradecylamino)ethyl)(tetradecyl)amino)ethan-l-ol (20-12), 2-((2-(Di((9Z,12Z)- octadeca-9, 12-dien- 1 -yl)amino)ethyl)(dodecyl)amino)ethan- 1 -ol (20- 13), 2-((2- (Di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)ethyl)((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)amino)ethan-l-ol (20-14), 2-((2-(Didodecylamino)ethyl)(hexyl)amino)ethan-l-ol (20- 15), 2-((2-(Dinonylamino)ethyl)(nonyl)amino)ethan-l-ol (20-16), 2-((2- (Didodecylamino)ethyl)(nonyl)amino)ethan-l-ol (20-17), 2-((2- (Dinonylamino)ethyl)(dodecyl)amino)ethan-l-ol (20-18), 2-((2- (Di dodecyl ami nojethyl Jami no)ethan- l -ol (20-19), Pentyl6-(dodecyl(2-(dodecyl(2- hydroxyethyl)amino)ethyl)amino)hexanoate (20-2), 2-((2- (Didodecylamino)ethyl)(dodecyl)amino)ethan-l-ol (20-20), 3-((2- (Didodecylamino)ethyl)(dodecyl)amino)propan-l-ol (20-21), 4-((2- (Didodecylamino)ethyl)(dodecyl)amino)butan- 1 -ol (20-22), (Z)-2-((2- (Didodecylamino)ethyl)(dodec-6-en- 1 -yl)amino)ethan- 1 -ol (20-23), 2-((2- (Didodecylamino)ethyl)(tetradecyl)amino)ethan- 1 -ol (20-24), 2-((2- (Didodecylamino)ethyl)((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethan-l-ol (20-25), Pentyl6-((2-(didodecylamino)ethyl)(2 -hydroxy ethyl)amino)hexanoate (20-3), Dipentyl6,6'-((2-(dodecyl(2-hydroxyethyl)amino)ethyl)azanediyl)dihexanoate (20-4), Diheptyl6,6'-((2-((6-(heptyloxy)-6- oxohexyl)(2hydroxyethyl)amino)ethyl)azanediyl)dihexanoate (20-5), Pentyl6-((2- (dinonylamino)ethyl)(2-hydroxyethyl)amino)hexanoate (20-6), Heptyl6-(dodecyl(2- (dodecyl(2-hydroxyethyl)amino)ethyl)amino)hexanoate (20-7), Nonyl8-((2- (didodecylamino)ethyl)(2-hydroxyethyl)amino)octanoate (20-8), Heptadecan-9-yl8-((2- (didodecylamino)ethyl)(2-hydroxyethyl)amino)octanoate (20-9), l-(2,2-Di((9Z,12Z)- octadeca-9,12-dien-l-yl)cyclopropyl)-N,N-dimethylmethanamine (21-1), 3,3- Di((9Z,12Z)-octadeca-9,12-dien-l-yl)cyclobutyl4-(dimethylamino)butanoate (21-2), 3,3- Di((9Z,12Z)-octadeca-9,12-dien-l-yl)cyclopentyl3-(dimethylamino)propanoate (21-3), 3 , 3 -Di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)cy clopentyl4-(dimethylamino)butanoate (21 -4), 1 -(2,3 -Di((8Z, 1 lZ)-heptadeca-8, 11 -dien- 1 -yl)cyclopropyl)-N,N-dimethylmethanamine (21-6), Unknown (75-016B), poly{4-((2-(dimethylamino)ethyl)thio)tetrahydro-2H-pyran- 2-one}-r-poly{4-(octylthio)tetrahydro-2H-pyran-2-one} (A7), (3aR5s,6aS)-N,N-dimethyl- 2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopentadl,3dioxol-5-amine (ALN100), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro- 3aHcyclopenta[d][l,3]dioxol-5-amine (ALN1001), ((3aR,5s,6aS)-N,N-dimethyl-2,2- di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine)) (ALNY-100), dimyristoyltrimethylammoniumpropane (Amino Lipid 6), Benzami7tdi7t- dialkyl-carboxylicacid (BADACA), N,N-dihydroxyethylmethyl-N-2- (cholesteryloxycarbonylamino)ethylammoniumbromide (BHEM-Chol), N,N-bis-(2- hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonylamino-ethyl)ammoniumbromide (BHEM-Chol 1), 2-{4-[(3P)-cholest-5-en-3-yloxy]butoxy}-iV?N-dimethyl-3-[(9Z,12Z)- octadeca-9! 12-dien-l-yloxy]propan-l -amine (Butyl-CLinDMA), (2JR)-2-{4-[(3P)-cholest- 5-en-3-yloxy]butoxy}-ArAdimethyl-3-[(9Z,12Z)-octadeca-9! 12-dien-l-yloxyjpropan-l- amine (Butyl-CLinDMA (2R)), (25)-2-{4-[(3P)-cholest-5-en-3-yloxy]butoxy}-iVy/V- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Butyl-CLinDMA (2S)), 1, l'-(2-(4-(2-((2-(bis(2 -hydroxy dodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodecan-2-ol (C 12-200), 1, l‘-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (Cl 2-200), Cholesteryl-succinyl Silane (C2), (9Z,9'Z,12Z,12'Z)-2-((4-(((3- (dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diylbis(octadeca-
9.12-dienoate) (Cationic Lipid A2), 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-di enoate (Cationic Lipid A3), l-(3-cholesteryl)-oxycarbonyl-aminomethylimidazole (CHIM), [(2- Morpholine-4-yl-ethylcarbamoyl)methyl]-carbamicacidcholesterylester (Chol-C3N-Mo2), [(2-Morpholine-4-yl-ethylcarbamoyl)-ethyl]-carbamicacidcholesterylesterChol-DMC3N- Mo2[l-Methyl-2-(2-morpholine-4-yl-ethylcarbamoyl)-propyl]- carbamicacidcholesterylester (Chol-C4N-Mo2), 1, 17-bis(2-octylcyclopropyl)heptadecan-9- yl4-(dimethylamino)butanoate (CL), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4- (dimethylamino)-butanoate (CL01), cholesteryl3-(dimethylamino)propanoate (CL06), cholesteryl2-(dimethylamino)acetate (CL08), N,N-dimethyl-2,3-bis(((9Z, 12Z)-octadeca-
9.12-dien- 1 -yl)oxy)propan- 1 -amine (CL- 1 ), N-methyl-2-(((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)oxy)-N-(2-((((9Z,12Z)-octadeca-9,12-diene-l-yl)oxy)ethyl)ethan-l -amine (CL- 11), (3R,4R)-3,4-bis(((Z)-hexadec-9-en-l-yl)oxy)-l-methylpyrrolidine(CompoundCL-12) (CL- 12), 2-(Dimethylamino)-N-((6Z,9Z,28Z,3 lZ)-Heptatriconta-6,9,28,3 l-tetraen-19- yl)acetamide (CL-13), 3-(Dimethylamino)propane-l,2-diyl(9Z,9'Z,12Z,12'Z)- bi s(octadeca-9, 12 -di enoate) (CL- 14), (9Z, 12Z)-di((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)amine (CL- 15), 7-Hydroxy7-(4-((l-methylpiperidine-4-carbonyl)oxy)butyl)tridecane- 1,13-diyldidodecanoate (CL15B6), 7-Hydroxy7-(4-((l-methylpiperidine-4- carbonyl)oxy)butyl)tridecane- 1,13 -diyl di tetradecanoate (CL 15C6), 7-Hydroxy7-(4-((l - methylpiperidine-4-carbonyl)oxy)butyl)tridecane-l,13-diyldipalmitate (CL15D6), 7- Hydroxy7-(4-((l-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-l,13-diyldioleate (CL15H6), Bis(2-(((9Z,12Z)-octadeca-9,12-dien-l-yl)oxy)ethyl)amine (CL-16), (9Z,12Z)- N-Methyl-N-(2-(((9Z,12Z)-octadeca-9,l 2-dien- l-yl)oxy)ethyl)octadeca-9,l 2-dien- 1- amine (CL- 17), (9Z, 12Z)-N-(3 -(((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)oxy)propyl)octadeca-
9.12-dien-l-amine (CL-18), (l-Methylpiperidin-3-yl)methyldi((l lZ,14Z)-icosa-l 1,14- dien- 1 -yl)carbamate (CL- 19), N-methyl-N,N-bis(2-((Z)-hexadec-9-enyloxy)ethyl)amine (CL-2), (13Z, 16Z)-N,N-Dimethyl-4-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)docosa-3 ,13,16- trien- 1 -amine (CL-20), (S)-2- Amino-3 -hydroxy -N,N-bis(2-(((Z)-octadeca-9-en- 1 - yl)oxy)ethyl)propanamide (CL-21), C2:N,N-dihexadecyl-N'-(3- triethoxysilylpropyl)succinamide (CL3), trans-l-Methyl-3,4-bis((((Z)-octadec-9-en-l- yl)oxy)methyl)pyrrolidine (CL-3), trans- 1 -methylpyrrolidine-3 ,4- diyl)bis(methylene)(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (CL-4), 7-(4- (Diisopropylamino)butyl)-7-hydroxytridecane-l,13-diylditetradecanoate (CL4C6), 7-(4- (Diisopropylamino)butyl)-7-hydroxytridecane-l,13-diyldipalmitate (CL4D6), 11-(4- (Diisopropylamino)butyl)-11-hy droxyhenicosane- 1,21 -diyldi oleate (CL4H10), 7-(4- (Diisopropylamino)butyl)-7-hydroxytridecane-l,13-diyldioleate (CL4H6), 9-(4- (Diisopropylamino)butyl)-7-hydroxyheptadecane-l,17-diyldi oleate (CL4H8), (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,3 l-tetraen-19-yl4-(dimethylamino)butanoate (CL- 5), 2-(Dimethylamino)-N-(2-(((Z)-octadeca-9-en-l-yl)oxy)ethyl)-N-((9Z,12Z)-octadeca-
9.12-diene-l-yl)acetamide (CL-53), 3-((2-(((Z)-octadeca-9-en-l-yl)oxy)ethyl)((9Z,12Z)- octadeca-9,12-dien-l-yl)amino)propane-l-All (CL-54), l-Methyl-3,3-bis((((9Z,12Z)- octadeca-9,12-dien- l-yl)oxy)methyl)azeti dine (CL-55), l-Methyl-3,3-bis(2-(((9Z,12Z)- octadeca-9,12-dien- l-yl)oxy)ethyl)azeti dine (CL-56), l-Methyl-3,3-bis(2-(((9Z,12Z)- octadeca-9,12-dien- l-yl)oxy)propyl)azeti dine (CL-57), 2-(3,3-di((9Z,12Z)-octadeca-9,12- dien- 1 -yl)azetidin- 1 -yl)ethan- 1 -ol (CL-58), 2-(3 ,3 -di((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)azetidin- 1 -yl)propan- 1 -ol (CL-59), 3 -(Di((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)amino)propan- 1 -o (CL-6), 3 -(Dimethyl amino)propyl3 ,3 -di ((9Z, 12Z)-octadeca-9, 12- dien- 1 -yl)azetidine- 1 -carboxylate (CL-60), 2-(Di((Z)-octadeca-9-en- 1 -yl)amino)ethane- 1 - ol (CL-61), 3-(Di((Z)-octadeca-9-en-l-yl)amino)propan-l-ol (CL-62), (HZ,14Z)-2- ((Dimethylamino)methyl)-2-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)icosa- 11,14-dien- 1 -ol (CL-63), (1 lZ,14Z)-2-(Dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dien-l-yl)icosa-l 1,14- dien- 1 -ol (CL-64), 3 -(Dimethylamino)-2,2-bis((((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)oxy)methyl)propan-l-ol (CL-65), (9Z,12Z)-N-(2-(((Z)-Octadeca-9-en-l- yl)oxy)ethyl)octadeca-9, 12-dien- 1 -amine (CL-7), l-Methyl-3,3-di((9Z, 12Z)-octadeca-
9.12-dien- 1 -yl)azetidine (CL-8), N,2-Dimethyl- 1 ,3 -bis(((9Z, 12Z)-octadeca-9, 12-dien- 1 - yl)oxy)propan-2-amine (CL-9), 3-Dimethylamino-2-(Cholest-5-en-3B-oxybutan-4-oxy)-l - (cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-oxy)-3'- oxapentoxy)-3-dimethy-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), cetyltrimethylammoniumbromide (CTAB), lA-Diarachidonyloxy-ATV-dimethyA-propyl-S- amine (DAraDMA), 0,0’-ditetradecanoyl-N-(a- trimethylammonioacetyl)di ethanolaminechloride (DC-6-14), 3 -[N-(N',N'- dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDA), dimethyldioctadecylammoniumbromide (DDA ), N,N-distearyl-N,N- dimethylammoniumbromide (DDAB), l,2-Didocosahexaenyloxy-(7V,N-dimethyl)-propyl- 3-amine (DDocDMA), N-(2-(dimethylamino)ethyl)-4,5-bis(dodecylthio)pentanamide (DEDPA), 3-Dimethylamino-2-(Cholest-5-en-3P-oxypent-3-oxa-an-5-oxy)-l-(cis,cis-
9.12-octadecadienoxy)propane (DEG-CLinDMA), 1,6-DileoylTri ethylenetetrami de (dio- TETA), Nl,N19-bis((S,23E,25E,27E,29E)-16-((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6- trimethylcyclo-hex-l-en-l-yl)nona-2,4,6,8-tetraenamido)-24,28-dimethyl-15,22-dioxo-30- (2,6,6-trimethylcyclohex-l-en-l-yl)-4,7,10-trioxa-14,21-diazatriaconta-23,25,27,29-tetraen- 1 -y l)-4, 7, 10,13,16-pentaoxanonadecane- 1 , 19-diamide (di VA-PEG-di VA), DiLin-N- Methylpiperazine (DL-033), DiLin-N,N-DimethylGlycine (DL-036), Dioleyl-N,N- DimethylGly cine (DL-048), 3 -(( 1 , 3 -bi s(((9Z, 12Z)-octadeca-9, 12-dienoyl)oxy)propan-2- yl)amino)propanoicacid (DLAPA), l,2-dilinolenyloxy-3 -dimethylaminopropane (DLenDMA), l-Linoleoyl-2-linoleyloxy-3 -dimethylaminopropane (DLin-2-DMAP), 3- (N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), l,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), l,2-Dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoley oxy-3 - (dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA ), 1,2- dilinoleyloxy-3-dimethylaminopropane (DLinDMA 1), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), dilinoleoyl-4-aminobutyricacid (DLinFAB), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-di oxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[ 1 ,3 ]-di oxolane (DLin-K-DMA), 1 ,2- Dilinoleyoxy-3-morpholinopropane (DLin-MA), (6Z,9Z,28Z,3 lZ)-heptatriacont- 6,9,28,31 -tetraene- 19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2- Dilinoleyloxy-3-(N-methylpiperazino)propane (DLinMPZ), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), Dilinoleyloxy3-piperidinopropylamine (DLinPip), 1 ,2Dilinoleyloxy3-(3'-hvdroxypiperidino)-propylamine (DLinPip-3OH), l,2Dilinoleyloxy3-(4'-hvdroxypiperidino)-propylamine (DLinPip-4OH), 1,2- Dilinoleyloxy-3-hvdroxypropane (DLinPO), l,2-Dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), l,2-Dilinoleoyl-3-trimethylaminopropane (DLinTAP), l,2-Dilinoleoyl-3- trimethylaminopropanechloridesalt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3- trimethylaminopropane (DLinTMA), l,2-Dilinoleyloxy-3- trimethylaminopropanechloridesalt (DLin-TMA.Cl), 3-((l,3-bis(((9Z,12Z.15Z)-octadeca- 9,12,15-trienoyl)oxy)propan-2-yl)amino)propanoicacid (DLLAPA), l,2Dilinoleyloxy3- (N,NdimethyD-propylamme (DLmDEA), l,2-Dilauroyl-sn-Glicero-3- Phosphoethanolamine (DLPE), l,2-Dilauroyl-sn-Glicero-3 -Glycerol (DLPG), N,N- Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), dimyristoylphosphatidylserine (DMPS), N-[l-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2 -hydroxy ethyl)ammoniumbromide (DMRIE), l,2-Dimyristyloxypropyl-3-dimethyl-hydroxy ethylammoniumbromide (DMRIE1), l,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), 3-(N,N- Dioleylamino)-l,2-propanedio (DOAP), 3-((l,3-bis(oleoyloxy)propan-2- yl)amino)propanoicacid (DOAP A), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), l,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP), N,N- dioleyl-N,N-dimethylammoniumchloride (DODAC), l,2-Dioleoyl-3 -Dimethylammoniumpropane (DODAP), N,N-dihydroxyethylN,N-dioctadecylammoniumchloride (DODEAC), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), dioleoyl-4-aminobutyricacid (DOF AB), Dioctadecylamidoglycylspermine (DOGS), l,2-Dioleoyl-3-methyl-
(m ethoxy carbonyl-ethyl)ammonium -Propane (DOMCAP), l,2-Dioleoyl-3-N-pyrrolidine- propane (DOP5P), l,2-Dioleoyl-3-N-pyrridinium-propane,bromidesalt (DOP6P), 1,2- dioleoyl-3-dimethyl-hydroxy ethylammoniumbromide (DORI), l,2-dioleyloxypropyl-3- dimethyl-hydroxy ethylammoniumbromide (DORIE), l,2-dioleyloxypropyl-3-dimethyl- hydroxybutylammoniumbromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimetyl- hydroxypropylammoniumbromide (DORIE-HP), l,2-dioleyloxypropyl-3-dimethyl- hydroxypentylammoniumbromide (DORIE-Hpe), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), 1,3- dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), N-(l-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP1), N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'- trimethylammoniumtosylate (DOTAU), l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)- heptadecenyl-3-(2-hydroxyethyl)imidazoliniumchloride (DOTIM), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride (DOTMA), di oleylphosphatidyluridinephosphatidylcholine (DOUPC), 1 ,2-Diphvtanyloxy-W.N- dimemyl)-butyl-4-amme (DPan-C2-DMA), l,2-Diphytanyloxy-3-(iV,7V-dimethyl)- propylamine (DPanDMA), 2,3-bis(dodecylthio)propyl(2-(dimethylamino)ethyl)carbamate (DPDEC), dipalmitoyl-4-aminobutyricacid (DPFAB), l,2-dipalmityloxypropyl-3- dimethyl-hydroxy ethylammoniumbromide (DPRIE), l,2-dipalmitoyl-3- trimethylammoniumpropane (DPTAP), l-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2- hydroxyethyl)imidazoliniumchloride (DPTIM), 3-((l,3-bis(stearoyloxy)propan-2- yl)amino)propanoicacid (DSAPA), distearyldimethylammonium (DSDMA), 1,2- distearloxy-N,N-dimethylaminopropane (DSDMA1), l,2-disteryloxypropyl-3-dimethyl- hydroxyethylammoniumbromide (DSRIE), l,2-disteroyl-3-trimethylammoniumpropane (DSTAP), ditetradecyltrimethylammonium (DTDTMA), l,2-dioleoyl-sn-glycero-3- ethylphosphocholine (EDOPC), N2- [N2,N5 -bi s(3 -aminopropyl)-L-ormithyl] -N,N- dioctadecyl-L-glutaminetetrahydrotrifluoroacetate (GC33), Cholest-5-en-3-ol(3P)-,3-[(3- aminopropyl)[4-[(3-aminopropyl)amino]butyl]carbamate] (GL67), glycerylmono-oleate (GMO), Guanidino-dialkyl-carboxylicacid (GUADACA), 2-(bis(2- (tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan- aminiumbromide (HEDC), 2,2'-(tert-butoxycarbonylazanediyl)bis(ethane-2, 1- diyl)ditetradecanoate (HEDC-BOC-TN), 1 -(2-(((3 S, 1 OR, 13R)- 10, 13 -dimethyl- 17-((R)-6- methylheptan-2-yl)-2, 3 ,4,7,8,9,10,11,12,13,14,15,16,17 -tetradecahydro- 1 H- cyclopenta[a]phenanthren-3-yldisulfanyl)ethyl)guanidine (HGT4002), (15Z, 18Z)-N,N- dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-l-yl)tetracosa-15,18-dien-l-amine (HGT5000), (15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien-l-yl)tetracosa-4, 15,18-trien-l- amine (HGT5001), Histaminyl-Cholesterolhemisuccinat (HisChol), histidinylcholesterolhemisuccinate (Hist-Chol), HydroSoyPC (HSPC), imidazolecholesterolester (ICE), 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l- piperazineethanamine (KL 10), N1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4-tridodecyl- 1 ,4- piperazinedi ethanamine (KL22), 14,25-ditridecyl- 15, 18,21 ,24-tetraaza-octatriacontane (KL25), N,N-di-n-lctradecyl,N-methyl-N-(2-guanidinyl)cthylammonium (Lipid 1), N,N- di-n-octadecyl,N-mcthyl-N-(2-guanidinyl)cthylammoniumchloride (Lipid 2), 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-di enoate (Lipid A), (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-dienoate (Lipid Al), 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (Lipid A2), ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l-diyl)bis(decanoate) (Lipid B), 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyl9Z,9'Z,12Z,12'Z)-bis(octadeca-9, 12-dienoate) (Lipid C), 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)-l 3 -(octanoyloxy)tridecyl3 -octylundecanoate (Lipid D), (6Z, 16Z)- 12-((Z)-dec-4-en- 1 -yl)docosa-6, 16-dien-l 1 -y 15 - (dimethylamino)pentanoate (Lipid I), Dioctadecyl-(2-hydroxyl-3- propylamino)aminopolylysine (Lipid T), (3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yloxy)-N,N-dimethylpropan-l-amine (MC3 Ether), describedinU.S.ProvisionalApplicationNo.61/384, 050 (MC3 Thioester), (4- ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-l-amine (MC4 Ether), 3-((2-(((9Z, 12Z)-octadeca-9, 12-dienoyl)oxy)ethyl)amino)propanoicacid (MLAP A), 3 -((2-(((9Z, 12Z, 15Z)-octadeca-9, 12,15- trienoyl)oxy)ethvnamino)propanoicacid (MLLAPA), mon-omycolylglycerol (MMG), 3- ((2-(oleoyloxy)ethyl)amino)propanoicacid (MOAPA), 4-(2-Aminoethyl)-Morpholino- Cholesterolhemisuccinat (MoChol), l,2-Dioleoyl-3-N-morpholine-propane (MoDO), Methylpyridiyl-dialkyl-carboxylicacid (MPDACA), monopalmitoylphosphatidylcholin (MPPC), 3-((2-(stearoyloxy)ethyl)amino)propanoicacid (MSAPA), Nl-[2-((lS)-l-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4- di[oleyloxy]-benzamide (MVL5), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca- 9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA (2R)), phosphatidylcholines (PC), l,3-Bis-(l,2-bis-tetradecyloxy-propyl-3-dimemylethoxyammoniumbromide)-propane-2-ol (PCL-2), palmitoyi-oieoyl-nor-arginine (PONA), stearylamine (STA), 2-(((tert- Butyldimethylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-l,3-diol (Synthesis Example 1 (A)), 3-((tert-Butyl(dimethyl)silyl)oxy)-2,2-bis(((9Z)-tetradec-9- enoyloxy)methyl)propyl(9Z)-tetradec-9-enoate (Synthesis Example 1 (B)), 3-Hydroxy- 2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl(9Z)-tetradec-9-enoate (Synthesis Example 1 (C)), 3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9- enoyloxy)methyl)propyl(9Z)-tetradec-9-enoate (Synthesis Example 1 (D)), 3-(5-(bis(2- hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2- hydroxyundecyl)amino)pentan-2-yl)-l,4-dioxane-2, 5-dione) (Target 24), trehalose6'6'- dibehenate (TDB), 1, l'-(2-(4-(2-((2-(bis(2hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodecan-2-ol (Tech Gl), 3- (( 1 ,3 -bis(((9Z, 12Z)-octadeca-9, 12-dienoyl)oxy)-2-((((9Z, 12Z)-octadeca-9, 12- dienoyl)oxy)methyl)propan-2-yl)amino)propanoicacid (TLAPA), (l-(2,3- linoleyloxypropoxy)-2-(linoleyloxy)-(7V,A/-dimethyl)-propyl-3-amine) (TLinDMA), 3- (( 1 ,3 -bis(((9Z .12Z .15Z)-octadeca-9.12.15 -tri enoy l)oxy)-2-((((9Z .12Z .15E)-octadeca- 9, 12, 15-trienoyl)oxy)methyl)propan-2-yl)amino)propanoicacid (TLLAPA), N-(a- trimethylammonioacetyl)-didodecyl-D-glutamatechloride (TMAG), 3-((l,3-bis(((Z)- octadec-9-enoyl)oxy)-2-((((Z)-octadec-9-enoyl)oxy)methyl)propan-2- yl)amino)propanoicacid (TOAPA), 3-((l,3-bis(stearoyloxy)-2- ((stearoyloxy)methyl)propan-2-yl)amino)propanoicacid (TS AP A), 1 ,N19- bis((l 6E,18E,20E, 22E)-17, 21 -dimethyl-15-oxo-23 -(2,6, 6-trimethylcy cl ohex-l-en-l-yl)- 4,7, 10-trioxa- 14-azatricosa- 16,18,20,22-tetraen- 1 -yl)-4,7, 10,13,16-pentaoxanonadecane- 1 , 19-diamide (VA-PEG- VA), 2,2-Dillinoleyl-4-dimethylaminoethyl-[ 1 ,3 ]-di oxolane (XTC), disclosedinNon-PatentLiteraturel l (YSK05), l,2-di-y-linolenyloxy-N,N- dimethylaminopropane (y-DLenDMA), a-D-Tocopherolhemisuccinoyl, (9Z,9,Z,12Z,12,Z)-2-((2-(((3- (dimethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-l,3-diylbis(octadeca- 9,12-dienoate), 2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16- dienoyl)oxy)propane- 1 ,3 -diyldioctanoate, 2-(((l 3Z, 16Z)-4-(((3 - (dimethylamino)propoxy)carbonyl)oxy)docosa- 13,16-dienoyl)oxy)propane- 1,3- diyldioctanoate, 2-((4-(((3- (ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyldioctanoate, 2-((4-(((3- (ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diylbis(decanoate), 2-((4-(((3- (diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diylbis(decanoate), 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaicosan-20-yl)propane-l,3- diyldioctanoate, 2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2- ((octanoyloxy)methyl)propane-l,3-diyl(9Z,9'Z)bis-tetradec-9-enoate, (9Z,9'Z,12Z,12'Z)- 2-(((l-(cyclopropylmethyl)piperidine-4-carbonyl)oxy)methyl)propane-l,3- diylbis(octadeca-9,12-dienoate), ((2-(((l-isopropylpiperidine-4-carbonyl)oxy)methyl)-l,4- phenylene)bis(oxy))bis(octane-8, 1 -diyl)bis(decanoate), 2-((4-(((3-
(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyldidodecanoate, 2-((4-(((3-
(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diyldidodecanoate,
2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyldidodecanoate, 2-((4-(((3-
(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyl di tetradecanoate, 2-((4-(((3-
(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyl di tetradecanoate, 2-((4-(((3-
(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3- diyl di tetradecanoate, (Z)-2-((4-(((3-
(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diyl dioleate,
(9Z,9,Z, 12Z, 12,Z, 15Z, 15,Z)-2-((4-(((3 -
(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diylbis(octadeca- 9, 12, 15-trienoate), (9Z,9,Z, 12Z, 12,Z)-2-((4-(((3 -
(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diylbis(octadeca-
9, 12-dienoate), (9Z,9,Z, 12Z, 12,Z)-2-((4-(((3 -
(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diylbis(octadeca-
9,12-dienoate), N,N,N-trimethyl-5-oxo-5-(3-((3-pentyloctanoyl)oxy)-2,2-bis(((3- pentyloctanoyl)oxy)methyl)propoxy)pentane-l-Aminiumiodide3-((5- (dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3- pentyloctanoate, 3-dimethylaminopropylcarbonate(9Z,12Z)-octacosa-19,22-dien-l 1-yl, 2-
(((N,N-dimethyl-P-alanyl)oxy]methyl}-2-[(octanoyloxy)methyl)propane-l,3- diyl(9Z,9'Z)bis-tetradec-9-enoate, O'I,Ol-(2-(7-dodecyl-14-methyl-3,9-dioxo-2,4,8,10- tetraoxa-14-azapentadecyl)propane-l,3-diyl)8-dimethyldioctanedioate, 8-dimethylO'I,01- (2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diyl)dioctanedioate, l-(3- ((6,6-bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4-dimethylpiperidine-4- carbonyl)oxy)methyl)propyl)8-methyloctanedioate, (9Z,12Z)-5-(((3- (dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyloctadeca-9,12-dienoate, 5-(((3- (dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyloctanoate, l-(3-((6,6-bis((2- propylpentyl)oxy)hexanoyl)oxy)-2-(((l,4-dimethylpiperidine-4- carbonyl)oxy)methyl)propyl)10-octyldecanedioate, 3 -(((3- (dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyldecanoate, l-(16-(((4,4- bis(octyloxy)butanoyl)oxy)methyl)-9-dodecyl-2-methyl-7, 13-dioxo-6,8, 12, 14-tetraoxa-2- azaheptadecan-17-yl)8-methyloctanedioate, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2- bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl(9Z, 12Z)-octadec-9, 12-dienoate, 3-((5- (Dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl3- pentyloctanoate, (9Z,9'Z, 12Z, 12'Z)-2-(((3-(diethylamino)propanoyl)oxy)methyl)propane- l,3-diylbis(octadeca-9, 12-dienoate), ((2-(((4-(dimethylamino)butanoyl)oxy)m ethyl)- 1,4- phenylene)bis(oxy))bis(octane-8,l-diyl)bis(decanoate), l-(3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propyl)8- methyloctanedioate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2- ((palmitoyloxy)methyl)propyll-methylpyrrolidine-3 -carboxylate, 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((tetradecanoyloxy)methyl)propyll-methylpyrrolidine-3- carboxylate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl9- pentyltetradecanoate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2- ((dodecanoyloxy)methyl)propyll-methylpyrrolidine-3 -carboxylate, 3 -(((3- (dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl9-pentyltetradecanoate, 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl7-hexyltridecanoate, 2- (5-(3-((l-methylpyrrolidine-3-carbonyl)oxy)-2-((tetradecanoyloxy)methyl)propoxy)-5- oxopentyl)propane-l,3-diyldioctanoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)- 13-(octanoyloxy)tridecyl5-heptyldodecanoate, 2-(5-(3-((l-methylpyrrolidine-3- carbonyl)oxy)-2-((palmitoyloxy)methyl)propoxy)-5-oxopentyl)propane-l,3- diyldioctanoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl5- heptyldodecanoate, 2-(((l-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3- diylbis(6,6-bis(octyloxy)hexanoate), (9Z, 12Z)-3-(((3- dimethylamino)propoxy)carbonyl)oxy)-l 3 -(octanoyloxy)tridecyloctadeca-9,12-di enoate, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9- enoyloxy)methyl)propyl(9Z)-octadec-9-enoate, 2-(10-dodecyl-3-ethyl-8, 14-dioxo-7,9, 13- trioxa-3 -azanonadecan- 19-yl)propane- 1 ,3 -diyldioctanoate, ((2-((( 1 -methylpiperidine-4- carbonyl)oxy)methyl)-l,4-phenylene)bis(oxy))bis(octane-8,l-diyl)bis(decanoate), 2-(((3- (dimethylamino)propanoyl)oxy)methyl)propane-l,3-diylbis(4,4-bis(octyloxy)butanoate), (9Z, 12Z)-2-((( 11 Z, 14Z)-2-((3 -(dimethylamino)propanoyl)oxy)icosa- 11 , 14-dien- 1 - yl)oxy)ethyloctadeca-9,12-di enoate, 2-(((l,3-dimethylpyrrolidine-3- carbonyl)oxy)methyl)propane-l,3-diylbis(4,4-bis(octyloxy)butanoate), (13Z,16Z)-4-(((3- (dimethylamino)propoxy)carbonyl)oxy)docosa- 13,16-dien- 1 -ylheptadecan-9-yl succinate, 2,2-bis(heptyloxy)ethyl3 -((3 -ethyl- 10-((9Z, 12Z)-octadeca-9, 12-dien- 1 -y 1)- 8 , 15-dioxo- 7,9,14-trioxa-3 -azaheptadecan- 17 -y 1 )di sulfanyl)propanoate, 2-((( 1 -methyl pyrrolidine-3 - carbonyl)oxy)methyl)propane- 1 , 3 -diylbi s(4,4-bi s(octyloxy)buta, 1 -(3 -(( 1 ,3 - dimethylpyrrolidine-3-carbonyl)oxy)-2-(((9Z,12Z)-octadeca-9,12- dienoyloxy)methyl)propyl) 10-octyldecanedioate, ( 13Z, 16Z)-4-(((3 - (diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-l-yl2,2-bis(heptyloxy)acetate, ( 13Z, 16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa- 13 , 16-dien- 1 -yl2,2- bis(heptyloxy)acetate, Aceticacid(20,23R)-2-methyl-9-[(9Z,12Z)-octadeca-9,12-dien-l- yl]-7-oxo-6,8,l l-trioxa-2-azanonacosa-20-En-23-yl3- (dimethylamino)propylcarbonate(HZ,14Z)-l-{[(9Z,12R)-12-hydroxyoctadec-9-en-l-yl], (12Z,15Z)-l-((((9Z,12Z)-octadeca-9,12-dien-l-yloxy)carbonyl)oxy)henicosa-12,15-dien- 3-yl3-(dimethylamino)propanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (dimethylamino)propyl)carbamoyl)oxy)methyl)propyloctadeca-9, 12-dienoate, (12Z, 15Z)- 3-((4-(dimethylamino)butanoyl)oxy)henicosa-12,15-dien-l-yl9-pentyltetradecanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l,2,2,6,6-pentamethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-dienoate, (12Z, 15Z)-3-((4- (dimethylamino)butanoyl)oxy)henicosa- 12, 15-dien- 1 -yl7-hexyltridecanoate, (9Z, 12Z)-3 - ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)methoxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-di enoate, (12Z, 15Z)-3-((4- (dimethylamino)butanoyl)oxy)henicosa-12,15-dien-l-yl5-heptyldodecanoate, (9Z,12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-ethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-di enoate, (12Z, 15Z)-3-((4- (dimethylamino)butanoyl)oxy)henicosa-12,15-dien-l-yl3-octylundecanoate,formatesalt, 3- ((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl(9Z)- hexadec-9-enoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-methylazetidin-3- yl)oxy)carbonyl)oxy)methyl)propyloctadeca-9, 12 - di enoate, (9Z, 12Z)-( 12Z, 15Z)-3 -((3 - (dimethylamino)propanoyl)oxy)henicosa-12,15-dien-l-yloctadeca-9,12-dienoate, 2-(((3- (diethylamino)propoxy)carbonyl)oxy)tetradecyl4,4-bis((2-ethylhexyl)oxy)butanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-di enoate, (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((((l-methylpyrrolidin-3- yl)oxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-di enoate, (9Z, 12Z)-3-(((2- (dimethylamino)ethoxy)carbonyl)oxy)pentadecyloctadeca-9, 12-di enoate, (9Z, 12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3 -(4-methylpiperazin- 1 - yl)propoxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-di enoate, 3- (Dimethylamino)propyltriacontan-l 1-ylcarbonateTriacontan-l l-ol, (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(pyrrolidin-l- yl)propoxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-di enoate, (9Z,12Z)-3-(((3- (ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyloctadeca-9, 12-di enoate, 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl4- ((diethylamino)methyl)benzoate, (9Z,12Z)-3-(((3-
(diethylamino)propoxy)carbonyl)oxy)pentadecyloctadeca-9, 12-di enoate, 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl3- ((dimethylamino)methyl)benzoate, (9Z,12Z)-3-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentadecyloctadeca-9, 12-di enoate, 3 -((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyll- methylpiperidine-3 -carboxylate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9,12-dienoyloxy)methyl)propyll-methylpiperidine-4-carboxylate, 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyll,4- dimethylpiperidine-4-carboxylate, 3-((4-(dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)- tetradec-9-enoyloxy)methyl)propyl(9Z)-hexadec-9-enoate, 2-(10-dodecyl-3-ethyl-8,14- dioxo-7, 9, 13-trioxa-3-azahexadecan-16-yl)propane-l,3-diyldi octanoate, (9Z,9'Z,12Z,12'Z)-2-(((4-(piperidin-l-yl)butanoyl)oxy)methyl)propane-l,3- diylbis(octadeca-9, 12-dienoate), 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z, 12Z)- octadeca-9,12-dienoyloxy)methyl)propyl4-methylmorpholine-2-carboxylate, (2R)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyll- methylpyrrolidine-2-carboxylate, (2S)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)- octadeca-9,12-dienoyloxy)methyl)propyll-methylpyrrolidine-2-carboxylate, (9Z,9'Z,12Z,12'Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((9Z,12Z)- octadeca-9,12-dienoyloxy)methyl)propane-l,3-diylbis(octadeca-9, 12-dienoate), (9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((l-ethylpiperidin-3- yl)methoxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-dienoate, 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyll- (cyclopropylmethyl)piperidine-4-carboxylate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2- (((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyll-isopropylpiperidine-4-carboxylate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3- (dimethylamino)propanoyl)oxy)methyl)propyloctadeca-9, 12-dienoate, 4- (dimethylamino)butylcarbonate(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-yl, 3-((6-(dimethylamino)hexanoyl)oxy)-2,2-bis(((9Z)-tetradec-9- enoyloxy)methyl)propyl(9Z)-tetradec-9-enoate, 2,5-bis((9Z, 12Z)-octadeca-9, 12- dienyloxy)benzyl3-(dimethylamino)propylcarbonate, (9Z,9'Z,12Z,12'Z)-2-(((4- (pyrrolidin-l-yl)butanoyl)oxy)methyl)propane-l,3-diylbis(octadeca-9, 12-dienoate), 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentadecyl5-heptyldodecanoate,
Aceticacid(7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacosa-9-en-7-yl, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl9-pentyltetradecanoate, (9Z,12Z)-3-((6,6-bis(octyloxy)hexanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-di enoate, 3 -(((3- (dimethylamino)propoxy)carbonyl)oxy)pentadecyl7-hexyltridec-6-enoate, (9Z,12Z)-3- (2,2-bis(heptyloxy)acetoxy)-2-((((2- (dimethylamino)ethoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-di enoate, 3 -(((3- (dimethylamino)propoxy)carbonyl)oxy)pentadecyl3-octylundec-2-enoate, (9Z,12Z)-3- (((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((5- heptyldodecanoyl)oxy)methyl)propyloctadeca-9,12-di enoate, 3 -(((3- dimethylamino)propoxy)carbonyl)oxy)pentadecyl3 octylundecanoate, (9Z,12Z)-3-(((3- (diethylamino)propoxy)carbonyl)oxy)-2-(((9- pentyltetradecanoyl)oxy)methyl)propyloctadeca-9,12-di enoate, Diaceticacid(7R,9Z,26Z,29R)-18-({[3- (dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl, 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((7- hexyltridecanoyl)oxy)methyl)propyloctadeca-9,12-di enoate, 3-(((3- (ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2- propylpentyl)oxy)octanoate, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2- (((3 -octylundecanoyl)oxy)methyl)propyloctadeca-9,12-di enoate, 3 -(((3- (diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis((2-propylpentyl)oxy)octanoate, 3- (((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-dibutoxyoctanoate, 3-((5- (dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl(9Z)- tetradec-9-enoate, 3-(Dimethylamino)propylcarbonate(6Z,9Z,26Z,29Z)-pentatriacontour- 6,9,26,29-tetraen- 18-yl, 2,5-bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)benzyl3- (dimethylamino)propanoate, (9Z,9'Z,12Z,12'Z)-2-(((3-(4-methylpiperazin-l- yl)propanoyl)oxy)methyl)propane- 1 , 3 -diylbi s(octadeca-9, 12 - di enoate), 3 -(((3 - (diethylamino)propoxy)carbonyl)oxy)pentadecyl8,8-bis(octyloxy)octanoate, 3- (Dimethylamino)propyloctacosane-l 1-ylcarbonate, 2,4-bis((9Z, 12Z)-octadeca-9, 12- dienyloxy)benzyl4-(dimethylamino)butanoate, (9Z, 12Z)-3-(((3- (diethylamino)propoxy)carbonyl)oxy)-2-(((2- heptylundecanoyl)oxy)methyl)propyloctadeca-9,12-di enoate, 3 -(((3- (diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-ethylhexyl)oxy)hexanoate, 2- ((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propane-l,3-diylbis(2- heptylundecanoate), 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6- bis(hexyloxy)hexanoate, 4-methyl-2,5-bis((9Z, 12Z)-octadeca-9, 12-dien-l-yloxy)benzyl4- (dimethylamino)butanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6- bis(octyloxy)hexanoate, 4-(dimethylamino)butyl4-methyl-2,5-bis((9Z, 12Z)-octadeca-9, 12- dienyl oxy )benzyl carb onate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pendadecyl4,4- bis((2-propylpentyl)oxy)butanoate, 2-(12-dodecyl-3-ethyl-8, 14-dioxo-7,9, 13-trioxa-3- azaoctadecan- 18-yl)propane- 1 ,3 -diyldioctanoate, 2-(5-oxo-5 -((3 -(((3 -(piperidin- 1 - yl)propoxy)carbonyl)oxy)pentadecyl)oxy)pentyl)propane-l,3-diyldioctanoate, 3- (dimethylamino)propyl4-methyl-2,5-bis((9Z,12Z)-octadeca-9,12-dien-l- yloxy)benzyl carb onate, 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl4,4- bis((2-propylpentyl)oxy)butanoate, 2-(l l-dodecyl-3-ethyl-9,15-dioxo-8,10,14-trioxa-3- azanonadecan- 19-yl)propane- 1 ,3 -diyldioctanoate, 2-(l 0-dodecyl-3 -ethyl-8, 15-dioxo- 7,9, 14-trioxa-3 -azanonadecan- 19-yl)propane- 1 ,3 -diyldioctanoate, 2-(5-((4-(((( 1 - methylpiperidin-4-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-l,3- diyldioctanoate, 2-(5-((4-((((l-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)hexadecyl)oxy)- 5-oxopentyl)propane-l,3-diyldioctanoate, 2-(5-((4-(((((R)-l-methylpyrrolidin-3- yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-l,3-diyldioctanoate, 2-(5-((4- (((((S)-l-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane- 1,3-diyldioctanoate, 2-(5-oxo-5-((4-(((S)-pyrrolidine-2- carbonyl)oxy)hexadecyl)oxy)pentyl)propane- 1,3 -diyldioctanoate, 2-(5-((4-((l,3- dimethylpyrrolidine-3-carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-l,3- diyldioctanoate, 2-(5-((4-((l,4-dimethylpiperidine-4-carbonyl)oxy)hexadecyl)oxy)-5- oxopentyl)propane- 1,3 -diyldioctanoate, 4,4-bis(octyloxy)butyl(3- (diethylamino)propyl)pentadecane- 1 ,3 -diyl di carbonate, 3 -(((3 - (diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-propylpentyl)oxy)butanoate, ((2-((((3 -(di ethyl ami no)propoxy (carbonyl )oxy (methyl )- l , 4- phenylene)bis(oxy))bis(octane-8, 1 -diyl)bis(decanoate), 4,4-bis(octyloxy)butyl5-(((3- (diethylamino)propoxy)carbonyl)oxy)heptadecanoate, 6-((6,6- bis(octyloxy)hexanoyl)oxy)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexyloctanoate, (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)henicosa-12,15-dien-l-yl6,6- bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl6,6- bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl6,6- bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl5-(4,6- diheptyl-l,3-dioxan-2-yl)pentanoate, 3-((5-(diethylamino)pentanoyl)oxy)pentadecyl6,6- bis(octyloxy)hexanoate, l-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yll,4- dimethylpiperidine-4-carboxylate, 3-((3-(l-methylpiperidin-4- yl)propanoyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate, l-((6,6- bis(octyloxy)hexanoyl)oxy)pentadecan-3-yll,3-dimethylpyrrolidine-3-carboxylate, 3-(((3- (diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis((2-ethylhexyl)oxy)butanoate, 2- (((l,3-dimethylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diylbis(8- (octanoyloxy)octanoate), ((2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)-l,4- phenylene)bis(oxy))bis(octane-8, 1 -diyl)bis(decanoate), (2R)-1 -((6,6- bis(octyloxy)hexanoyl)oxy)pentadecan-3-ylpyrrolidine-2-carboxylate, (2S)-l-((6,6- bis(octyloxy)hexanoyl)oxy)pentadecan-3 -yl 1 -methylpyrrolidine-2-carboxylate, (2R)- 1 - ((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yll-methylpyrrolidine-2-carboxylate, 3- (((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((3- ethylpentyl)oxy)hexanote, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl6,6- bis((2-propylpentyl)oxy)hexanoate, 3 -(((3- (diethylamino)propoxy)carbonyl)oxy)pentadecyl6,6-bis((2-propylpentyl)oxy)hexanoate, 3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate, 3-(((3- morpholinoproproxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate, 3-((((l- methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate, 3- (((3-(4-methylpiperazin-l-yl)propoxy)carbonyl)oxy)pentadecyl6,6- bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl4,4- bis(octyloxy)butanoate, 2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2- ((dodecanoyloxy)methyl)propane-l,3-diyl(9Z,9'Z)bis-tetradec-9-enoate, (9Z,9'Z,12Z,12'Z)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propane-l,3- diylbi s(octadeca-9, 12 - di enoate), 3 -(((4- (diethylamino)butoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanote, 3-(((3- (piperazin-l-yl)propoxy)carbonyl)oxy)pentadecyl6,6-bis(octyloxy)hexanoate, 3-(((3- piperidin-l-yl)propoxy)carbonyl)oxy)pentadecyl6.6-bis(octyloxy)hexanoate, 3-(((3- (dimethylamino)propoxy)carbonyl)oxy)pentadecyl4,4-bis(octyloxy)butanoate, (9Z,9'Z,12Z,12'Z)-2-(9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azatetradecan-14- yl)propane- 1 ,3 -diylbis(octadeca-9, 12-di enoate), (9Z, 12Z)- 10-dodecyl-3 -ethyl- 14-(2- ((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)-8,13-dioxo-7,9-dioxa-3,14-diazahexadecan- 16-yloctadeca-9, 12-dienoate, 2-((2-(((3 - (diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-l,3-diyldioctanoate, 2- (9-dodecyl-2-methyl-7, 13 -dioxo-6,8, 12-trioxa-2-azanonadecan- 19-yl)propane- 1,3- diyldioctanoate, 2-((decanoyloxy)methyl)-2-(((4- (dimethylamino)butanoyl)oxy)methyl)propane-l,3-diyl(9Z,9'Z)bis-tetradec-9-enoate, (9Z,9'Z,12Z,12'Z)-2-(((3-morpholinopropanoyl)oxy)methyl)propane-l,3-diylbis(octadeca- 9, 12-dienoate), 3-(Dimethylamino)propylcarbonate(6Z,9Z,28Z,3 lZ)-heptatriconta- 6,9,28,31-tetraen- 19-yl, 2,5-bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)benzyl4- (dimethylamino)butanoate, 2-(10-dodecyl-3-ethyl-8, 14-dioxo-7,9, 13-trioxa-3- azaoctadecan- 18-yl)propane- 1 ,3 -diyldioctanoate, (9Z,9'Z, 12Z, 12'Z)-2-((( 1,3- dimethylpyrrolidine-3-carbonyl)oxy)methyl)propane-l,3-diylbis(octadeca-9,12-dienoate), ((5-((dimethylamino)methyl)benzene- 1 ,2,3-triyl)tris(oxy))tris(decanel 0, 1 - diyl)trioctanoate, 0',0-(((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(propane- 3,l-diyl))9-dioctyldinonanedioate, (9Z,12Z)-3-(3-((dimethylamino)methyl)-5-(3-((3- octylundecanoyl)oxy)propoxy)phenoxy)propyloctadeca-9,12-dienoate, ((((5- ((dimethylamino)methyl)- 1 , 3 -phenylene)bi s(oxy))bi s(propane-3 , 1 -diyl))bis(oxy))bis(4- oxobutane-4,l-diyl)bis(decanoate), (R)-4-(3-((R)-3,4-bis(octanoyloxy)butoxy)-5- ((dimethylamino)methyl)phenoxy)butane-l,2-diyldioctanoate, (S)-4-(3-((S)-3,4- bis(octanoyloxv)butoxv)-5-((dimethylamino)methyl)phenoxy)butane-l,2-diyldioctanoate, (R)-4-(3-((S)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane-
1.2-diyldioctanoate, 4,4'-((5-((dimethylamino)methyl)-l,3- phenylene)bis(oxy))bis(butanel,2-diyl)tetraoctanoate, didodecyl6,6'-((5-
((dimethylamino)methyl)-l,3-phenylene)bis(oxy))dihexanoate, di((9Z,12Z)-octadeca- 9,12-dien-l-yl)5,5'-((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))dipentanoate, (((5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene))bis(oxy))bis(6-oxohexane- 6,l-diyl)bis(decanoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene)bis(8- (octanoyloxy)octanoate), (5-((dimethylamino)methyl)-l,3- phenylene)bis(methylene)bis(10-(octanoyloxy)decanoate), (((5-((dimethylamino)methyl)-
1.3-phenylene)bis(methylene))bis(oxy))bis(6-oxohexane-6,l-diyl)dioctanoate, (((5- ((dimethylamino)methyl)-l,3-phenylene)bis(methylene))bis(oxy))bis(8-oxooctane-8,l- diyl)bis(decanoate), (9Z,9'Z,12Z,12'Z)-(((5-((dimethylamino)methyl)-l,3- phenylene)bis(methylene))bis(oxy))bis(4-oxobutane-4,l-diyl)bis(octadeca-9,12-dienoate), O', 0-((5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene))8-dinonyl di octanedi oate, 0,0'-((5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene))bis(10- (octanoyloxy)decyl)disuccinate, 0,0'-((5-((dimethylamino)methyl)-l,3- phenylene)bis(methylene))di((9Z,12Z)-octadeca-9,12-dien-l-yl)disuccinate, (9Z,9'Z,12Z,12'Z)-(5-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-l,3- phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,12Z)-4-(3- ((dimethylamino)methyl)-5-(4-(oleoyloxy)butoxy)phenoxy)butyloctadeca-9,12-dienoate, (9Z,9'Z, 12Z, 12'Z, 15Z, 15 'Z)-((5 -((dimethvlamino)methyl)- 1,3- phenylene)bis(oxy))bis(butane-4,l-diyl)bis(octadeca-9,12,15-trienoate), ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane-4,l-diyl)ditetradecanoate, (Z)-((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane-4,l-diyl)dioleate, ((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(hexane-6,l-diyl)didodecanoate, (9Z,9'Z,12Z,12'Z)-((((5-((diethylamino)methyl)-l,3-phenylene)bis(oxy))bis(ethane-2,l- diyl))bis(oxy))bis(ethane-2, l-diyl)bis(octadeca-9, 12-dienoate), didecyl8,8'-((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))dioctanoate, ((5-
I l l ((dimethylamino)methyl)- 1 , 3 -phenylene)bi s(oxy))bi s(propane-3 , 1 - diy 1 )b i s(3 - octylundecanoate), (9Z.9'Z.12Z.12'Z)-((5-((diethvlamino)methvn-2-methvl-l .3- phenylene)bi s(oxy))bi s(butane-4, 1 - diy 1 )b i s(octadeca-9, 12 - di enoate), ((5 - ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l-diyl)didodecanoate, ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l-diyl)bis(decanoate), (9Z.9'Z.12Z.12'Z)-((5-((dimethvlarnino)methvn-2-methvl-l .3- phenylene)bi s(oxy))bi s(butane-4, 1 - diy 1 )b i s(octadeca-9, 12 - di enoate), (8Z, 8'Z)-((5 - ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(hexane-bis(dodec-8-enoate), (9Z,9'Z,12Z,12'Z)-((5-((3-hydroxyazetidin-l-yl)methyl)-l,3- phenylene)bi s(oxy))bi s(butane-4, 1 - diy 1 )b i s(octadeca-9, 12 - di enoate), ((5 - ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(hexane-6,l-diyl)dioctanoate, ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(hexane-6,l-diyl)bis(decanoate), (9Z.9'Z.12Z.12'Z)-((5-((dimethvlamino)methvn-1.3-phenylene)bis(oxy))bis(octane-8,l- diyl)bis(octadeca-9, 12-dienoate), (9Z,9'Z, 12Z, 12'Z)-((5-((dimethvlamino)methyl)-l,3- phenylene)bis(oxy))bis(hexane-6, 1 -diyl)bis(octadeca-9, 12-dienoate), ((5- ((dimethylamino)methyl)- 1 , 3 -phenylene)bi s(oxy))bi s(decane- 10,1 -diyl)dihexanoate, ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(decane-10,l-diyl)dioctanoate, ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l-diyl)dioctanoate, ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l-diyl)dihexanoate, (9Z,9'Z,12Z,12'Z)-((5-((dimethvlamino)methyl)-l,3-phenylene)bis(oxy))bis(ethane-2,l- diyl)bis(octadeca-9, 12-dienoate), (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-l,3- phenylene)bis(oxy))bis(propane-3,l-diyl)bis(octadeca-9, 12-dienoate), (9Z,9'Z,12Z,12'Z)- ((5 -((dimethylamino)methyl)- 1 , 3 -phenylene)bi s(oxy))bi s(butane-4, 1 - diy 1 )b i s(octadeca- 9, 12-dienoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene)ditridecanoate, (9Z,9'Z,12Z,12'Z)-(5-((dimethylamino)methyl)-l,3- phenylene)bis(methylene)bis(octadeca-9, 12-dienoate), (2,6-bis((9Z,12Z)-octadeca-9,12- dien-l-yloxy)pyridin-4-yl)methyl3-(dimethylamino)propanoate, (9Z,9'Z,12Z,12'Z)-5-(((3- (dimethylamino)propanoyl)oxy)methyl)-l,3-phenylenebis(octadeca-9, 12-dienoate), 1- (3 , 5 -bi s((9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy)phenyl)-N,Ndimethylmethanamine, 3,5- bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)benzyl3-(dimethylamino)propanoate, l-(3,5- bis(4,4-bis(octyloxy)butoxy)phenyl)-N,N-dimethylmethanamine, ((((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane-4,l- diyl))bis(oxy))bis(propane-3,2,l-triyl)tetraoctanoate, ((5-(((4- (dimethylamino)butanoyl)oxy)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l- diyl)bis(decanoate), ((5-(((3-(dimethylamino)propanoyl)oxy)methyl)-l,3- phenylene)bi s(oxy))bi s(octane-8, 1 -diy 1 )b i s(decanoate), (9Z, 9'Z, 12Z, 12'Z)-((5 -(3 - morpholinopropyl)-l,3-phenylene)bis(oxy))bis(butane4,l-diyl)bis(octadeca-9,12- dienoate), (9Z,9'Z,12Z,12'Z)-((5-(3-(dimethvlamino)propyl)-l,3- phenylene)bis(oxy))bis(butane-4,l-diyl)bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12'Z)- ((5 -(3 -(piperidin- 1 -yl)propyl)- 1 , 3 -phenylene)bi s(oxy))bi s(butane-4, 1 -diy 1 )b i s(octadeca- 9,12-dienoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene)bis(9- pentyltetradecanoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene)bis(7- hexyltridecanoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene)bis(5- heptyldodecanoate), ((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane-4,l- diy 1 )b i s(3 -octylundecanoate), ((5 -((dimethylamino)methyl)- 1,3- phenylene)bi s(oxy))bi s(butane-4, 1 - diy 1 )b i s(5 -heptyldodecanoate), ((5 - ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane-4,l-diyl)bis(9- pentyltetradecanoate), ((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane- 4,l-diyl)bis(7-hexyltridecanoate), (9Z,9'Z,12Z,12'Z)-((5-(pyrrolidin-l-ylmethyl)-l,3- phenylene)bis(oxy))bis(butan4, l-diyl)bis(octadeca-9, 12-dienoate), (((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(methylene))bis(propane-3,2,l- triyl)tetraoctanoate, (((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butane-4,l- diyl))bis(propane-3,2,l-triyl)tetraoctanoate, (9Z.12Z)-4-(3-((dimethvlamino)methvn-5-(4- ((3 -octylundecanoyl)oxy)butoxy)phenoxy)butyloctadeca-9, 12-dienoate, bis(l,3- bis(octanoyloxy)propan-2-yl)0,0'-((5-((dimethylamino)methyl)-l,3- phenylene)bis(methylene))disuccinate, (5-((dimethylamino)methyl)-l,3- phenylene)bis(methylene)bis(6-(((nonyloxy)carbonyl)oxy)hexanoate), 2-(3-(4-(5- ((dimethylamino)methyl)-2-methyl-3-((9Z,12Z)-octadeca9,12-dien-l- yloxy)phenoxy)butoxy)-3-oxopropyl)propane-l,3-diyldihexanoate, 3- ((dimethylamino)methyl)-5-(((8-(octanoyloxy)octanoyl)oxy)methyl)benzyl3- octylundecanoate, ((5-((diethylamino)methyl)benzene-l,2,3-triyl)tris(oxy))tris(decane- 10,1 -diyl)trioctanoate, 1 -(3 , 5-bis((Z)-octadec-9-en- 1 -yloxy)phenyl)-N,N- dimethylmethanamine, N’-methyl-N’,N”.N”-tris((2E.6E)-3.7.1 l-trimethyldodeca-2.6.10- trien- 1 -vnpropane- 1 ,3 -diamine, 1, 17-bis(2-((2- pentylcyclopropyl)methyl)cyclopropyl)heptadecan-9-yl4-(dimethylamino)butanoate, ethyl(7Z)-17-{[4-(dimethylamino)butanoyl]oxy}hexacos-7-enoate, (Z)-methyl6-(2- (dimethylamino)-3-(octadec-9-en-l-yloxy)propoxy)hexanoate, 2-(Didodecylamino)-l-(4- (N-(2-(dinonylamino)ethyl)-N-dodecylglycyl)piperazin-l-yl)ethan-l-one, 3-((3-(l-(3-((2- (Dinonylamino)ethyl)(nonyl)amino)propanoyl)piperidin-4- yl)propyl)(nonyl)amino)propylhexanoate, 3 -((3 -(4-(3 -((2- (Dinonylamino)ethyl)(nonyl)amino)propanoyl)piperazin-l-yl)-3- oxopropyl)(nonyl)amino)propylhexanoate, 3 -((2-(Dinonylamino)ethyl)(nonyl)amino)- 1 - (4-(3 -(dinonylamino)propyl)piperidin- 1 -yl)propan- 1 -one, Pentyl4-((3 -(1 -(3 -((2- (dinonylamino)ethyl)(nonyl)amino)propanoyl)piperidin-4-yl)propyl)(nonyl)amino)butano, Pentyl4-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4- yl)ethyl)(nonyl)amino)butanoate, Pentyl4-(((l-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)pyrrolidin-3-yl)methyl)(nonyl)amino)butanoate, Pentyl4-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)butanoate, Pentyl4-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-3- yl)ethyl)(nonyl)amino)butanoate, 2-(Didodecylamino)- 1 -(4-(N-(2-(dinonylamino)ethyl)- N-nonylglycyl)piperazin- 1 -yl)ethan- 1 -one, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)- 1 - (3-(2-(dinonylamino)ethyl)piperidin-l-yl)ethan-l-one, Dipentyl4,4'-((2-(4-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2-oxoethyl)azanediyl)dibutyrate, Pentyl4-(nonyl(2-(4-(N-nonyl-N-(2-(nonyl(4-oxo-4- (penlyloxy)buryl)amino)ethyl)glycyl)piperazin-l-yl)-2-oxoethyl)amino)butanoate, 2-((2- (Dinonylamino)ethyl)(nonyl)amino)- 1 -(3 -((dinonylamino)methyl)pyrrolidin- 1 -yl)ethan- 1 - one, 2-((2-(Didodecylamino)ethyl)(dodecyl)amino)-l-(4-(dinonylglycyl)piperazin-l- yl)ethan- 1 -one, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)- 1 -(3 -(2- (dinonylamino)ethyl)pyrrolidin- 1 -yl)ethan- 1 -one, Pentyl4-((3 -(4-(3 -((2- (dinonylamino)ethyl)(nonyl)amino)propanoyl)piperazin- 1 -y l)-3 - oxopropyl)(nonyl)amino)butanoate, 3-((2-(l-(N-(2-(Dinonylamino)ethyl)-N- nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)propylhexanoate, Butyl5-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)pentanoate, 2-((2- (Didodecylamino)ethyl)(nonyl)amino)-l-(4-(dinonylglycyl)piperazin-l-yl)ethan-l-one, Propyl6-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4- yl)ethyl)(nonyl)amino)hexanoate, Ethyl7-((2-(l-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)heptanoate, Methyl8-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)octanoate, 3-((2- (4-(N-(2-(Dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(nonyl)amino)propylhexanoate, Butyl5-((2-(4-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)pentanoate, Propyl6-((2-(4-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperazin-2-oxoethyl)(nonyl)amino)hexanoate, Ethyl7-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(nonyl)amino)heptanoate, 3 -(Dinonylamino)- 1 - (4 - (3 -((2- (dinonylamino)ethyl)(nonyl)amino)propanoyl)piperazin-l-yl)propan-l-one, 2-((2- (Dinonylamino)ethyl)(nonyl)amino)- 1 -(4-(ditetradecylgly cyl)piperazin- 1 -yl)ethan- 1 -one, 2-(Dinonylamino)-l-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl)piperidin-l- yl)ethan- 1 -one, 2-(Dinonylamino)-l-(4-(N-(2-(dinonylamino)ethyl)-N- dodecylglycyl)piperazin- 1 -yl)ethan- 1 -one, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)- 1 - (4-(2-(dinonylamino)ethyl)piperidin-l-yl)ethan-l-one, Methyl8-((2-(4- (dinonylglycyl)piperazin- 1 -yl)-2-oxoethyl)(2-((8-methoxy-8- oxooctyl)(nonyl)amino)ethyl)amino)octanoate, Methyl8-((2-(dinonylamino)ethyl)(2-(4- (dinonylglycyl)piperazin-l-yl)-2-oxoethyl)amino)octanoate, Methyl8-((2-((2-(4- (dinonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoate, Pentyl4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-2- oxoethyl)(nonyl)amino)butanoate, Methyl8-((2-(4-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperazin- 1 -yl)-2-oxoethyl)(nonyl)amino)octanoate, 2-((2- (Didodecylamino)ethyl)(dodecyl)amino)-l-(5-(dinonylglycyl)-2,5- diazabicy clo[2.2.1 ]heptan-2-yl)ethan- 1 -one3 , 2-(Dinonylamino)- 1 -(5-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethan-l-one, N1 ,N1 ,N2-Tri((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)-N2-(2-(piperazin- 1 -yl)ethyl)ethane- 1,2- diamine, N 1 ,N1 ,N2-Tri((Z)-octadec-9-en- 1 -yl)-N2-(2-(piperazin- 1 -yl)ethyl)ethane- 1,2- diamine, 2-(Dinonylamino)-l-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l- yl)ethan-l-one, N1 ,N1 ,N2-Tridodecyl-N2-(2-(piperazin- 1 -yl)ethyl)ethane- 1 ,2-diamine, N1 ,N1 ,N2-Trinonyl-N2-(2-(piperazin- 1 -yl)ethyl)ethane- 1 ,2-diamine, N1 ,N1 ,N2-Trihexyl- N2-(2-(piperazin-l-yl)ethyl)ethane-l,2-diamine, Nl-(2-(4-(2- (Didodecylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tri((9Z,12Z)-octadeca-9,l 2-dien- 1 -yl)ethane- 1 ,2-diamine, N 1 -(2-(4-(2-(Didodecylamino)ethyl)piperazin- 1 -yl)ethyl)- N 1 ,N2,N2-tri((Z)-octadec-9-en- 1 -yl)ethane- 1 ,2-diamine, N 1 -(2-(4-(2- (Ditetradecylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tritetradecylethane-l,2- diamine, N 1 -(2-(4-(2-(Didodecylamino)ethyl)piperazin- 1 -yl)ethyl)-N 1 ,N2,N2- tritetradecylethane- 1 ,2-diamine, N 1 -(2-(4-(2-(Dinonylamino)ethyl)piperazin- 1 -yl)ethyl)- N 1 ,N2,N2-tritetradecylethane- 1 ,2-diamine, 2-(Didodecylamino)-l-(4-(2-((2- (didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l-yl)ethan-l-one, Nl-(2-(4-(2- (Di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)ethyl)piperazin- 1 -yl)ethyl)-N 1 ,N2,N2- tri dodecyl ethane- 1 ,2-diamine, N 1 -(2-(4-(2-(Di((Z)-octadec-9-en- 1 - yl)amino)ethyl)piperazin- 1 -yl)ethyl)-N 1 ,N2,N2-tridodecylethane- 1 ,2-diamine, N1 ,N1 ,N2- Tridodecyl-N2-(2-(4-(2-(dodecyl((9Z,12Z)-octadeca-9,12-dien-l- yl)amino)ethyl)piperazin- 1 -yl)ethyl)ethane- 1 ,2-diamine, N 1 -(2-(4-(2- (Ditetradecylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tridodecylethane-l,2-diamine, Nl-(2-(4-(2-(Di((Z)-dodec-6-en-l-yl)amino)ethyl)piperazin-l-yl)ethyl)-
N 1 ,N2,NAtridodecylethane- 1 ,2-diamine, (Z)-N 1 -(2-(4-(2-(Dodec-6-en-l- yl(dodecyl)amino)ethyl)piperazin-l-yl)ethyl)-N,N2,N2-tridodecylethane- 1 ,2-diamine, N1 - (2-(4-(2-(Dinonylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tridodecylethane-l,2- diamine, Nl-(2-(4-(2-(Dioctylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2- tri dodecyl ethane- 1 ,2-diamine, N 1 -(2-(4-(2-(Dihexylamino)ethyl)piperazin-l-yl)ethyl)- N 1 ,N2,N2-tridodecylethan- 1 ,2-diamine, N 1 -(2-(4-(2-(Ditetradecylamino)ethyl)piperazin- l-yl)ethyl)-N 1 ,N2,N2-trinonylethane- 1 ,2-diamine, 2-((2- (Didodecylamino)ethyl)(dodecyl)amino)-l-(4-(2-(didodecylamino)ethyl)piperazin-l- yl)ethan-l-one, N 1 -(2-(4-(2-(Didodecylamino)ethyl)piperazin-l-yl)ethyl)-N 1 ,N2,N2- trinonylethane-l,2-diamine, Nl-(2-(4-(2-(Dinonylamino)ethyl)piperazin-l-yl)ethyl)- Nl,N2,N2-trinonylethane-l,2-diamine, Nl-(2-(4-(2-(Didodecylamino)ethyl)piperazin-l- yl)ethyl)-N 1 ,N2,N2-trihexylethane- 1 ,2-diamine, Dimethyl 12,12'-((2-(4-(2-((2- (didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl)didodecanoate, Methyl 12-((2-(4-(2-((2 - (didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l- yl)ethyl)(dodecyl)amino)dodecanoate, Dipentyl6,6'-((2-(4-(2-((2- (didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)dihexanoate, Pentyl6-((2-(4-(2-((2-(ditetradecylamino)ethyl)(tetradecyl)amino)ethyl)piperazin-l- yl)ethyl)(dodecyl)amino)hexanoate, Pentyl6-((2-(4-(2-((2- (didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l- yl)ethyl)(dodecyl)amino)hexanoate, 2-(Didodecylamino)-l-(4-(N-(2- (didodecylamino)ethyl)-N-dodecylglycyl)piperazin-l-yl)ethan-l-one, 2- (Didodecylamino)-l-(4-(N-(2-(didodecylamino)ethyl)-N-nonylglycyl)piperazin-l- yl)ethan-l-one, 2-(Didodecylamino)-N-(2-(4-(2-(didodecylamino)ethyl)piperazin-l- yl)ethyl)-N-dodecylacetamide, ((2-((3,S',4R)-3,4-dihydroxypyrrolidin-l- yl)acetyl)azanediyl)bi s(ethane-2, 1 -diyl)(9Z, 9'Z, 12Z, 12'Z)-bi s(octadeca-9, 12 - di enoate), 2- amino-N,N-dihexadecyl-3-(lH-imidazol-5-yl)propanamide, (2-amino-N,N-dihexadecyl-3- (lH-imidazol-5-yl)propanamide, methyl(9Z)-19-[2-(dimethylamino)ethyl]heptacos-9- enoate, methyl8-(2-{9-[2-(dimethylamino)ethyl]octadecyl}cyclopropyl)octanoate, methyl(9Z)-19-[2-(dimethylamino)ethyl]octacos-9-enoate, ethyl8-(2-{ll- [(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoate, ethyl8-(2-{ll- [(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate, di((9Z,12Z)-octadeca-9,12- dien-l-yl)3-(((2-(dimethylamino)ethoxy)carbonyl)amino)pentanedioate, Heptyl6-((2-(l-(N- (2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)hexanoate, ethyl8-(2-{ll-[(dimethylamino)methyl]nonadecyl}cyclopropyl)octanoate, Pentyl8-((2-(l- (N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4- yl)ethyl)(tetradecyl)amino)octanoate, ethyl8-(2-{ll- [(dimethylamino)methyl]icosyl}cyclopropyl)octanoate, ethyl8-(2-{9- [(dimethylamino)methyl]pentadecyl}cyclopropyl)octanoate, 3-((2-(l-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)piperidin-4- yl)ethyl)(tetradecyl)amino)propyldecanoate, Heptyl6-((2-(4-(N-(2-(dinonylamino)ethyl)- N-nonylglycyl)piperazin-l-yl)-2-oxoethyl)(tetradecyl)amino)hexanoate, ethyl8-(2-{9- [(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, Pentyl8-((2-(4-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperazin-2-oxoethyl)(tetradecyl)amino)octanoate, ethyl8-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoate, methyl6-(2-(8- (2-(dimethylamino)-3-(nonyloxy)propoxy)octyl)cyclopropyl)hexanoate, methyl(9Z)-21- (dimethylamino)heptacos-9-enoate, methyl(9Z)-21-{[4-
(dimethylamino)butanoyl]oxy }heptacos-9-enoate, (2R)-N,N-dimethyl-l -[(9Z, 12Z)- octadeca-9, 12-dien-l -yloxy]dodecan-2-amine, (15Z, 18Z)-N,N-dimethyltetracoda- 15,18- dien-5-amine, ethyl8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate, 3- ((2-(4-(N-(2-(Dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(tetradecyl)amino)propyldecanoate, ethyl4-(2-{ll- [(dimethylamino)methyl]icosyl}cyclopropyl)butanoate, ethyl8-(2-{7- [(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, 3-((3-(l-(3-((2- (Dinonylamino)ethyl)(nonyl)amino)propanoyl)piperidin-4- yl)propyl)(nonyl)amino)propylhexanoate, ethyl6-(2-{9- [(dimethylamino)methyl]pentadecyl}cyclopropyl)hexanoate, 3-((3-(4-(3-((2- (Dinonylamino)ethyl)(nonyl)amino)propanoyl)piperazin-l-yl)-3- oxopropyl)(nonyl)amino)propylhexanoate, ethyl6-(2-{9- [(dimethylamino)methyl]hexadecyl}cyclopropyl)hexanoate, 3-((2- (Dinonylamino)ethyl)(nonyl)amino)-l-(4-(3-(dinonylamino)propyl)piperidin-l-yl)propan- 1-one, Pentyl4-((3-(l-(3-((2-(dinonylamino)ethyl)(nonyl)amino)propanoyl)piperi din-4- yl)propyl)(nonyl)amino)butaA, ethyl6-(2-{9- [(dimethylamino)methyl]heptadecyl}cyclopropyl)hexanoate, Pentyl4-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)butanoate, ethyl6- (2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)hexanoate, Pentyl4-(((l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)methyl)(nonyl)amino)butanoate, ethyl(9Z)-21-[(dimethylamino)methyl]heptacos-9-enoate, Pentyl4-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)ethyl)(nonyl)amino)butanoate, ethyl(9Z)-21-[(dimethylamino)methyl]octacos-9-enoate, ((2-((3,S',4R)-3,4- dihydroxypyrrolidin-l-yl)acetyl)azanediyl)bis(ethane-2,l-diyl)(9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-di enoate), Pentyl4-((2-(l-(N-(2-(dinonylamino)ethyl)-N- nonylgly cyl)piperi din-3 -yl)ethyl)(nonyl)amino)butanoate, ethyl(9Z)-21- [(dimethylamino)methyl]nonacos-9-enoate, methyl6-(2-(8-(2-(dimethylamino)-3- (heptyloxy)propoxy)octyl)cyclopropyl)hexanoate, methyl(9Z)-21-{[4-
(dimethylamino)butanoyl]oxy}octacos-9-enoate, methyl(9Z)-21-(dimethylamino)octacos- 9-enoate, 2-(Didodecylamino)- 1 -(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin- 1 -yl)ethan- 1 (2S)-N.N-dimethyl- 1 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy ]nonan-2- amine, (18Z,2 lZ)-N,N-dimethylheptacosa- 18,21 -dien- 10-amine, ethyl(9Z)-21 - [(dimethylamino)methyl]triacont-9-enoate, ethyl(9Z)- 19- [(dimethylamino)methyl]pentacos-9-enoate, ethyl(9Z)- 19- [(dimethylamino)methyl]hexacos-9-enoate, ethyl(9Z)- 19- [(dimethylamino)methyl]heptacos-9-enoate, ethyl(9Z)- 19- [(dimethylamino)methyl]octacos-9-enoate, ethyl(5Z)-17- [(dimethylamino)methyl]hexacos-5-enoate, ethyl(9Z)-17- [(dimethylamino)methyl]hexacos-9-enoate, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)-l- (3-(2-(dinonylamino)ethyl)piperidin-l-yl)ethan-l-one, ethyl(7Z)-17-
[(dimethylamino)methyl]tricos-7-enoate, Dipentyl4,4'-((2-(4-(N-(2-(dinonylarnino)ethyl)- N-nonylglycyl)piperazin-l-yl)-2-oxoethyl)azanediyl)dibutyrate, Pentyl4-(nonyl(2-(4-(N- nonyl-N-(2-(nonyl(4-oxo-4-(pentyloxy)butyl)amino)ethyl)glycyl)piperazin-l-yl)-2- oxoethyl)amino)butanoate, ethyl(7Z)- 17-[(dimethylamino)methyl]tetracos-7-enoate, ethyl(7Z)-17-[(dimethylamino)methyl]pentacos-7-enoate, 2-((2- (Dinonylamino)ethyl)(nonyl)amino)-l-(3 -((dinonylamino)methyl)pyrrolidin- 1 -yl)ethan- 1 - one, trans-3 -[(3 } 7-dimethyloctyl)oxy]- 1 -methyl-4~[(9Z, 12Z)-octadeca-9512-dien- 1 - yloxyj pyrrolidine, methyl6-(2-(8-(2-(dimethylamino)-3- (hexyloxy)propoxy)octyl)cyclopropyl)hexanoate, methyl(9Z)-21 -{ [4- (dimethylamino)butanoyl]oxy}nonacos-9-enoate, methyl(9Z)-21- (dimethylamino)nonacos-9-enoate, (2S)-N,N-dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]tridecan-2-amine, (15Z, 18Z)-N,N-dimethyltetracosa-l 5, 18-dien-7-amine, ethyl(7Z)- 17-[(dimethylamino)methyl]hexacos-7-enoate, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)- l-(3 -(2-(dinonylamino)ethyl)pyrrolidin- 1 -yl)ethan- 1 -one, methyl6-(2-{ll- [(dimethylamino)methyl]icosyl}cyclopropyl)hexanoate, methyll0-(2-{7- [(dimethylamino)methyl]hexadecyl}cyclopropyl)decanoate, methyl8-(2-{ll- [(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoate, methyl8-(2-{ll- [(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate, methyl8-(2-{ll- [(dimethylamino)methyl]nonadecyl}cyclopropyl)octanoate, methyl8-(2-{ll- [(dimethylamino)methyl]icosyl}cyclopropyl)octanoate, Pentyl4-((3-(4-(3-((2- (dinonylamino)ethyl)(nonyl)amino)propanoyl)piperazin-l-yl)-3- oxopropyl)(nonyl)amino)butanoate, methyl8-(2-{9- [(dimethylamino)methyl]pentadecyl}cyclopropyl)octanoate, methyl8-(2-{9- [(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoate, 3-((2-(l-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)propylhexanoate, methyl8-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoate, methyl8-(2- (dimethylamino)-3-((6-((2-octylcyclopropyl)methoxy)-6- oxohexyl)oxy)propoxy)octanoate, Butyl5-((2-(l-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)pentanoate, trans-l-methyl-3-[(12Z)- octadec-12-en-l-yloxy]-4-(octyloxy)pyrrolidine, methyl(9Z)-21-{[4- (dimethylamino)butanoyl]oxy}triacont-9-enoate, methyl(9Z)-21-(dimethylamino)triacont- 9-enoate, 2-((2-(Didodecylamino)ethyl)(nonyl)amino)- 1 -(4-(dinonylglycyl)piperazin- 1 - yl)ethan- 1 -oneStep 1 :MethylN-(2-(didodecylamino)ethyl)-N-nonylglycinate, 1 - ((2R,3S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin- 1 (2H)-yl)tetrahydrofum ethanesulfonate, (Z)-m ethyl 16-(3 -(decyl oxy )-2- (dimethylamino)propoxy)hexadec-7-enoate, (2S)- 1 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 - yloxy]nonan-2-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, Propyl6-((2- (l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4- yl)ethyl)(nonyl)amino)hexanoate, methyl7-(2-(dimethylamino)-3-((6-((2- octylcyclopropyl)methoxy)-6-oxohexyl)oxy)propoxy)heptanoate, methyl(7Z)- 19- [(dimethylamino)methyl]octacos-7-enoate, methyl(HZ)-19- [(dimethylamino)methyl]octacos-ll-enoate, Ethyl7-((2-(l-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)heptanoate, (2-octylcyclopropyl)methyl6- (2-(dimethylamino)-3-((5-methoxy-5-oxopentyl)oxy)propoxy)hexanoate, Methyl8-((2-(l- (N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)octanoate, methyl(9Z)-21-[(dimethylamino)methyl]heptacos-9-enoate, (2-octylcyclopropyl)methyl6- (2-(dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)hexanoate, methyl(9Z)-21- [(dimethylamino)methyl]octacos-9-enoate, 3-((2-(4-(N-(2-(Dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)propylhexanoate, (Z)-methyl8-(2- (dimethylamino)-3-((6-oxo-6-(undec-2-en-l-yloxy)hexyl)oxy)propoxy)octanoate, methyl(9Z)-21-[(dimethylamino)methyl]nonacos-9-enoate, Butyl5-((2-(4-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)pentanoate, (Z)-methyl7-(2-(dimethylamino)-3-((6-oxo-6-(undec-2-en-l- yloxy)hexyl)oxy)propoxy)heptanoate, Propyl6-((2-(4-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)hexanoate, methyl(9Z)-21 - [(dimethylamino)methyl]triacont-9-enoate, (Z)-undec-2-en-l-yl6-(2-(dimethylamino)-3- ((5 -m ethoxy-5 -oxopentyl)oxy)propoxy)hexanoate, methyl(9Z)-19- [(dimethylamino)methyl]pentacos-9-enoate, Ethyl7-((2-(4-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)heptanoate, (Z)-undec-2-en- 1 -y 16- (2-(dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)hexanoate, methyl6-(2- (dimethylamino)-3-((6-((2-octylcyclopropyl)methoxy)-6- oxohexyl)oxy)propoxy)hexanoate, methyl(9Z)-19-[(dimethylamino)methyl]hexacos-9- enoate, 3-(Dinonylamino)-l-(4-(3-((2- (dinonylamino)ethyl)(nonyl)amino)propanoyl)piperazin-l-yl)propan-l-one, methyl(9Z)- 19-[(dimethylamino)methyl]heptacos-9-enoate, 2-((2- (Dinonylamino)ethyl)(nonyl)amino)-l-(4-(ditetradecylglycyl)piperazin-l-yl)ethan-l-one, (Z)-methyl6-(2-(dimethylamino)-3-((6-oxo-6-(undec-2-en-l- yloxy)hexyl)oxy)propoxy)hexanoate, methyl8-(2-(dimethylamino)-3-((8-(2-(6-methoxy-6- oxohexyl)cyclopropyl)octyl)oxy)propoxy)octanoate, methyl8-(2-{9- [(dimethylamino)methyl]octadecyl}cyclopropyl)octanoate, 2-(Dinonylamino)-l-(4-(2-((2- (dinonylamino)ethyl)(nonyl)amino)ethyl)piperidin- 1 -yl)ethan- 1 -one, trans-l-methyl-3 - [(9Z)-octadec-9-en-l-yloxy]-4-(octyloxy)pyrrolidine, methyl(9Z)-19-{[4- (dimethylamino)butanoyl]oxy}pentacos-9-enoate, methyl(9Z)-19- (dimethylamino)pentacos-9-enoate, (Z)-m ethyl 16-(2-(dimethylamino)-3 - (nonyloxy)propoxy)hexadec-7-enoate, (2S)-l-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]decan-2-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, methyl7-(2- (dimethylamino)-3-((8-(2-(6-methoxy-6- oxohexyl)cyclopropyl)octyl)oxy)propoxy)heptanoate, methyl(9Z)-19- [(dimethylamino)methyl]octacos-9-enoate, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)-l- (4-(2-(dinonylamino)ethyl)piperidin-l-yl)ethan-l-one, Methyl8-((2-(4- (dinonylglycyl)piperazin-l-yl)-2-oxoethyl)(2-((8-methoxy-8- oxooctyl)(nonyl)amino)ethyl)amino)octanoate, methyl6-(2-(8-(2-(dimethylamino)-3-((5- methoxy-5-oxopentyl)oxy)propoxy)octyl)cyclopropyl)hexanoate, ethyl8-{2-[ll- (dimethylamino)heptadecyl]cyclopropyl}octanoate, Methyl8-((2-(dinonylamino)ethyl)(2- (4-(dinonylglycyl)piperazin-l-yl)-2-oxoethyl)amino)octanoate, methyl6-(2-(8-(2- (dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)octyl)cyclopropyl)hexanoate, ethyl8-{2-[ll-(dimethylamino)octadecyl]cyclopropyl}octanoate, Methyl8-((2-((2-(4- (dinonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoate, ethyl8-{2-[ll-(dimethylamino)nonadecyl]cyclopropyl}octanoate, (Z)-methyll6-(2- (dimethylamino)-3-((8-methoxy-8-oxooctyl)oxy)propoxy)hexadec-7-enoate, Pentyl4-((2- (4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(nonyl)amino)butanoate, ethyl 8- { 2- [11- (dimethylamino)icosyl]cyclopropyl}octanoate, (Z)-methyll6-(2-(dimethylamino)-3-((7- methoxy-7-oxoheptyl)oxy)propoxy)hexadec-7-enoate, Methyl8-((2-(4-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)octanoate, ethyl8-{2-[9-(dimethylamino)pentadecyl]cyclopropyl}octanoate, (Z)-methyll6-(2- (dimethylamino)-3-((5-methoxy-5-oxopentyl)oxy)propoxy)hexadec-7-enoate, (1 lE,20Z,23Z)-N,N-dimethylnonacosa-l l,20,23-trien-10-amine, N,N-dimethyl-l- [(1 S,2R)-2-octylcyclopropyl]pentadecan-8-amine, ethyl 8- { 2- [9- (dimethylamino)hexadecyl]cyclopropyl}octanoate, 2-((2- (Didodecylamino)ethyl)(dodecyl)amino)-l-(5-(dinonylglycyl)-2,5- diazabicy clo[2.2.1 ]heptan-2-yl)ethan- 1 -one3 , (Z)-methyl 16-(2-(dimethylamino)-3 -(4- methoxy-4-oxobutoxy)propoxy)hexadec-7-enoate, methyl6-(2-(8-(2-(dimethylamino)-3- ((6-methoxy-6-oxohexyl)oxy)propoxy)octyl)cyclopropyl)hexanoate, ethyl8-{2-[9- (dimethylamino)heptadecyl]cyclopropyl}octanoate, 2-(Dinonylamino)-l-(5-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethan-l-one, 1- [( 1 S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N1 ,N1 ,N2- Tri((9Z,12Z)-octadeca-9,12-dien-l-yl)-N2-(2-(piperazin-l-yl)ethyl)ethane-l,2-diamine, ethyl 8- { 2- [9-(dimethylamino)octadecyl] cyclopropyl } octanoate, 1 - [( 1 R,2 S)-2- heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, (Z)-methyll6-(2-(dimethylamino)-3- ((6-methoxy-6-oxohexyl)oxy)propoxy)hexadec-7-enoate, Nl,Nl,N2-Tri((Z)-octadec-9- en-l-yl)-N2-(2-(piperazin-l-yl)ethyl)ethane-l,2-diamine, N,N-dimethyl-3-{7-[(lS,2R)-2- octylcyclopropyl]heptyl } dodecan- 1 -amine, methyl8-(2-(dimethylamino)-3 -((8-(2-((2- pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)octanoate, ethyl4-{2-[ll- (dimethylamino)icosyl]cyclopropyl}butanoate, trans-l-Methyl-3-[((9Z,12Z)-octadeca- 9,12-dienyl)oxy]-4-octyloxy-pyrrolidine, methyl(9Z)-19-(dimethylamino)hexacos-9- enoate, methyl(9Z)-19-{[4-(dimethylamino)butanoyl]oxy}hexacos-9-enoate, (Z)- methyll6-(2-(dimethylamino)-3-(heptyloxy)propoxy)hexadec-7-enoate, (2R)-1-[(9Z,12Z)- octadeca-9, 12-dien- 1 -yloxy]dodecan-2-amine, ( 13Z, 16Z)-N,N-dimethyldocosa- 13,16- dien-5-amine, N,N-dimethyl-l-[(lR,2S)-2-undecylcyclopropyl]tetradecan-5-amine, methyl7-(2-(dimethylamino)-3-((8-(2-((2- pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)heptanoate, ethyl8-{2-[7- (dimethylamino)hexadecyl]cyclopropyl}octanoate, 2-(Didodecylamino)-N-dodecyl-N-(2- (piperazin-l-yl)ethyl)acetamide, N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]hexadecan- 8-amine, N 1 -(2-(Piperazin-l-yl)ethyl)-N 1 ,N2,N2-tritetradecylethane-l,2-diamine, methyl6- (2-(dimethylamino)-3-((8-(2-((2- pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)hexanoate, ethyl6-{2-[9- (dimethylamino)pentadecyl]cyclopropyl}hexanoate, N,N-dimethyl-l-[(lS,2S)-2-
{ [(lR,2R)-2-pentylcyclopropyl]methyl } cyclopropyl]nonadecan- 10-amine, NN 1 ,N2- Tridodecyl-N2-(2-(piperazin-l-yl)ethyl)ethane-l,2-diamine, methyl5-(2-(dimethylamino)- 3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)pentanoate, ethyl6- {2-[9-(dimethylamino)hexadecyl]cyclopropyl}hexanoate, N,N-dimethyl-21-[(lS,2R)-2- octylcyclopropyl]henicosan-l O-amine, NNN2-Trinonyl-N2-(2-(piperazin-l- yl)ethyl)ethane-l,2-diamine, methyl4-(2-(dimethylamino)-3-((8-(2-((2- pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)butanoate, ethyl6-{2-[9- (dimethylamino)heptadecyl]cyclopropyl}hexanoate, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl]nonadecan- 1 O-amine, N1 ,N1 ,N2-Trihexyl-N2-(2-(piperazin-l- yl)ethyl)ethane-l,2-diamine, methyl8-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-
1-yloxy)propoxy)octanoate, ethyl6-{2-[9- (dimethylamino)octadecyl]cyclopropyl}hexanoate, Nl-(2-(4-(2- (Didodecylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tri((9Z,12Z)-octadeca-9,12-dien-l- yl)ethane-l,2-diamine, methyl7-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-l- yloxy)propoxy)heptanoate, ethyl(9Z)-21-(dimethylamino)heptacos-9-enoate, 1-[(1S,2R)-
2-hexylcyclopropyl]-N,N-dimethylnonadecan- 1 O-amine, 1 -methyl 18-[(2Z)-non-2-en- 1 - yl]9-{[4-(dimethylamino)butanoyl]oxy}octadecanedioate, Nl-(2-(4-(2- (Didodecylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tri((Z)-octadec-9-en-l-yl)ethane- 1,2-diamine, N,N-dimethyl-l-[(l S,2R)-2-octylcyclopropyl]heptadecan-8-amine, methyl6- (2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-l-yloxy)propoxy)hexanoate, ethyl(9Z)-21 -(dimethylamino)octacos-9-enoate, dimethyl(9Z)- 19-{ [4- (dimethylamino)butanoyl]oxy}heptacos-9-enedioate, Nl-(2-(4-(2- (Ditetradecylamino)ethyl)piperazin-l-yl)ethyl)-Nl,N2,N2-tritetradecylethane-l,2-diamine, methyl5-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-l-yloxy)propoxy)pentanoate, ethyl8-{[4-(dimethylamino)butanoyl]oxy}-15-(2-octylcyclopropyl)pentadecanoate, ethyl(9Z)-21 -(dimethylamino)nonacos-9-enoate, (13Z, 16Z)-N,N-dimethyl-3 -nonyldocosa- 13,16-dien- 1 -amine, N 1 -(2-(4-(2-(Didodecylamino)ethyl)piperazin-l-yl)ethyl)-N 1 ,N2,N2- tritetradecylethane- 1 ,2-diamine, methyl9-{ [4-(dimethylamino)butanoyl]oxy } - 16-(2- octylcyclopropyl)hexadecanoate, methyl4-(2-(dimethylamino)-3-((9Z, 12Z)-octadeca-9, 12- dien- 1 -yloxy)propoxy)butanoate, ethyl(9Z)-21 -(dimethylamino)triacont-9-enoate, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine, methyl8-(2- (dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)octanoate, ethyl(9Z)-19- (dimethylamino)pentacos-9-enoate, ethyl(18Z,21Z)-8-{[4- (dimethylamino)butanoyl]oxy}heptacosa-l 8, 21-di enoate, (16Z)-N,N-dimethylpentacos- 16-en-8-amine, methyl(9Z)-19-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-enoate, methyl(9Z)-19-(dimethylamino)heptacos-9-enoate, 2-(Didodecylamino)-l-(4-(2-((2- (didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l-yl)ethan -1-one, (Z)-m ethyl 16-(2- (dimethylamino)-3-(hexyloxy)propoxy)hexadec-7-enoate, (2S)-l-[(9Z,12Z)-octadeca- 9, 12-dien- 1 -yloxy]dodecan-2-amine, ( 16Z, 19Z)-N,N-dimethylpentacosa~ 16,19-dien-8- amine, N 1 -(2-(4-(2-(Dinonylamino)ethyl)piperazin-l-yl)ethyl)-N 1 ,N2AV2- tritetradecylethane-l,2-diamine, methyl7-(2-(dimethylamino)-3-((8-(2- octylcyclopropyl)octyl)oxy)propoxy)heptanoate, methyl(19Z,22Z)-9-{[4- (dimethylamino)butanoyl]oxy}octacosa-19,22-dienoate, ethyl(9Z)-19- (dimethylamino)hexacos-9-enoate, (22Z)-N,N-dimethylhentriacont-22-en- 10-amine, N1 - (2-(4-(2-(Di((Z)-octadec-9-en-l-yl)amino)ethyl)piperazin-l-yl)ethyl)- !AA- tridodecylethane-l,2-diamine, methyl5-(2-(dimethylamino)-3-((8-(2- octylcyclopropyl)octyl)oxy)propoxy)pentanoate, ethyl(9Z)-19-(dimethylamino)heptacos- 9-enoate, (2-butylcyclopropyl)methyl 12-{ [4- (dimethylamino)butanoyl]oxy}henicosanoate, (20Z)-N,N-dimethylnonacos-20-en-10- amine, Nl,Nl,N2-Tridodecyl-N2-(2-(4-(2-(dodecyl((9Z,12Z)-octadeca-9,12-dien— yl)amino)ethyl)piperazin-l-yl)ethyl)ethane-l,2-diamine, methyl4-(2-(dimethylamino)-3- ((8-(2-octylcyclopropyl)octyl)oxy)propoxy)butanoate, ethyl(9Z)- 19- (dimethylamino)octacos-9-enoate, (2-octylcyclopropyl)methyl8-{[4-
(dimethylamino)butanoyl]oxy}heptadecanoate, (24Z)-N,N-dimethyltritriacont-24-en-10- amine, N 1 -(2-(4-(2-(Ditetradecylamino)ethyl)piperazin-l-yl)ethyl)-N 1 ,N2,N2- tridodecylethane-l,2-diamine, ethyl(5Z)-17-(dimethylamino)hexacos-5-enoate, (Z)- methyl8-(2-(dimethylamino)-3-(octadec-9-en-l-yloxy)propoxy)octanoate, (2Z)-hept-2-en- l-yll2-{[4-(dimethylamino)butanoyl]oxy}henicosanoate, (17Z)-N,N-dimethylnonacos-17- en-10-amine, Nl-(2-(4-(2-(Di((Z)-dodec-6-en-l-yl)amino)ethyl)piperazin-l-yl)ethyl)- N 1 ,N2,N2-tridodecylethane- 1 ,2, -diamine, ethyl(9Z)- 17-(dimethylamino)hexacos-9- enoate, (Z)-methyl7-(2-(dimethylamino)-3-(octadec-9-en-l-yloxy)propoxy)heptanoate, (2Z)-undec-2-en-l-yl8-{[4-(dimethylamino)butanoyl]oxy}heptadecanoate, (14Z)-N,N- dimethylnonacos- 14-en- 10-amine, ethyl(7Z)- 17-(dimethylamino)tricos-7-enoate, (Z)-N 1 - (2-(4-(2-(Dodec-6-en-l-yl(dodecyl)amino)ethyl)piperazin-N!AA-tridodecylethane-l,2- diamine, (Z)-methyl5-(2-(dimethylamino)-3-(octadec-9-en-l-yloxy)propoxy)pentanoate, (2-hexylcyclopropyl)methyll0-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate, (15Z)- N,N-dimethylheptacos-15-en-10-amine, ethyl(7Z)-17-(dimethylamino)tetracos-7-enoate, (Z)-methyl4-(2-(dimethylamino)-3-(octadec-9-en-l-yloxy)propoxy)butanoate, (2Z)-non-2- en-l-yll0-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate, (20Z)-N,N- dimethylheptacos-20-en-l 0-amine, Nl-(2-(4-(2-(Dioctylamino)ethyl)piperazin-l-yl)ethyl)- Nl,N2AV2-tridodecylethane-l,2-diamine, methyl6-(2-(dimethylamino)-3-((8-(2- octylcyclopropyl)octyl)oxy)propoxy)hexanoate, ethyl 6-[2-(9- { [4- (dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]hexanoate, ethyl(7Z)-17- (dimethylamino)pentacos-7-enoate, 1-[(1 lZ,14Z)-l-nonylicosa-l 1,14-dien-l- yl]pyrrolidine, ethyl(7Z)-17-(dimethylamino)hexacos-7-enoate, (20Z,23Z)-N-ethyl-N- methylnonacosa-20,23-dien-l 0-amine, N,N-dimethylheptacosan-l 0-amine, methyl6-{2- [ll-(dimethylamino)icosyl]cyclopropyl}hexanoate, methyl6-[2-(ll-{[4- (dimethylamino)butanoyl]oxy}icosyl)cyclopropyl]hexanoate, (2- octylcyclopropyl)methyl6-(3-(decyloxy)-2-(dimethylamino)propoxy)hexanoate, methyl8- {2-[9-(dimethylamino)octadecyl]cyclopropyl}octanoate, methyl8-[2-(9-{[4- (dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]octanoate, methyl7-(2-(8-(2- (dimethylamino)-3-(octyloxy)propoxy)octyl)cyclopropyl)heptanoate, Heptadecan-9-yl8- ((2 -hydroxy ethyl)(tetradecyl)amino)octanoateRepresentative, 2-((2- (Didodecylamino)ethyl)(dodecyl)amino)-l-(4-(2-(didodecylamino)ethyl)piperazin-l- yl)ethan-l-one, (2S)-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]undecan-2-amine,
( 17Z,20Z)-N,N-dimemylhexacosa- 17,20-dien-9-amine, ( 18Z)-heptacos- 18-en- 10-yl4- (dimethylamino)butanoate, (2S)-l-({6-[3B))-cholest-5-en-3-yloxy]hexyl}oxy)-N,N- dimethyl-3-[(9Z)-octadec-9-en-l-yloxy]propan-2-amine, methyl 10- {2- [7- (dimethylamino)hexadecyl]cyclopropyl}decanoate, methyll0-[2-(7-{[4- (dimethylamino)butanoyl]oxy}hexadecyl)cyclopropyl]decanoate, (2S)-N,N-dimethyl-l- ({8-[(lR,2R)-2-{[(lS,2S)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)tridecan-2- amine, (2-octylcyclopropyl)methyl6-(2-(dimethylamino)-3-(nonyloxy)propoxy)hexanoate, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, 4-((N-(2-(Dinonylamino)ethyl)-N- nonylglycyl)oxy)pentan-2-yldinonylglycinate, 3-Hydroxybutan-2-ylN-(2- (dinonylamino)ethyl)-N-nonyl, Di(heptadecan-9-yl)8,8'-(26,28-dimethyl-ll,24,30,43- tetraoxo- 10,25,29,44-tetraoxa- 19,35-diazatripentacontane- 19,35 -diyl)dioctanoate, Di(heptadecan-9-yl)8,8'-(26,27-dimethyl-ll,24,29,42-tetraoxo-10,25,28,43-tetraoxa-19,34- diazadopentacontane-19,34-diyl)dioctanoate, Di(heptadecan-9-yl)8,8'-(ll,24,29,42- tetraoxo-10,25,28,43-tetraoxa-19,34-diazadopentacontane-19,34-diyl)dioctanoate, Di(heptadecan-9-yl)8,8'-((piperazine-l,4-diylbis(5-oxopentane-5,l-diyl))bis((8-(nonyloxy)- 8-oxooctyl)azanediyl))dioctanoate, Di(heptadecan-9-yl)15,18-dimethyl-9,24-bis(8- (nonyloxy)-8-oxooctyl)- 14,19-dioxo-9, 15,18,24-tetraazadotriacontanedioate, Di(heptadecan-9-yl)15,19-dimethyl-9,25-bis(8-(nonyloxy)-8-oxooctyl)-14,20-dioxo- 9, 15, 19, 25-tetraazatri triacontanedioate, Di(heptadecan-9-yl) 15,18-diethyl-9,24-bis(8- (nonyloxy)-8-oxooctyl)- 14,19-dioxo-9, 15,18,24-tetraazadotriacontanedioate, N,N- dimethyl-3-{[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]methyl}dodecan-l-amine, methyl8-[2- (ll-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]octanoate, methyl8-{2-[ll- (dimethylamino)heptadecyl]cyclopropyl}octanoate(Compoundl8);, Heptadecan-9-yl8-((2- hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, (2-octylcyclopropyl)methyl6- (2-(dimethylamino)-3-(heptyloxy)propoxy)hexanoate, (17Z)-N,N-dimethylhexacos-17-en-
9-amine, Nl-(2-(4-(2-(Didodecylamino)ethyl)piperazin-l-yl)ethyl)-NlAV2,N2- trihexylethane-l,2-diamine, N,N-dimethyl-2-{[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]methyl}undecan-l -amine, methyl8-{2-[ll- (dimethylamino)octadecyl]cyclopropyl}octanoate, (2-octylcyclopropyl)methyl6-(2- (dimethylamino)-3-(hexyloxy)propoxy)hexanoate, (18Z)-N,N-dimethylheptacos-l 8-en-
10-amine, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)ethyltetradecanoate, 2-((2- (Dinonylamino)ethyl)(nonyl)amino)ethylnonanoate, TetradecylN-(2- (dinonylamino)ethyl)-N-nonylglycinate, NonylN-(2-(dinonylamino)ethyl)-N- nonylglycinate, 4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)acetamido)butylpentanoate, l,l'-(Piperazine-l,4-diyl)bis(5-(didecylamino)pentan-l-one, 2-((2- (dinonylamino)ethyl)(nonyl)armno)-N-tetradecylacetamide, N-decyl-2-((2- (dinonylamino)ethyl)(nonyl)amino), Nl-(3-(3-(dinonylamino)propoxy)propyl)-Nl,N2,N2- trinonylethane-l,2-diamine, Nl-(2-(dinonylamino)ethyl)-N\N8,N8-trinonyloctane-l,8- diamine, methyl8-[2-(ll-{[4- (dimethylamino)butanoyl]oxy}nonadecyl)cyclopropyl]octanoate, methyl8-{2-[ll- (dimethylamino)nonadecyl]cyclopropyl}octanoate, (Z)-undec-2-en-l-yl6-(3-(decyloxy)-2- (dimethylamino)propoxy)hexanoate, (2R, 12Z, 15Z)-N,N-dimethyl- 1 - (undecyloxy)henicosa-12,15-dien-2-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien- 9-amine, 2-(dinonylamino)-N-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N- methylacetamido)butyl)-N-m ethyl acetamide, 7, 10-dimethyl- 13,16-dinonyl-6,ll-dioxo-4- tetradecyl-4,7, 10,13,16-pentaazapentacosyldecanoate, 2-(dinonylamino)-N-(2-(2-((2- (dinonylamino)ethyl)(nonyl)amino)-N-ethylacetamido)ethyl)-N-ethylacetamide, 2- (dinonylamino)-N-(3-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N- methylacetamido)propyl)-N-methylacetamide, 2-((2-(di((Z)-non-3-en-l- yl)amino)ethyl)((Z)-non-3-en-l-yl)amino)-N-(2-(2-(dinonylamino)-N- methylacetamido)ethyl)-N-methylacetamide, 2-(dinonylamino)-N-(2-(2-((2- (dinonylamino)ethyl)(nonyl)amino)acetamido)ethyl)acetamide, Pentyl8,ll-dimethyl- 5,14,17-trinonyl-7,12-dioxo-5,8,ll,14,17-pentaazahexacosanoate2-((2- (Dinonylamino)ethyl)(nonyl)aniino)-N-methyl-N-(2-(methylandno)ethyl)acetami, 2- (Dinonylamino)-N-(2-(2-((2-(dinonylamino)ethyl)(nonyl)amino)-N- methylacetamido)ethyl)-N-methylacetamide2-(Dinonylamino)-N-methyl-N-(2- (methylamino)ethyl)acetamide, 2-((N-(2-(Dinonylamino)ethyl)-N- nonylglycyl)oxy)ethyldinonylglycinate2 -Hydroxy ethyldinonylglycinate, methyl8-[2-(ll- {[4-(dimethylamino)butanoyl]oxy}icosyl)cyclopropyl]octanoate, methyl8-{2-[ll- (dimethylamino)icosyl]cyclopropyl}octanoate, (Z)-undec-2-en-l-yl6-(2-(dimethylamino)- 3-(nonyloxy)propoxy)hexanoate, (2R, 12Z, 15Z)-1 -(hexadecyloxy)-N,N-dimethylhenicosa- 12,15-dien-2-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, 1,1- (Piperazine-l,4-diyl)bis(4-(didecylamino)butan-l-one)fert-Butyl4-(didecylaminobutanoate, Heptyl5-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-5-oxopentanoate5- (Heptloxy)-5-oxopentanoicacid, Heptyl5-(4-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-5-oxopentanoate5-(Heptloxy)-5-oxopentanoic, (Z)-4-((2-(4- (N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(tetradecyl)amino)but-2-en-l-ylnonanoate(Z)-4-Hydroxybut-2-en-l-ylnonanoate, (Z)-3-((2-(4-(N-(2-(Dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(tetradec-9-en- 1 -yl)amino)propy 1 decanoate(Z)-T etradec-9-en-l- ylmethanesulfonate, methyl8-[2-(9-{[4- (dimethylamino)butanoyl]oxy}pentadecyl)cyclopropyl]octanoate, methyl8-{2-[9- (dimethylamino)pentadecyl]cyclopropyl}octanoate, (Z)-undec-2-en-l-yl6-(2- (dimethylamino)-3-(heptyloxy)propoxy)hexanoate, (2R,12Z,15Z)-l-(hexyloxy)-N,N- dimethylhenicosa- 12, 15-dien-2-amine, ( 16Z, 19Z)-N,N-dimethylpentacosa- 16,19-dien-6- amine, Methyl8-((2-(4-(N-(2-(Di((Z)-non-3-en-l-yl)amino)ethyl)-N-((Z)-non-3-en-l- yl)glycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)octanoatefert-Butyl4- (nonylglycyl)piperazine-l -carboxylate, 3-((2-(4-(N-(2-(Dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-2-oxoethyl)(tetradecyl)amino)propyl(Z)-dec-3-enoate(Z)-Dec- 3-en-l-ol, 2-((2-(Di((Z)-non-3-en-l-yl)amino)ethyl)((Z)-non-3-en-l-yl)amino)-l-(4- (dinonylglycyl)piperazin- 1 -yl)ethan- 1 -one(Z)- 1 -Bromonon-4-ene, 3 -((2-(4-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)piperazin- oxoethyl)(dodecyl)amino)propyloctanoatetot-Butyldodecylglycinate, S-Pentyl4-((2-(4-(N- (2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(nonyl)amino)butanethioate, 3-((2-(l-(N-(2-(Dinonylamino)ethyl)-N- nonylglycyl)piperidin-Ayl)ethyl)(nonyl)amino)propyl3-methylhexanoatefert-Butyl4-(2- ((3-((3-methylhexanoyl)oxy)propyl)(nonyl)amino)ethyl)piperidine-l-, 3-((2-(l-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)-2- methylpropylhexanoate, 3-((2-(4-(N-(2-(Dinonylamino)ethyl)-N-nonylglycyl)piperazin- oxoethyl)(nonyl)amino)propyl3-methylhexanoate, 3-((2-(4-(N-(2-(Dinonylamino)ethyl)- N-nonylglycyl)piperazin-oxoethyl)(nonyl)amino)-2-methylpropylhexanoate, methyl8-[2- (9-{[4-(dimethylamino)butanoyl]oxy}hexadecyl)cyclopropyl]octanoate, methyl8-{2-[9- (dimethylamino)hexadecyl]cyclopropyl}octanoate, (Z)-undec-2-en-l-yl6-(2- (dimethylamino)-3-(hexyloxy)propoxy)hexanoate, (2R,12Z,15Z)-l-(decyloxy)-N,N- dimethylhenicosa-12,15-dien-2-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7- amine, 2-((2-(Dinonylamino)ethyl)(nonyl)amino)ethyl 1 -(dinonylglycyl)piperidine-4- carboxylate, l-(2-(Dinonylamino)ethyl)4-(2-((2- (dinonylamino)ethyl)(nonyl)amino)ethyl)cyclohexane-l,4-dicarboxylate2- (Dinonylamino)ethan-l -ol, Methyll2-((2-(l-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)dodecanoatefert-Butyl3-(2-((12- methoxy-12-oxododecyl)(tetradecyl)amino)ethyl)pyrrolidine-l-carboxylate, 3-((2-(l-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3- yl)ethyl)(tetradecyl)amino)propyldecanoateter/-Butyl3-(2-((3- (decanoyloxy)propyl)(tetradecyl)amino)ethyl)pyrrolidine-l-carboxylate, "Heptyl6-((2-(l- (N-(2-(dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3- yl)ethyl)(tetradecyl)amino)hexanoatetot-Butyl3-(2-((6-(heptyloxy)-6- oxohexyl)(tetradecyl)amino)ethyl)pyrrolidine-l -carboxylate, ", Pentyl8-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3- yl)ethyl)(tetradecyl)amino)octanoate/er/-Butyl3-(2-(tetradecylamino)ethyl)pyrrolidine-l- carboxylate, Methyl 12-((2-(l-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperi din-3- yl)ethyl)(tetradecyl)amino)dodecanoate-Butyl3-(2-((12-methoxy-12- oxododecyl)(tetradecyl)amino)ethyl)piperidine-l-carboxylate, 3-((2-(l-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)piperidin-3- yl)ethyl)(tetradecyl)amino)propyldecanoate, Heptyl6-((2-(l-(N-(2-(dinonylamino)ethyl)- N-nonylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)hexanoate, Pentyl8-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)octanoate, Pentyl6-((2-(4-(2-((2-(didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-l- yl)ethyl)(dodecyl)amino)hexanoateStepl :Pentyl6-bromohexanoate, methyl8-[2-(9-{[4- (dimethylamino)butanoyl]oxy}heptadecyl)cyclopropyl]octanoate, methyl8-{2-[9- (dimethylamino)heptadecyl]cyclopropyl}octanoate, (2S,12Z,15Z)-N,N-dimethyl-l- (octyloxy)henicosa-12, 15-dien-2-amine, (2-octylcyclopropyl)methyl6-(2- (dimethylamino)-3-(octyloxy)propoxy)hexanoate, (18Z,21Z)-N,N-dimethylheptacosa- 18,21 -dien-8-amine, trans- 1 -methyl-3 ,4-bis(((Z)-hexadec-9-enoyloxy)methyl)pyrrolidine, (Z)-Non-2-en-l-yl4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2- oxoethyl)(tetradecyl)amino)butanoate, trans-l-methyl-3,4-bis(((9Z, 12Z)-octadeca-9, 12- dienoyloxy)methyl)pyrrolidine, Methyl 12-((2-(4-(N-(2-(dinonylamino)ethyl)-N- nonylglycyl)piperazin-l-yl)-2-oxoethyl)(tetradecyl)amino)dodecanoate, ethyl(7Z)-17-[2- (dimethylamino)ethyl]hexacos-7-enoate, trans-l-methyl-3,4-bis(((Z)-octadeca-9- enoyloxy)methyl)pyrrolidine, methyl6-(2-{]l l-A2- (dimethylamino)ethyl]icosyl}cyclopropyl)hexanoate, Methyl 12-((2-(l-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)dodecanoate, methyl 10-(2-V-A2-(dimethylamino)ethyl]hexadecyl}cyclopropyl)decanoate, methyl8-(2- { 11 l-;2-(dimethylamino)ethyl]heptadecyl}cyclopropyl)octanoate, 2-(l-(N-(2-
(Dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyldinonylglycinatefert-Butyl4-(2- ((dinonylglycyl)oxy)ethyl)piperidine-l -carboxylate, methyl8-(2-{lLl-;2- (dimethylamino)ethyl]octadecyl}cyclopropyl)octanoate, methyl8-(2-{ll 1- "2- (dimethylamino)ethyl]nonadecyl}cyclopropyl)octanoate, l,-(piperazine-l,4-diyl)bis(2- (dinonylamino)ethan-l-one), methyl8-[2-{]l l-A2- (dimethylamino)ethyl]icosyl}cyclopropyl)octanoate, methyl8-(2-{9-[2- (dimethylamino)ethyl]pentadecyl } cy clopropyl)octanoate, methyl(7Z)- 19-{ [4- (dimethylamino)butanoyl]oxy}octacos-7-enoate, methyl(7Z)-19-(dimethylamino)octacos- 7-enoate, cis-l-methyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-4-(octyloxy)pyrrolidine, 2-(Didodecylamino)-l-(4-(N-(2-(didodecylamino)ethyl)-N-dodecylglycyl)piperazin-l- yl)ethan-l-one, (Z)-undec-2-en-l-yl6-(2-(dimethylamino)-3-(octyloxy)propoxy)hexanoate, (2SN,N-dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]decan-2-amine(Compoundl l), (19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-9-amine, methyl8-(2-{9-[2- (dimethylamino)ethyl]hexadecyl}cyclopropyl)octanoate, 5-((2-(4-(N-(2- (Dinonylamino)ethyl)-N-nonylglycyl)piperazin- oxoethyl)(nonyl)amino)pentylmethylcarbonate, methyl8-(2-{9-[2- (dimethylamino)ethyl]heptadecyl}cyclopropyl)octanoate, methyl(7Z)-19-[2- (dimethylamino)ethyl]octacos-7-enoate, (Z)-Pent-2-en-l-yl4-((2-(4-(N-(2- (dinonylamino)ethyl)-N-nonylglycyl)piperazin-l-yl)-2-oxoethyl)(nonyl)amino)butanoate, methyl(HZ)-19-[2-(dimethylamino)ethyl]octacos-ll-enoate, methyl(9Z)-21-[2- (dimethylamino)ethyl]heptacos-9-enoate, methyl(9Z)-21 -[2- (dimethylamino)ethyl]octacos-9-enoate, methyl(9Z)-21-[2-(dimethylamino)ethyl]nonacos- 9-enoate, 2-(l-(N-(2-(Dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3- yl)ethyldinonylglycinate, methyl(9Z)-21-[2-(dimethylamino)ethyl]triacont-9-enoate, (1- (N-(2-(Dinonylamino)ethyl)-N-nonylglycyl)pyrrolidin-3-yl)methyldinonylglycinate, methyl(9Z)-19-[2-(dimethylamino)ethyl]pentacos-9-enoate, methyl(9Z)-19-[2- (dimethylamino)ethyl]hexacos-9-enoate, methyl6-(2-(8-(3-(decyloxy)-2- (dimethylamino)propoxy)octyl)cyclopropyl)hexanoate, methyl(HZ)-19-{[4- (dimethylamino)butanoyl] oxy } octacos-11 -enoate, methyl( 11Z)- 19- (dimethylamino)octacos-ll -enoate, (2S)-N,N-dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]dodecan-2-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, Methyldi((9Z,12Z)-octadeca-9,12-dienyl)amine, methyl(9Z)-19-{[4- (dimethylamino)butanoyl]oxy}octacos-9-enoate, methyl(9Z)-19-(dimethylamino)octacos- 9-enoate, (Z)-methyll7-(2-(dimethylamino)-3-(octyloxy)propoxy)heptadec-8-enoate, (3R,4R)-3,4-bis((Z)-hexadec-9-enyloxy)-l-methylpyrrolidine, (2S)-N,N-dimethyl-l- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]undecan-2-amine, (20Z,23Z)-nonacosa-20,23-dien- 10-yl4-(dimethylamino)butanoate, (20Z,23Z)-N,N-dimethylnonacosa-20,23 -dien- 10- amine, 3-((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N,N- dimethylpropan-l-amine, 3-((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)- N,N-dimethylpropan-l -amine, (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl4- (dimethylamino)butanoate), (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-ll-yl5- (dimethylamino)pentanoate, (6Z, 16Z)- 12-((Z)-dec-4-enyl)docosa-6, 16-dien-l 1 -yl 5 - (dimethylamino)pentanoat, (6Z, 16Z)- 12-((Z)-dec-4-enyl)docosa-6, 16-dien- 11 -y 15 - (dimethylamino)pentanoate, L-arginine-alpha-(2,3-dilauryloxy)propylamide, L-lysine- alpha-(2,3-dilauryloxy)propylamide, 2,3-dioleyloxypropylamine, 2,3- distearyloxypropylamine, 2,3-dilauryloxypropylamine, dilinoleylmethyl4- (dimethylamino)propylether), dilinoleylmethyl4-(dimethylamino)butylether), and 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane.
[0313] In some embodiments, the at least one non-cationic lipid comprises at least one phospholipid, at least one fusogenic lipid, at least one anionic lipid, at least one helper lipid, at least one neutral lipid, or any combination thereof. In some embodiments, the LNP may be essentially devoid of the at least one non-cationic lipid. In some embodiments, the LNP may contain no amount of the at least one non-cationic lipid.
[0314] In some embodiments, at least one non-cationic lipid may be selected from, but is not limited to, at least one of l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), DSPCbutwith3unsaturateddoublebondspertail (18:3 PC), Acylcamosine (AC), l-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), N-oleoyl-SPM (C 18 :1), N-lignocerylSPM (C24:0), N-nervacylC (C24:l), carbamoyl]cholesterol (Cet-P), cholesterolhemisuccinate (CHEMS), cholesterol (Choi), Cholesterolhemidodecanedicarboxylicacid (Chol-C12), 12- Cholesteryloxycarbonylaminododecanoicacid (Chol-C 13N), Cholesterolhemioxalate (Chol-C2), Cholesterolhemimalonate (Chol-C3), N-(Cholesteryl-oxycarbonyl)glycine (Chol-C3N), Cholesterolhemiglutarate (Chol-C5), Cholesterolhemiadipate (Chol-C6), Cholesterolhemipimelate (Chol-C7), Cholesterolhemisuberate (Chol-C8), Cardiolipid (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetylphosphate (DCP), dihexadecylphosphate (DCP1), 1,2-Dipalmitoy glycerol-3 - hemisuccinate (DGSucc), short-chainbis-n-heptadecanoylphosphatidylcholine (DHPC), dihexadecoylphosphoethanolamine (DHPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), Dimyristoylglycerolhemi succinate (DMGS), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), di oleyloxybenzyl alcohol (DOBA), l,2-dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2~(2-{2-[2-(2,3-Bis-octadec-9- enyloxy-propoxy)-ethoxy]-ethoxy } -ethoxy )-ethy 1 ] -3 -(3 ,4,5 - 1 rihydroxy-6-hydroxymethyl- 1 etrahydro-pyran-2-ylsulfanyl)-propionamide (D0GP4aMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), di oleoylphosphatidylglycerol (DOPG), l,2-dioleoyl-sn-glycero-3-(phospho- L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoylphosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), eggphosphatidylcholine (EPC), N-histidinylcholesterolcarbamate (HCChol), histaminedi stearoylglycerol (HDSG), N-histidinylcholesterolhemisuccinate (HistChol), 1,2-Dipalmitoylglycerol-hemisuccinate- Na-Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-Oa- histidinyl-Na-hemisuccinate (IsohistsuccDG), mannosializeddipalmitoylphosphatidylethanolamine (ManDOG), 1,2-Dioleoyl-sn-Glycero- 3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC- PE), l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), l-myristoyl-2- hydroxy-sn-glycero-phosphocholine (MHPC), athiol- reactivemaleimideheadgrouplipide.g. l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [4-(p-maleimidophenyl)but-yramid (MPB-PE), NervonicAcid (NA), sodiumcholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N-dodecanoyl (NC12-DOPE), DefinedbysynthesisexampleinW02008042973A2 (ND98), "N- glutarylphosphatidylethanolamine(s)ofFormulal " (NG-PE), N-hydroxysulfosuccinimide (NHS-'x1), "N~(co)-dicarboxylicacid- derivatizedphosphatidylethanolaminesencompassedbyFormulal " (NcoPE-'x1), OleicAcid (OA), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidicacid (PA), phosphatidylethanolaminelipid (PE), PElipidconjugatedwithpolyethyleneglycol(PEG).OneexampleofPEG- PEcanbepolyethyleneglycol-distearoylphosphatidylethanolaminelipid (PEG-PE), phosphatidylglycerol (PG), partiallyhydrogenatedsoyphosphatidylchloline (PHSPC), phosphatidylinositollipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissaminerhodamineB-phosphatidylethanolaminelipid (Rh-PE), purifiedsoy- derivedmixtureofphospholipids (SIOO), phosphatidylcholine (SM), 18-l-transPE,l- stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybeanphosphatidylcholine (SPC), sphingomyelins (SPM), alpha.alpha'-trehalose6,6'-dibehenate (TDB), 1,2-dielaidoyl-sn- glycero-3-phophoethanol amine (transDOPE ), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)- 5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2/-/)-yl)tetrahydrofuran-2- yl)methylmethylphosphate, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dilinolenoyl-sn-glycero-3 - phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dioleyl-sn- glycero-3 -phosphoethanolamine, l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 16-0- monomethylPE, 16-0-dimethylPE, and di oleylphosphatidylethanolamine.
[0315] In some embodiments, the LNP comprises at least one of the cationic lipids described in International Patent Publication No. WO2018118102 (the contents of which are herein incorporated reference in its entirety) such as, but not limited to ATX-2, ATX- 43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, and ATX-88. Methods of making these cationic lipids are also provided in International Patent Publication No. WO2018118102 and the contents of which are herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid in the LNP is ATX-88. As a non-limiting example, the LNP comprises about 20-60% of ATX-2, ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, or ATX-88, about 5-25% of a noncationic lipid, about 25-55% of sterol and about 0.5-15% of a PEG-modified lipid.
[0316] In some embodiments, the LNP comprises at least one of the cationic lipids described in International Patent Publication No. WO2018119163 (the contents of which are herein incorporated reference in its entirety) such as, but not limited to ATX-0002, ATX-0043, ATX-0057, ATX-0061, ATX-0063, ATX-0058, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0086, ATX-0087, ATX-0088, ATX-0091, ATX-0092, ATX-0093, ATX-0094, ATX-0095, ATX-0096, ATX-0097, ATX-0098, ATX-0100, ATX-0101, ATX-0102, ATX-0106, ATX-0107, ATX-0108, ATX-0109, ATX-0110, ATX-0111, ATX-0114, ATX-0115, ATX-0117, ATX-0118, ATX-0121, ATX-0122, ATX-0123, ATX-0124, ATX-0125, ATX-0126, ATX-0129, ATX-0132, and ATX-0134. Methods of making these cationic lipids are also provided in International Patent Publication No. WO2018119163 and the contents of which are herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid in the LNP is ATX-88. As a non-limiting example, the LNP comprises about 20-60% of ATX-0002, ATX-0043, ATX-0057, ATX-0061, ATX-0063, ATX-0058, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0086, ATX-0087, ATX-0088, ATX-0091, ATX-0092, ATX-0093, ATX-0094, ATX-0095, ATX-0096, ATX-0097, ATX-0098, ATX-0100, ATX-0101, ATX-0102, ATX-0106, ATX-0107, ATX-0108, ATX-0109, ATX-0110, ATX-0111, ATX-0114, ATX-0115, ATX-0117, ATX-0118, ATX-0121, ATX-0122, ATX-0123, ATX-0124, ATX-0125, ATX-0126, ATX-0129, ATX-0132, or ATX-0134, about 5-25% of a non-cationic lipid, about 25-55% of sterol and about 0.5- 15% of a PEG-modified lipid.
[0317] In some embodiments, the LNP comprises at least one of the cationic lipids described in International Patent Publication Nos. W02012019168. W02012045082.
WO2012135805, WO2012158736, WO2013039857, WO2013039861, WO2013052523, W02013090186, W02013090648, W02013096709, WO2013101690, WO2013106496, W02013130161, WO2013151663, WO2013151664, WO2013151665, WO2013151666, WO2013151667, WO2013151668, WO2013151669, W02013151670, WO2013151671, WO2013151672, WO2013151736, WO2014028429, W02014081507, WO2014093574, WO2014093924, WO2014113089, WO2014144039, WO2014144711, WO2014144767, WO2014152027, W02014152030, W02014152031, WO2014152211, WO2014152540, WO2014158795, WO2014159813, WO2014164253, WO2015006747, WO2015034925, WO2015034928, WO2015038892, WO2015048744, W02015051169, W02015051173, W02015051214, WO2015058069, WO2015085318, W02015089511, WO2015105926, WO2015164674, WO2015196118, WO2015196128, W02015196130, WO2015199952, W02016011222, WO2016011226, W02016011306, WO2016014846, WO2016022914, W02016036902, WO2016077123, WO2016077125, W02016100812, WO2016118724, WO2016118725, WO2016164762, WO2016176330, WO2016201377, W02017004143, WO2017015457, WO2017015463, W02017015630, WO2017019935, WO2017020026, WO2017031232, WO2017049074, WO2017049245, WO2017049275, WO2017049286, WO2017062513, WO2017070601, W02017070613, WO2017070616, WO2017070618, WO20 17070620, WO2017070622, W02017070623, WO2017070624, WO2017070626, WO2017075531, WO2017099823, WO2017106799, WO2017112865, WO2017112943, WO2017117528, W02017120612, WO2017127750, WO2017180917, W02017201317, WO2017201325, WO2017201328, WO2017201332, WO2017201333, WO2017201340, WO20 17201342, WO2017201346, WO2017201347, WO2017201348, WO2017201349, W02017201350, WO2017201352, WO2017214175, WO2017218704, WO2017223135, WO20 17223176, WO2018053209, W02018075980, W02018078053, WO2018081459, W02018081480, WO2018081638, W02018089540, WO2018089851, WO2018107026, W02018107088, WO2018144082, WO2018144775, WO2018144778, WO2018151816, WO2018157009, WO2018170245, WO2018170256, WO2018170260, WO2018170270, W02018170306, WO2018170336, WO2018170347, WO2018175783, WO2018187590, WO2018191657, WO2018191719, W02018200737, W02018200943, WO2018213731, WO2018213789, WO2018231990, W02018232006, WO2018232120, WO2018232355, WO2018232357, WO2019018765, WO2019023179, W02019036000, W02019036008, W02019036028, W02019036030, W02019036670, WO2019036682, W02019046809, W02019055807, WO2019089818, WO2019089828, WO2019103993, W02019104152, W02019104160, W02019104195, WO2019136241, W02019143910, W02019148101, WO2019152557, W02019200171, WO2019226650, W02020023390, W02020047201, W02020056147, W02020056155, W02020056239, W02020056304, W02020056370, W02020061284, W02020061295, W02020061317, W02020061332, W02020061367, W02020061426, W02020061457, W0202006242, W02020069169, W02020081938, W02020097291, W02020097409, W02020146805, W02020160397, W02020160430, W02020185811, W02020190750, W02020227510, WO2020227537, WO2020227615, WO2020227642, W02020227690, WO2020243561, WO2020263883, WO2020263985, WO2021022173, WO2021026358, W02021030701, W02021050864, W02021050986, WO2021055833, WO2021055835, WO2021055849, W02021076805, W02021076811, WO2021142280, WO2021154763, WO2021155243, WO2021155267, WO2021155274, WO2021159040, and WO2021159130. As a non-limiting example, the LNP comprises about 20-60% of a cationic lipid, about 5-25% of a non-cationic lipid, about 25-55% of sterol and about 0.5-15% of a PEG-modified lipid.
[0318] In some embodiments, the LNP comprises an ionizable lipid or lipid-like material. As a non-limiting example, the ionizable lipid may be C12-200, CKK-E12, 5A2- SC8, BAMEA-016B, or 7C1. Other ionizable lipids are known in the art and are useful herein.
[0319] In some embodiments, the LNP comprises a phospholipid. As a non-limiting example, the phospholipid (helper) may be DOPE, DSPC, DOTAP, or DOTMA. [0320] In some embodiments, the LNP comprises a PEG derivative. As a non-limiting example, the PEG derivative may be a lipid-anchored such as PEG is C14-PEG2000, C14- PEG1000, C14-PEG3000, C14-PEG5000, C12-PEG1000, C12-PEG2000, C12-PEG3000, C12-PEG5000, C16-PEG1000, C16-PEG2000, C16-PEG3000, C16-PEG5000, C18- PEG1000, C18-PEG2000, C18-PEG3000, or C18-PEG5000.
[0321] In some embodiments, the at least one sterol comprises at least one cholesterol or cholesterol derivative. In some embodiments, the LNP may be essentially devoid of an at least one sterol. In some embodiments, the LNP may contain no amount of the at least one sterol.
[0322] In some embodiments, the at least one additional LNP functional component comprises at least one component that reduced aggregation of particles, at least one component that decreases clearing of the LNP from circulation in a subject, at least component that increases the LNP’s ability to traverse mucus layers, at least one component that decreases a subjects immune response to administration of the LNP, at least one component that modifies membrane fluidity of the LNP, at least one component that contributes to the stability of the LNP, or any combination thereof. In some embodiments, the LNP may be essentially devoid of the at least one additional LNP functional component. In some embodiments, the LNP may contain no amount of the at least one additional LNP functional component.
[0323] In some embodiments, the additional LNP functional component may be comprised of a polymer. In some embodiments, the polymer comprising the additional LNP functional component may be comprised of at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.
[0324] In some embodiments, the average molecular weight of the polymer moiety (e.g., PEG) may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 0,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000, 9,000 to 20,000, 9,500 to0,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to0,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to0,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000 to0,000, 17,500 to 20,000, 18,000 to 20,000, 18,500 to 20,000, 19,000 to 20,000, 19,500 to0,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to 19,500,,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500, 5,500 19,500, 6,000 to 19,500, 6,500 to 19,500, 7,000 to 19,500, 7,500 to 19,500, 8,000 to9,500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to 19,500, 10,500 to9,500, 11,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500, 13,000 to9,500, 13,500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 to9,500, 16,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to9.500, 18,500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2,000 to 19,000, 2,500 to9,000, 3,000 to 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to9,000, 5,500 to 19,000, 6,000 to 19,000, 6,500 to 19,000, 7,000 to 19,000, 7,500 to9,000, 8,000 to 19,000, 8,500 to 19,000, 9,000 to 19,000, 9,500 to 19,000, 10,000 to9,000, 10,500 to 19,000, 11,000 to 19,000, 11,500 to 19,000, 12,000 to 19,000, 12,500 to9,000, 13,000 to 19,000, 13,500 to 19,000, 14,000 to 19,000, 14,500 to 19,000, 15,000 to9,000, 15,500 to 19,000, 16,000 to 19,000, 16,500 to 19,000, 17,000 to 19,000, 17,500 to9,000, 18,000 to 19,000, 18,500 to 19,000, 1,500 to 18,500, 2,000 to 18,500, 2,500 to8.500, 3,000 to 18,500, 3,500 to 18,500, 4,000 to 18,500, 4,500 to 18,500, 5,000 to8,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500, 7,000 to 18,500, 7,500 to8,500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to 18,500, 10,000 to8,500, 10,500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500, 12,500 to8,500, 13,000 to 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to 18,500, 15,000 to8,500, 15,500 to 18,500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to 18,500, 17,500 to8,500, 18,000 to 18,500, 1,500 to 18,000, 2,000 to 18,000, 2,500 to 18,000, 3,000 to8,000, 3,500 to 18,000, 4,000 to 18,000, 4,500 to 18,000, 5,000 to 18,000, 5,500 to8,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 to 18,000, 7,500 to 18,000, 8,000 to ,000, 8,500 to 18,000, 9,000 to 18,000, 9,500 to 18,000, 10,000 to 18,000, 10,500 to,000, 11,000 to 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 to 18,000, 13,000 to,000, 13,500 to 18,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to 18,000, 15,500 to,000, 16,000 to 18,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000, 1,500 to,500, 2,000 to 17,500, 2,500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, 4,000 to,500, 4,500 to 17,500, 5,000 to 17,500, 5,500 to 17,500, 6,000 to 17,500, 6,500 to,500, 7,000 to 17,500, 7,500 to 17,500, 8,000 to 17,500, 8,500 to 17,500, 9,000 to,500, 9,500 to 17,500, 10,000 to 17,500, 10,500 to 17,500, 11,000 to 17,500, 11,500 to,500, 12,000 to 17,500, 12,500 to 17,500, 13,000 to 17,500, 13,500 to 17,500, 14,000 to,500, 14,500 to 17,500, 15,000 to 17,500, 15,500 to 17,500, 16,000 to 17,500, 16,500 to.500, 17,000 to 17,500, 1,500 to 17,000, 2,000 to 17,000, 2,500 to 17,000, 3,000 to,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to 17,000, 5,000 to 17,000, 5,500 to,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 to 17,000, 8,000 to,000, 8,500 to 17,000, 9,000 to 17,000, 9,500 to 17,000, 10,000 to 17,000, 10,500 to,000, 11,000 to 17,000, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 to,000, 13,500 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 to.500, 3,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to,500, 5,500 to 16,500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500, 7,500 to,500, 8,000 to 16,500, 8,500 to 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to,500, 10,500 to 16,500, 11,000 to 16,500, 11,500 to 16,500, 12,000 to 16,500, 12,500 to,500, 13,000 to 16,500, 13,500 to 16,500, 14,000 to 16,500, 14,500 to 16,500, 15,000 to,500, 15,500 to 16,500, 16,000 to 16,500, 1,500 to 16,000, 2,000 to 16,000, 2,500 to,000, 3,000 to 16,000, 3,500 to 16,000, 4,000 to 16,000, 4,500 to 16,000, 5,000 to,000, 5,500 to 16,000, 6,000 to 16,000, 6,500 to 16,000, 7,000 to 16,000, 7,500 to,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000, 9,500 to 16,000, 10,000 to,000, 10,500 to 16,000, 11,000 to 16,000, 11,500 to 16,000, 12,000 to 16,000, 12,500 to,000, 13,000 to 16,000, 13,500 to 16,000, 14,000 to 16,000, 14,500 to 16,000, 15,000 to,000, 15,500 to 16,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 to 15,500, 3,000 to ,500, 3,500 to 15,500, 4,000 to 15,500, 4,500 to 15,500, 5,000 to 15,500, 5,500 to,500, 6,000 to 15,500, 6,500 to 15,500, 7,000 to 15,500, 7,500 to 15,500, 8,000 to,500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to 15,500, 10,000 to 15,500, 10,500 to,500, 11,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500, 12,500 to 15,500, 13,000 to.500, 13,500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 15,000 to 15,500, 1,500 to,000, 2,000 to 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000, 4,000 to,000, 4,500 to 15,000, 5,000 to 15,000, 5,500 to 15,000, 6,000 to 15,000, 6,500 to,000, 7,000 to 15,000, 7,500 to 15,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to,000, 9,500 to 15,000, 10,000 to 15,000, 10,500 to 15,000, 11,000 to 15,000, 11,500 to,000, 12,000 to 15,000, 12,500 to 15,000, 13,000 to 15,000, 13,500 to 15,000, 14,000 to,000, 14,500 to 15,000, 1,500 to 14,500, 2,000 to 14,500, 2,500 to 14,500, 3,000 to.500, 3,500 to 14,500, 4,000 to 14,500, 4,500 to 14,500, 5,000 to 14,500, 5,500 to,500, 6,000 to 14,500, 6,500 to 14,500, 7,000 to 14,500, 7,500 to 14,500, 8,000 to,500, 8,500 to 14,500, 9,000 to 14,500, 9,500 to 14,500, 10,000 to 14,500, 10,500 to,500, 11,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500, 13,000 to.500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000, 2,000 to 14,000, 2,500 to,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to 14,000, 5,000 to,000, 5,500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000, 7,500 to,000, 8,000 to 14,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to,000, 10,500 to 14,000, 11,000 to 14,000, 11,500 to 14,000, 12,000 to 14,000, 12,500 to,000, 13,000 to 14,000, 13,500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to.500, 3,000 to 13,500, 3,500 to 13,500, 4,000 to 13,500, 4,500 to 13,500, 5,000 to,500, 5,500 to 13,500, 6,000 to 13,500, 6,500 to 13,500, 7,000 to 13,500, 7,500 to,500, 8,000 to 13,500, 8,500 to 13,500, 9,000 to 13,500, 9,500 to 13,500, 10,000 to,500, 10,500 to 13,500, 11,000 to 13,500, 11,500 to 13,500, 12,000 to 13,500, 12,500 to,500, 13,000 to 13,500, 1,500 to 13,000, 2,000 to 13,000, 2,500 to 13,000, 3,000 to,000, 3,500 to 13,000, 4,000 to 13,000, 4,500 to 13,000, 5,000 to 13,000, 5,500 to,000, 6,000 to 13,000, 6,500 to 13,000, 7,000 to 13,000, 7,500 to 13,000, 8,000 to,000, 8,500 to 13,000, 9,000 to 13,000, 9,500 to 13,000, 10,000 to 13,000, 10,500 to 3,000, 11,000 to 13,000, 11,500 to 13,000, 12,000 to 13,000, 12,500 to 13,000, 1,500 to2,500, 2,000 to 12,500, 2,500 to 12,500, 3,000 to 12,500, 3,500 to 12,500, 4,000 to2,500, 4,500 to 12,500, 5,000 to 12,500, 5,500 to 12,500, 6,000 to 12,500, 6,500 to2,500, 7,000 to 12,500, 7,500 to 12,500, 8,000 to 12,500, 8,500 to 12,500, 9,000 to2,500, 9,500 to 12,500, 10,000 to 12,500, 10,500 to 12,500, 11,000 to 12,500, 11,500 to2.500, 12,000 to 12,500, 1,500 to 12,000, 2,000 to 12,000, 2,500 to 12,000, 3,000 to2,000, 3,500 to 12,000, 4,000 to 12,000, 4,500 to 12,000, 5,000 to 12,000, 5,500 to2,000, 6,000 to 12,000, 6,500 to 12,000, 7,000 to 12,000, 7,500 to 12,000, 8,000 to2,000, 8,500 to 12,000, 9,000 to 12,000, 9,500 to 12,000, 10,000 to 12,000, 10,500 to2,000, 11,000 to 12,000, 11,500 to 12,000, 1,500 to 11,500, 2,000 to 11,500, 2,500 to 1.500, 3,000 to 11,500, 3,500 to 11,500, 4,000 to 11,500, 4,500 to 11,500, 5,000 to 1,500, 5,500 to 11,500, 6,000 to 11,500, 6,500 to 11,500, 7,000 to 11,500, 7,500 to 1,500, 8,000 to 11,500, 8,500 to 11,500, 9,000 to 11,500, 9,500 to 11,500, 10,000 to 1.500, 10,500 to 11,500, 11,000 to 11,500, 1,500 to 11,000, 2,000 to 11,000, 2,500 to 1,000, 3,000 to 11,000, 3,500 to 11,000, 4,000 to 11,000, 4,500 to 11,000, 5,000 to 1,000, 5,500 to 11,000, 6,000 to 11,000, 6,500 to 11,000, 7,000 to 11,000, 7,500 to 1,000, 8,000 to 11,000, 8,500 to 11,000, 9,000 to 11,000, 9,500 to 11,000, 10,000 to 1,000, 10,500 to 11,000, 1,500 to 10,500, 2,000 to 10,500, 2,500 to 10,500, 3,000 to0.500, 3,500 to 10,500, 4,000 to 10,500, 4,500 to 10,500, 5,000 to 10,500, 5,500 to0,500, 6,000 to 10,500, 6,500 to 10,500, 7,000 to 10,500, 7,500 to 10,500, 8,000 to0.500, 8,500 to 10,500, 9,000 to 10,500, 9,500 to 10,500, 10,000 to 10,500, 1,500 to0,000, 2,000 to 10,000, 2,500 to 10,000, 3,000 to 10,000, 3,500 to 10,000, 4,000 to0,000, 4,500 to 10,000, 5,000 to 10,000, 5,500 to 10,000, 6,000 to 10,000, 6,500 to0,000, 7,000 to 10,000, 7,500 to 10,000, 8,000 to 10,000, 8,500 to 10,000, 9,000 to0,000, 9,500 to 10,000, 1,500 to 9,500, 2,000 to 9,500, 2,500 to 9,500, 3,000 to 9,500,,500 to 9,500, 4,000 to 9,500, 4,500 to 9,500, 5,000 to 9,500, 5,500 to 9,500, 6,000 to.500, 6,500 to 9,500, 7,000 to 9,500, 7,500 to 9,500, 8,000 to 9,500, 8,500 to 9,500, 9,000 9,500, 1,500 to 9,000, 2,000 to 9,000, 2,500 to 9,000, 3,000 to 9,000, 3,500 to 9,000,,000 to 9,000, 4,500 to 9,000, 5,000 to 9,000, 5,500 to 9,000, 6,000 to 9,000, 6,500 to 9,000, 7,000 to 9,000, 7,500 to 9,000, 8,000 to 9,000, 8,500 to 9,000, 1,500 to 8,500, 2,000 to 8,500, 2,500 to 8,500, 3,000 to 8,500, 3,500 to 8,500, 4,000 to 8,500, 4,500 to 8,500, 5,000 to 8,500, 5,500 to 8,500, 6,000 to 8,500, 6,500 to 8,500, 7,000 to 8,500, 7,500 to
8.500, 8,000 to 8,500, 1,500 to 8,000, 2,000 to 8,000, 2,500 to 8,000, 3,000 to 8,000, 3,500 to 8,000, 4,000 to 8,000, 4,500 to 8,000, 5,000 to 8,000, 5,500 to 8,000, 6,000 to 8,000,
6.500 to 8,000, 7,000 to 8,000, 7,500 to 8,000, 1,500 to 7,500, 2,000 to 7,500, 2,500 to
7.500, 3,000 to 7,500, 3,500 to 7,500, 4,000 to 7,500, 4,500 to 7,500, 5,000 to 7,500, 5,500 to 7,500, 6,000 to 7,500, 6,500 to 7,500, 7,000 to 7,500, 1,500 to 7,000, 2,000 to 7,000,
2.500 to 7,000, 3,000 to 7,000, 3,500 to 7,000, 4,000 to 7,000, 4,500 to 7,000, 5,000 to 7,000, 5,500 to 7,000, 6,000 to 7,000, 6,500 to 7,000, 1,500 to 6,500, 2,000 to 6,500, 2,500 to 6,500, 3,000 to 6,500, 3,500 to 6,500, 4,000 to 6,500, 4,500 to 6,500, 5,000 to 6,500,
5.500 to 6,500, 6,000 to 6,500, 1,500 to 6,000, 2,000 to 6,000, 2,500 to 6,000, 3,000 to 6,000, 3,500 to 6,000, 4,000 to 6,000, 4,500 to 6,000, 5,000 to 6,000, 5,500 to 6,000, 1,500 to 5,500, 2,000 to 5,500, 2,500 to 5,500, 3,000 to 5,500, 3,500 to 5,500, 4,000 to 5,500,
4.500 to 5,500, 5,000 to 5,500, 1,500 to 5,000, 2,000 to 5,000, 2,500 to 5,000, 3,000 to 5,000, 3,500 to 5,000, 4,000 to 5,000, 4,500 to 5,000, 1,500 to 4,500, 2,000 to 4,500, 2,500 to 4,500, 3,000 to 4,500, 3,500 to 4,500, 4,000 to 4,500, 1,500 to 4,000, 2,000 to 4,000,
2.500 to 4,000, 3,000 to 4,000, 3,500 to 4,000, 1,500 to 3,500, 2,000 to 3,500, 2,500 to
3.500, 3,000 to 3,500, 1,500 to 3,000, 2,000 to 3,000, 2,500 to 3,000, 1,500 to 2,500, 2,000 to 2,500, and 1,500 to 2,000 daltons.
[0325] In some embodiments the polymer (e.g., PEG) is conjugated to at least one lipid. In some embodiments the lipid conjugated to the polymer comprised of at least one neutral lipid, at least one phospholipid, at least one anionic lipid, at least one cationic lipid, at least one cholesterol, at least one cholesterol derivative, or any combination thereof.
[0326] In some embodiments, the lipid conjugated to the polymer may be selected from, but is not limited to, at least one of the cationic, non-cationic, or sterol lipids listed previously.
[0327] In some embodiments, the at least one PEG-lipid conjugate may be selected from, but is not limited to at least one of Siglec-IL-PEG-DSPE, R)-2,3- bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S- DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG Cl 4, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k- DMG, PEG2k-Cl l, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE Cl 8, PEG DMPE Cl 4, PEG DLPE Cl 2, PEG Click DMG Cl 4, PEG Click Cl 2, PEG Click CIO, N(Carbonyl-methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3- phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG- 2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000- DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE- PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE- PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG- PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, C18PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14 : 0-PEG2KPE, 1 ,2-di stearoyl-sn-gly cero-3 -phosphoethanol amine-PEG2000, (R)-2, 3 - bis(octadecyloxy)propyl- 1 -(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)- C-DOMG, PEG-C-DMA, and DSPE-PEG-X.
[0328] The amounts and ratios of LNP components may be varied by any amount dependent on the desired form, structure, function, cargo, target, or any combination thereof. The amount of each component may be expressed in various embodiments as percent of the total molar mass of all lipid or lipid conjugated components accounted for by the indicated component (mol%), The amount of each component may be expressed in various embodiments as the relative ratio of each component based on molar mass (Molar Ratio). The amount of each component may be expressed in various embodiments as the weight of each component used to formulate the LNP prior to fabrication (mg or equivalent). The amount of each component may be expressed in various embodiments by any other method known in the art. Any formulation given in one representation of component amounts (“units”) is expressly meant to encompass any formulation expressed in different units of component amounts, wherein those representations are effectively equivalent when converted into the same units. In some embodiments, “effectively equivalent” means two or more values within about 10% of one another.
[0329] In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 20 to 60 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 50 to 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of more than about 60 mol% or about 85 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 95 mol% or less. In some embodiments, the LNP comprises a cationic lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount from about 20 to 30 mol%, 20 to 35 mol%, 20 to 40 mol%, 20 to 45 mol%, 20 to 50 mol%, 20 to 55 mol%, 20 to 60 mol%, 20 to 65 mol%, 20 to 70 mol%, 20 to 75 mol%, 20 to 80 mol%, 20 to 85 mol%, 20 to 90 mol%, 25 to 35 mol%, 25 to 40 mol%, 25 to 45 mol%, 25 to 50 mol%, 25 to 55 mol%, 25 to 60 mol%, 25 to 65 mol%, 25 to 70 mol%, 25 to 75 mol%, 25 to 80 mol%, 25 to 85 mol%, 25 to 90 mol%, 30 to 40 mol%, 30 to 45 mol%, 30 to 50 mol%, 30 to 55 mol%, 30 to 60 mol%, 30 to 65 mol%, 30 to 70 mol%, 30 to 75 mol%, 30 to 80 mol%, 30 to 85 mol%, 30 to 90 mol%, 35 to 40 mol%, 35 to 45 mol%, 35 to 50 mol%, 35 to 55 mol%, 35 to 60 mol%, 35 to 65 mol%, 35 to 70 mol%, 35 to 75 mol%, 35 to 80 mol%, 35 to 85 mol%, 35 to 90 mol%, 40 to 45 mol%, 40 to 50 mol%, 40 to 55 mol%, 40 to 60 mol%, 40 to 65 mol%, 40 to 70 mol%, 40 to 75 mol%, 40 to 80 mol%, 40 to 85 mol%, 40 to 90 mol%, 45 to 55 mol%, 45 to 60 mol%, 45 to 65 mol%, 45 to 70 mol%, 45 to 75 mol%, 45 to 80 mol%, 45 to 85 mol%, 45 to 90 mol%, 50 to 60 mol%, 50 to 65 mol%, 50 to 70 mol%, 50 to 75 mol%, 50 to 80 mol%, 50 to 85 mol%, 50 to 90 mol%, 55 to 65 mol%, 55 to 70 mol%, 55 to 75 mol%, 55 to 80 mol%, 55 to 85 mol%, 55 to 90 mol%, 60 to 70 mol%, 60 to 75 mol%, 60 to 80 mol%, 60 to 85 mol%, 60 to 90 mol%, 65 to 75 mol%, 65 to 80 mol%, 65 to 85 mol%, 65 to 90 mol%, 70 to 80 mol%, 70 to 85 mol%, 70 to 90 mol%, 75 to 85 mol%, 75 to 90 mol%, 80 to 90 mol% or 85 to 95 mol%.
[0330] In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one non-one cationic lipid in an amount of about 5 to 35 mol%. In some embodiments, the LNP comprises at least one cationic lipid in an amount of about 5 to 25 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of less than about 5 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of more than about 25 mol% or about 35 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of about 95 mol% or less. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%. In some embodiments, the LNP comprises at least one non-cationic lipid in an amount from about 5 to 15 mol%, 5 to 25 mol%, 5 to 35 mol%, 5 to 45 mol%, 5 to 55 mol%, 10 to 20 mol%, 10 to 30 mol%, 10 to 40 mol%, 10 to 50 mol%, 15 to 25 mol%, 15 to 35 mol%, 15 to 45 mol%, 20 to 30 mol%, 20 to 40 mol%, 20 to 50 mol%, 25 to 35 mol%, 25 to 45 mol%, 30 to 40 mol%, 30 to 50 mol%, and 35 to 45 mol%. [0331] In some embodiments, the LNP comprises at least one sterol in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 20 to 45 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 25 to 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of less than about 20 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of more than about 45 mol% or about 55 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of about 95 mol% or less. In some embodiments, the LNP comprises at least one sterol in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one sterol in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%. In some embodiments, the LNP comprises at least one sterol in an amount from about 10 to 20 mol%, 10 to 30 mol%, 10 to 40 mol%, 10 to 50 mol%, 10 to 60 mol%, 15 to 25 mol%, 15 to 35 mol%, 15 to 45 mol%, 15 to 55 mol%, 15 to 65 mol%, 20 to 30 mol%, 20 to 40 mol%, 20 to 50 mol%, 20 to 60 mol%, 25 to 35 mol%, 25 to 45 mol%, 25 to 55 mol%, 25 to 65 mol%, 30 to 40 mol%, 30 to 50 mol%, 30 to 60 mol%, 35 to 45 mol%, 35 to 55 mol%, 35 to 65 mol%, 40 to 50 mol%, 40 to 60 mol%, 45 to 55 mol%, 45 to 65 mol%, 50 to 60 mol%, and 55 to 65 mol%.
[0332] In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 0.1 to 100 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 0.5 to 15 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 15 to 40 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of less than about 0.1 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of more than about 15 mol% or about 40 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of about 95 mol% or less. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the LNP comprises at least one additional LNP functional component in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.
[0333] In some embodiments, the LNP comprises at least one additional LNP functional component in an amount from about 0.1 to 1 mol%, 0.1 to 2 mol%, 0.1 to 3 mol%, 0.1 to 4 mol%, 0.1 to 5 mol%, 0.1 to 6 mol%, 0.1 to 7 mol%, 0.1 to 8 mol%, 0.1 to
9 mol%, 0.1 to 10 mol%, 0.1 to 15 mol%, 0.1 to 20 mol%, 0.1 to 25 mol%, 1 to 2 mol%, 1 to 3 mol%, 1 to 4 mol%, 1 to 5 mol%, 1 to 6 mol%, 1 to 7 mol%, 1 to 8 mol%, 1 to 9 mol%, 1 to 10 mol%, 1 to 15 mol%, 1 to 20 mol%, 1 to 25 mol%, 2 to 3 mol%, 2 to 4 mol%, 2 to 5 mol%, 2 to 6 mol%, 2 to 7 mol%, 2 to 8 mol%, 2 to 9 mol%, 2 to 10 mol%, 2 to 15 mol%, 2 to 25 mol%, 3 to 4 mol%, 3 to 5 mol%, 3 to 6 mol%, 3 to 7 mol%, 3 to 8 mol%, 3 to 9 mol%, 3 to 10 mol%, 3 to 15 mol%, 3 to 20 mol%, 3 to 25 mol%, 4 to 5 mol%, 4 to 6 mol%, 4 to 7 mol%, 4 to 8 mol%, 4 to 9 mol%, 4 to 10 mol%, 4 to 15 mol%, 4 to 20 mol%, 4 to 25 mol%, 5 to 10 mol%, 5 to 15 mol%, 5 to 20 mol%, 5 to 25 mol%,
10 to 15 mol%, 10 to 20 mol%, 10 to 25 mol%, 15 to 20 mol%, 15 to 25 mol%, and 20 to 25 mol%.
[0334] In some embodiments, the LNP is comprised of about 30-60 mol% of at least one cationic lipid, about 0-30 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one sterol (e.g., cholesterol), and about 0- 10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid). [0335] In some embodiments, the LNP is comprised of about 35-55 mol% of at least one cationic lipid, about 5-25 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mol% of at least one sterol (e.g., cholesterol), and about 0-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0336] In some embodiments, the LNP is comprised of about 35-45 mol% of at least one cationic lipid, about 25-35 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 20-30 mol% of at least one sterol (e.g., cholesterol), and about 0-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid). [0337] In some embodiments, the LNP is comprised of about 45-65 mol% of at least one cationic lipid, about 5-10 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 25-40 mol% of at least one sterol (e.g., cholesterol), and about 0.5-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0338] In some embodiments, the LNP is comprised of about 40-60 mol% of at least one cationic lipid, about 5-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 35-45 mol% of at least one sterol (e.g., cholesterol), and about 0.5-3 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0339] In some embodiments, the LNP is comprised of about 30-60 mol% of at least one cationic lipid, about 0-30 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 15-50 mol% of at least one sterol (e.g., cholesterol), and about 0.01- 10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid). [0340] In some embodiments, the LNP is comprised of about 10-75 mol% of at least one cationic lipid, about 0.5-50 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 5-60 mol% of at least one sterol (e.g., cholesterol), and about 0.1-20 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0341] In some embodiments, the LNP is comprised of about 50-65 mol% of at least one cationic lipid, about 3-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mol% of at least one sterol (e.g., cholesterol), and about 0.5-2 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0342] In some embodiments, the LNP is comprised of about 50-85 mol% of at least one cationic lipid, about 3-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 30-40 mol% of at least one sterol (e.g., cholesterol), and about 0.5-2 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0343] In some embodiments, the LNP is comprised of about 25-75 mol% of at least one cationic lipid, about 0.1-15 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 5-50 mol% of at least one sterol (e.g., cholesterol), and about 0.5-20 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid). [0344] In some embodiments, the LNP is comprised of about 50-65 mol% of at least one cationic lipid, about 5-10 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 25-35 mol% of at least one sterol (e.g., cholesterol), and about 5-10 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0345] In some embodiments, the LNP is comprised of about 20-60 mol% of at least one cationic lipid, about 5-25 mol% of at least one non-cationic lipid (e.g., a phospholipid), about 25-55 mol% of at least one sterol (e.g., cholesterol), and about 0.5-15 mol% of at least one additional LNP functional component (e.g., a PEGylated lipid).
[0346] In some embodiments, the nucleic acid vaccine described here is formulated in a lipid nanoparticle comprising a cationic lipid, a phospholipid, a sterol and a PEGylated lipid as described herein. The LNP may comprise about 20-70% (mole) of a cationic lipid in the formulation , e.g., about 30-60%, or about 30-50%, or 50%. The LNP may comprise about 5-20% (mole) of a phospholipid in the formulation, e.g., 5-10%, 5-15%, 10-15% or about 10% of the phospholipid. The LNP may comprise about 20-45% (mole) of a sterol in the formulation, e.g., 20-40%, 30-40%, or about 38% of the sterol. The LNP may comprise about 0.5 to 5.0% of a PEGylated lipid, e.g., about 0.5-2.0%, 1.0-3.0%, 1.5-2.0% or about 2.0% of the PEGylated lipid.
[0347] As a non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, a phospholipid, a sterol and a PEGylated lipid.
[0348] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, DSPC, a sterol and a PEGylated lipid.
[0349] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, a phospholipid, a sterol and PEG-DMA.
[0350] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, a phospholipid, cholesterol and PEGylated lipid. [0351] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, DSPC, a sterol and a PEGylated lipid.
[0352] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, a phospholipid, cholesterol and a PEGylated lipid.
[0353] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, a phospholipid, a sterol and PEG- DMA.
[0354] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, a phospholipid, cholesterol and PEG-DMA.
[0355] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, DSPC, a sterol and PEG- DMA.
[0356] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising a cationic lipid, DSPC, cholesterol and a PEGylated lipid.
[0357] As another non-limiting example, the nucleic acid vaccine described here is formulated a lipid nanoparticle comprising 3D-P-DMA, DSPC, cholesterol and PEG- DMA.
[0358] In some embodiments, the nucleic acid vaccine described here is formulated in a lipid nanoparticle comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 1.6% of a PEGylated lipid in the formulation.
[0359] As non-limiting examples, the cationic lipid, sterol, phospholipid and PEGylated lipid is 3D-P-DMA, DSPC, Cholesterol and PEG-DMA, respectively.
[0360] In some embodiments, the nucleic acid vaccine described here is formulated in a lipid nanoparticle comprising about 53% (by weight) of 3D-P-DMA, about 14% (by weight of DSPC, about 26% (by weight) of cholesterol and about 7% (by weight) of a PEG-DMA in the formulation.
[0361] In some embodiments, the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 27 that is formulated in a LNP comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 2.0 % of a PEGylated lipid. As a non-limiting example, the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 27 that is formulated in a LNP comprising about 50% (by mole) of 3D-P-DMA, about 10% (by mole) of DSPC, about 38% (by mole) of cholesterol and about 2.0 % of a PEG-DMA.
[0362] In some embodiments, the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 43 that is formulated in a LNP comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 2.0 % of a PEGylated lipid. As a non-limiting example, the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 43 that is formulated in a LNP comprising about 50% (by mole) of 3D-P-DMA, about 10% (by mole) of DSPC, about 38% (by mole) of cholesterol and about 2.0 % of a PEG-DMA.
[0363] In some embodiments, the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 39 or SEQ ID NO: 41 that is formulated in a LNP comprising about 50% (by mole) of a cationic lipid, about 10% (by mole) of a phospholipid, about 38% (by mole) of a sterol and about 2.0 % of a PEGylated lipid. As a non-limiting example, the nucleic acid vaccine compositions described here may comprise at least one nucleic acid vaccine comprising a polynucleotide of SEQ ID NO: 39 or SEQ ID NO: 41 that is formulated in a LNP comprising about 50% (by mole) of 3D-P-DMA, about 10% (by mole) of DSPC, about 38% (by mole) of cholesterol and about 2.0 % of a PEG-DMA. [0364] In some embodiments, the LNPs can be characterized by their shape. In some embodiments, the LNPs are essentially spherical. In some embodiments, the LNPs are essentially rod-shaped (i.e., cylindrical). In some embodiments, the LNPs are essentially disk shaped.
[0365] In some embodiments, the LNPs can be characterized by their size. In some embodiments, the size of an LNP can be defined as the diameter of its largest circular cross section, referred to herein simply as its diameter. In some embodiments the LNPs may have a diameter between 30 nm to about 150 nm. In some embodiments, the LNP may have diameters ranging between about 40 to 150 nm 50 to 150 nm, 60 to 150 nm, about 70 to 150 nm, or 80 to 150 nm, 90 to 150 nm, 100 to nm, 110 to 150 nm, 120 to 150 nm, 130 to 150 nm, or 140 to 150 nm.
[0366] In some embodiments, a population of LNPs, such as those resulting from the same formulation, may be characterized by measuring the uniformity of size, shape, or mass of the particles in the population, uniformity may be expressed in some embodiments as the poly dispersity index (PI) of the population. In some embodiments uniformity may be expressed in some embodiments as the disparity (D) of the population. The terms “polydispersity index” and “disparity” are understood herein to be equivalent and may be used interchangeably. In some embodiments, a population of LNPs resulting from a given formulation will have a PI of between about 0.1 and 1. In some embodiments, a population of LNPs resulting from a giving formulation will have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, a population of LNPs resulting from a given formulation will have a PI of between about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1. [0367] In some embodiments, the LNP may fully or partially encapsulate a cargo, such as nucleic acid constructs of the present disclosure. In some embodiments, essentially 0% of the cargo present in the final formulation is exposed to the environment outside of the LNP (i.e., the cargo is fully encapsulated. In some embodiments, the cargo is associated with the LNP but is at least partially exposed to the environment outside of the LNP. In some embodiments, the LNP may be characterized by the% of the cargo not exposed to the environment outside of the LNP, e.g., the encapsulation efficiency. For the sake of clarity, an encapsulation efficiency of about 100% refers to an LNP formulation where essentially all the cargo is fully encapsulated by the LNP, while an encapsulation rate of about 0% refers to an LNP where essential none of the cargo is encapsulated in the LNP, such as with an LNP where the cargo is bound to the external surface of the LNP. On some embodiments, an LNP may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%. less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15% less than about 10%, or less than 5%. In some embodiments, an LNP may have an encapsulation efficiency of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%,
30 to 70%, 20 to 70%, 10 to 70%, 40 to 50%, 30 to 50%, 20 to 50%, 10 to 50%, 30 to
40%, 20 to 40%, 10 to 40%, 20 to 30%, 10 to 30%, and 10 to 20%.
[0368] In some embodiments, the at least one targeting agent may be presented on the external surface of the nanoparticle. In some embodiments, the at least one targeting agent may be conjugated to a lipid-component of the nanoparticle. In some embodiments, the at least one targeting agent may be conjugated to a polymer component of the nanoparticle. In some embodiments, the at least one targeting agent may be anchored to the nanoparticle via hydrophobic ad hydrophilic interactions among the at least one targeting agent, the nanoparticle membrane, and the aqueous environments inside or outside the nanoparticle. In some embodiments, the at least one targeting agent is conjugated to a peptide/protein component of the nanoparticle membrane. In some embodiments, the at least one targeting agent is conjugated to a suitable linker moiety which is conjugated to a component of the nanoparticle membrane. In some embodiments, any combination of forces and bonds can result in the targeting agent being associated with the nanoparticle.
[0369] The LNPs described herein may be formed using techniques known in the art. As a non-limiting example, an organic solution containing the lipids is mixed together with an acidic aqueous solution containing the nucleic acid compositions in a microfluidic channel resulting in the formation of targeting system (delivery vehicle and the nucleic acid vaccine).
[0370] In some embodiments, the lipid compositions are described according to the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol- %. As a non-limiting example, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol- %. As a non-limiting example, the ionizable lipid mol-% of the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In some embodiments, transfer vehicle variability between lots will be less than 15%, less than 10% or less than 5%.
[0371] In some embodiments, the mol-% of the helper lipid may be from about 1 mol- % to about 50 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 5 mol-% to about 30 mol-%. In some embodiments, the mol-% of the helper lipid may be from about 10 mol-% to about 20 mol- %. In some embodiments, the helper lipid mol-% of the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. [0372] In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%. In some embodiments, the structural lipid mol-% of the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%.
[0373] In some embodiments, the mol-% of the PEG modified lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG modified lipid may be about 1.5 mol-%. In some embodiments, the PEG modified lipid mol-% of the transfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%.
[0374] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication Nos. WO2011127255 or W02008103276, the contents of each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, the contents of which is herein incorporated by reference in its entirety.
[0375] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. WO2020237227, the contents of each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. WO2020237227, the contents of which is herein incorporated by reference in its entirety. [0376] In some embodiments, the lipid may be a cleavable lipid such as those described in PCT Patent Application Publication No. WO2012170889, the contents of which are herein incorporated by reference in their entirety.
[0377] In some embodiments, the nanoparticles described herein may comprise at least one cationic polymer described herein and/or known in the art.
[0378] In some embodiments, the cationic lipid may be synthesized by methods known in the art and/or as described in PCT Patent Application Publication Nos.
W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO201 1022460, WO2012061259, WO2012054365, WO2012044638, W02010080724 and W0201021865; the contents of each of which are herein incorporated by reference in their entirety.
[0379] In some embodiments, the pharmaceutical compositions of the nucleic acid vaccine compositions may include at least one of the PEGylated lipids described in PCT Patent Application Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.
[0380] In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations. As a non-limiting example, LNP formulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In some embodiments, the LNP formulations of the nucleic acid vaccine compositions may contain PEG-c-DOMG at 3% lipid molar ratio. In some embodiments, the LNP formulations of the nucleic acid vaccine compositions may contain PEG-c-DOMG at 1.5% lipid molar ratio.
[0381] In some embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene 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, C 12-200 and DLin-KC2-DMA. [0382] In some embodiments, the LNP formulation may contain PEG-DMG 2000 (1,2- dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(poly ethylene glycol)-2000), a cationic lipid known in the art. In some embodiments, the LNP formulation may contain PEG-DMG 2000and 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, the LNP formulation may contain PEG- DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS, 2012, 109(36): 14604-14609; herein incorporated by reference in its entirety).
[0383] As another non-limiting example, the nucleic acid vaccine compositions described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Patent Application Publication No. US20120207845; the contents of which are herein incorporated by reference in their entirety.
[0384] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a plurality of cationic lipids, such as a first and a second cationic lipid as described in US 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 can be selected on the basis of a first property and the second cationic lipid can be selected on the basis of a second property, where the properties may be determined as outlined in US20130017223. In some embodiments, the first and second properties are complementary.
[0385] The nucleic acid vaccine compositions described herein may be formulated with a lipid particle comprising one or more cationic lipids and one or more second lipids, and one or more nucleic acids, wherein the lipid particle comprises a solid core, as described in US Patent Publication No. US20120276209 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.
[0386] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be complexed with a cationic amphiphile in an oil-in-water (o/w) emulsion such as described in European Publication No.: EP2298358 to Satishchandran et al., the contents of which are incorporated herein by reference in their entirety. The cationic amphiphile may be a cationic lipid, modified or unmodified spermine, bupivacaine, or benzalkonium chloride and the oil may be a vegetable or an animal oil. As a non-limiting example, at least 10% of the nucleic acid-cationic amphiphile complex is in the oil phase of the oil-in-water emulsion (see e.g., the complex described in. EP2298358 to Satishchandran et al.), the contents of which are incorporated herein by reference in its entirety.
[0387] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a composition comprising a mixture of cationic compounds and neutral lipids. As a non-limiting example, the cationic compounds may be 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 diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide and sphingomyelin. In another non-limiting example, the lipid formulation may comprise a cationic lipid of formula A, a neutral lipid, a sterol and a PEG or PEG-modified lipid disclosed in US Patent Publication No.: US 20120101148 to Akinc et al., the contents of which are incorporated herein by reference in their entirety.
[0388] In some embodiments, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or W02008103276. As a non-limiting example, the nucleic acid vaccine compositions of the present disclosure may be encapsulated in any of the lipid nanoparticle (LNP) formulations described in WO201 1127255 and/or W02008103276; the contents of each of which are herein incorporated by reference in their entirety.
[0389] 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 formula 1-60 of US Patent Publication No. US20050222064; the contents of which are herein incorporated by reference in their entirety. The LNP formulations comprising a polycationic composition may be used for the delivery of the nucleic acid vaccine compositions described herein in vivo and/or in vitro.
[0390] In some embodiments, the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; the contents of which are herein incorporated by reference in their entirety.
[0391] The nanoparticle formulations 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, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., PCT Patent Application Publication No. W02012109121; the contents of which are herein incorporated by reference in their entirety).
[0392] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). 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 a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
[0393] In some embodiments, the nucleic acid vaccine compositions is formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. The lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8): 1696-1702; the contents of which are herein incorporated by reference in their entirety).
[0394] In some embodiments, formulations comprising the nucleic acid vaccine compositions described herein may also be constructed or altered such that they passively or actively are directed to 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 Q aU Pidm 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; the contents of each of which are incorporated herein by reference in their entirety). One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3 -DMA-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010, 18: 1357-1364; the contents of which are herein incorporated by reference in their entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011, 8: 197-206; Musacchio and T orchilin, 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., Afo/ Ther. 2010, 18: 1357-1364; Srinivasan et al., Methods Mol Biol. 2012, 820: 105-116; Ben-Arie et < ., Methods Mol Biol. 2012, 757:497-507; Peer J Control Release. 2010, 20:63-68; Peer et al., Proc Natl Acad Sci USA. 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; the contents of each of which are incorporated herein by reference in their entirety).
[0395] In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated for controlled release and/or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a therapeutic outcome. In some embodiments, the nucleic acid vaccine compositions may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround, or encase. As it relates to the formulation of the compositions of the disclosure, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulated” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent.
Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition of the disclosure using fluorescence and/or electron micrograph. 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 disclosure are encapsulated in the delivery agent.
[0396] The nucleic acid vaccine compositions may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EV Ac), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc., Deerfield, IL), PEG- based sealants, and COSEAL® (Baxter International, Inc., Deerfield, IL).
[0397] In some embodiments, the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
[0398] In some embodiments, the formulations comprising the 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 ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
[0399] In some embodiments, the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains. Degradable 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 degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[0400] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a targeting lipid with a targeting moiety such as the targeting moieties disclosed in US Patent Application Publication No.: US20130202652 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the targeting moiety of formula I of US 20130202652 to Manoharan et al. may be selected in order to favor the lipid being localized with a desired organ, tissue, cell, cell type or subtype, or organelle. Non-limiting targeting moieties that are contemplated in the present disclosure include transferrin, anisamide, an RGD peptide, prostate specific membrane antigen (PSMA), fucose, an antibody, or an aptamer.
[0401] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be encapsulated in a therapeutic nanoparticle. Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, PCT Patent Application Publication Nos. W02010005740, W02010030763, W02010005721, W02010005723, and WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286 and US20120288541 and US Pat. No. 8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are herein incorporated by reference in their entirety. Therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which are herein incorporated by reference in their entirety.
[0402] In some embodiments, the therapeutic nanoparticle may be formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the nucleic acid vaccine compositions of the present disclosure (see PCT Patent Application Publication No. W02010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, the contents of each of which are herein incorporated by reference in their entirety).
[0403] In some embodiments, the therapeutic nanoparticles may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticles may include a corticosteroid (see PCT Patent Application Publication No. WO2011084518; the contents of which are herein incorporated 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 in nanoparticles described in PCT Patent Application Publication No. WO2008121949, W02010005726, W02010005725, and WO2011084521, and US Pub No. US20100069426, US20120004293 and US20100104655, the contents of each of which are herein incorporated by reference in their entirety.
[0404] In some embodiments, the nanoparticles of the present disclosure may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, poly acrylates, polymethacrylates, poly cyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
[0405] In some embodiments, the therapeutic nanoparticle comprises a diblock copolymer. In some embodiments, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
[0406] As a non-limiting example, the therapeutic nanoparticle comprises a PLGA- PEG block copolymer (see US Pub. No. US20120004293 and US Pat. No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat. No 8,246,968 and PCT Patent Application Publication No. WO2012166923, the contents of each of which are herein incorporated by reference in their entirety). [0407] In some embodiments, the therapeutic nanoparticle may comprise a multiblock copolymer such as, but not limited to the multiblock copolymers described in U.S. Pat. Nos. 8,263,665 and 8,287,910; the contents of each of which are herein incorporated by reference in their entirety.
[0408] In some embodiments, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer. (See e.g., U.S. Pub. No. US20120076836; the contents of which are herein incorporated by reference in their entirety).
[0409] In some embodiments, the nanoparticles for delivery of the nucleic acid vaccines described herein include block co-polymers. Non-limiting examples of block copolymers include those of formula I, formula II, formula III, formula IV, formula V, formula VI and formula VII of PCT Patent Application Publication No. W02015017519, the contents of which are herein incorporated by reference in their entirety.
[0410] In some embodiments, the therapeutic nanoparticle 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 methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof. [0411] In some embodiments, the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No. 8,287,849; the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
[0412] In some embodiments, the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains. Degradable 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. The degradable polyesters may include a PEG conjugation to form a PEGylated polymer. [0413] In some embodiments, the therapeutic nanoparticle may include a conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006, 66:6732-6740; the contents of which are herein incorporated by reference in their entirety).
[0414] In some embodiments, the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see PCT Patent Application Publication No. WO2011084513 and US Pub No. US20110294717, the contents of each of which are herein incorporated by reference in their entirety).
[0415] In some embodiments, the nucleic acid vaccine compositions may be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in PCT Patent Application Publication Nos. W02010005740, W02010030763, W0201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, W02012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US20120244222, the contents of each of which are herein incorporated by reference in their entirety. The synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in PCT Patent Application Publication Nos. W02010005740, W02010030763 and W0201213501and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US2012024422, the contents of each of which are herein incorporated by reference in their entirety. The synthetic nanocarrier formulations may be lyophilized by methods described in PCT Patent Application Publication Pub. No. WO2011072218 and US Pat. No. 8,211,473; the contents of each of which are herein incorporated by reference in their entirety.
[0416] In some embodiments, the synthetic nanocarriers may contain reactive groups to release the nucleic acid vaccine compositions described herein (see PCT Patent Application Publication No. WO20120952552 and US Pub No. US20120171229, the contents of each of which are herein incorporated by reference in their entirety). [0417] In some embodiments, the synthetic nanocarriers may be formulated for targeted release. In some embodiments, the synthetic nanocarrier may be formulated to release the nucleic acid vaccine compositions at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle may be formulated to release the nucleic acid vaccine compositions after 24 hours and/or at a pH of 4.5 (see PCT Patent Application Publication Nos. W02010138193 and W02010138194 and US Pub Nos. US20110020388 and US20110027217, the contents of each of which are herein incorporated by reference in their entireties).
[0418] In some embodiments, the synthetic nanocarriers may be formulated for controlled and/or sustained release of the nucleic acid vaccine compositions described herein. As a non-limiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in PCT Patent Application Publication No. W02010138192 and US Pub No. US20100303850, the contents each of which are herein incorporated by reference in their entirety.
[0419] In some embodiments, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Pub. No. US20120282343; the contents of which are herein incorporated by reference in their entirety.
[0420] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a modular composition such as described in US Pat. No. US 8,575,123 to Manoharan et al., the contents of which are herein incorporated by reference in their entirety. As a non-limiting example, the modular composition may comprise a nucleic acid, e.g., the nucleic acid vaccine compositions of the present disclosure, at least one endosomolytic component, and at least one targeting ligand. The modular composition may have a formula such as any formula described in US 8,575,123 to Manoharan et al..
[0421] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be encapsulated in the lipid formulation to form a stable nucleic acid-lipid particle (SNALP) such as described in US Pat. No. US8,546,554 to de Fougerolles et al., the contents of which are incorporated here by reference in their entirety. The lipid may be cationic or non-cationic. In one non-limiting example, the lipid to nucleic acid ratio (mass/mass ratio) (e.g., lipid to nucleic acid vaccine compositions ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from 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, the SNALP includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (Lipid A), 10% di oleoylphosphatidylcholine (DSPC), 40% cholesterol, 10% polyethylene glycol (PEG)-C- DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 nucleic acid/lipid ratio.
[0422] The nucleic acid vaccine compositions of the present disclosure may be formulated with a nucleic acid-lipid particle comprising an endosomal membrane destabilizer as disclosed in US Pat. No. US 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 membrane destabilizer may be a Ca2+ ion.
[0423] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with formulated lipid particles (FLiPs) disclosed in US Pat. No. US 8,148,344 to Akinc et al., the contents of which are herein incorporated by reference in their entirety. Akinc et al. teach that FLiPs may comprise at least one of a single or double-stranded oligonucleotide, where the oligonucleotide has been conjugated to a lipophile and at least one of an emulsion or liposome to which the conjugated oligonucleotide has been aggregated, admixed or associated. These particles have surprisingly been shown to effectively deliver oligonucleotides to heart, lung and muscle as disclosed in US 8148344 to Akinc et al.. [0424] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be delivered to a cell using a composition comprising an expression vector in a lipid formulation as described in US Pat. No. US 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 comprising first and second inverted repeated sequences from an adeno associated virus (AAV), a rep gene from AAV, and a nucleic acid fragment. The expression vector in Tam is complexed with lipids.
[0425] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a lipid formulation disclosed in US Pub. No. US 20120270921 to de Fougerolles et al., the contents of which are incorporated herein by reference in their entirety. In one non-limiting example, the lipid formulation may include a cationic lipid having the formula A described in US 20120270921. In another nonlimiting example, the compositions of exemplary nucleic acid-lipid particles disclosed in Table A of US20120270921 may be used with the nucleic acid vaccine compositions of the present disclosure.
[0426] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be fully encapsulated in a lipid particle disclosed in US Pub. No. US 20120276207 to Maurer et al., the contents of which are incorporated herein by reference in their entirety. The particles may comprise a lipid composition comprising preformed lipid vesicles, a charged therapeutic agent, and a destabilizing agent to form a mixture of preformed vesicles and therapeutic agent in a destabilizing solvent, wherein the destabilizing solvent is effective to destabilize the membrane of the preformed lipid vesicles without disrupting the vesicles.
[0427] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a conjugated lipid. In a non-limiting example, the conjugated lipid may have a formula such as described in US Pub. No. US 20120264810 to Lin et al., the contents of which are incorporated herein by reference in their entirety. The conjugate lipid may form a lipid particle which further comprises a cationic lipid, a neutral lipid, and a lipid capable of reducing aggregation. [0428] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a neutral liposomal formulation such as disclosed in US Pub. No. US 20120244207 to Fitzgerald et al., the contents of which are incorporated herein by reference in their entirety. The phrase “neutral liposomal formulation” refers to a liposomal formulation with a near neutral or neutral surface charge at a physiological pH. Physiological pH can be, e.g., about 7.0 to about 7.5, or, e.g., about 7.5, or, e.g., 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or, e.g., 7.3, or, e.g., 7.4. An example of a neutral liposomal formulation is an ionizable lipid nanoparticle (iLNP). A neutral liposomal formulation can include an ionizable cationic lipid, e.g., DLin-KC2-DMA.
[0429] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with a charged lipid or an amino lipid. As used herein, the term "charged lipid" is meant to include those lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amino head group. The quaternary amine carries a permanent positive charge. The head group can optionally include an ionizable group, such as a primary, secondary, or tertiary amine that may be protonated at physiological pH. The presence of the quaternary amine can alter the pKa of the ionizable group relative to the pKa of the group in a structurally similar compound that lacks the quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine) In some embodiments, a charged lipid is referred to as an "amino lipid." In a non-limiting example, the amino lipid may be any amino lipid described in US Pub. 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 structure disclosed in Tables 3-7 of Hope, such as structure (II), DLin-K-C2-DMA, DLin-K2-DMA, DLin-K6-DMA, etc. The resulting pharmaceutical preparations may be lyophilized according to Hope. In another non-limiting example, the amino lipids may be any amino lipid described in US 20110117125 to Hope et al., the contents of which are incorporated herein by reference in their entirety, such as a lipid of 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 the structure (I), (II), (III), or (IV), or 4-(R)-DLin-K-DMA (VI), 4-(S)-DLin-K-DMA (V) as described in PCT Patent Application Publication No. W02009132131 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. In another non-limiting example, the charged lipid used in any of the formulations 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.
[0430] In some embodiments, the nucleic acid vaccine composition s of the present disclosure may be formulated with an association complex. In a non-limiting example, the association complex comprises one or more compounds each having a structure defined by formula (I), a PEG-lipid having a structure defined by formula (XV), a steroid and a nucleic acid disclosed in US Pat. No. US8,034,376 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. The nucleic acid vaccine compositions may be formulated with any association complex described in US Pat. No. US8,034,376., the contents of which are herein incorporated by reference in its entirety. [0431] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with reverse head group lipids. As a non-limiting example, the nucleic acid vaccine compositions may be formulated with a zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the head group, such as a lipid having structure (A) or structure (I) described in PCT Patent Application Publication No. WO201 1056682 to Leung et al., the contents of which are incorporated herein by reference in their entirety.
[0432] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated in a lipid bilayer carrier. As a non-limiting example, the nucleic acid vaccine compositions may be combined with a lipid-detergent mixture comprising a lipid mixture of an aggregation-preventing agent in an amount of about 5 mol% to about 20 mol%, a cationic lipid in an amount of about 0.5 mol% to about 50 mol%, and a fusogenic lipid and a detergent, to provide a nucleic acid-lipid-detergent mixture; and then dialyzing the nucleic acid-lipid-detergent mixture against a buffered salt solution to remove the detergent and to encapsulate the nucleic acid in a lipid bilayer carrier and provide a lipid bilayer-nucleic acid composition, wherein the buffered salt solution has an ionic strength sufficient to encapsulate of from about 40 % to about 80 % of the nucleic acid, described in PCT Patent Application Publication No. WO1999018933 to Cullis et al., the contents of which are incorporated herein by reference in their entirety. [0433] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may comprise (a) a nucleic acid; (b) 1.0 mole % to 45 mole % of a cationic lipid; (c) 0.0 mole % to 90 mole % of another lipid; (d) 1.0 mole % to 10 mole % of a bilayer stabilizing component; (e) 0.0 mole % to 60 mole % cholesterol; and (f) 0.0 mole % to 10 mole % of cationic polymer lipid as described in EP1328254 to Cullis et al., the contents of which are incorporated herein by reference in their entirety.
[0434] In some embodiments, the nucleic acid vaccine may be delivered using smaller LNPs. Such particles may comprise a diameter from below 0.1 pm up to 100 nm such as, but not limited to, less than 0.1 pm, less than 1.0 pm, less than 5 pm, less than 10 pm, less than 15 pm, less than 20 pm, less than 25 pm, less than 30 pm, less than 35 pm, less than 40 pm, less than 50 pm, less than 55 pm, less than 60 pm, less than 65 pm, less than 70 pm, less than 75 pm, less than 80 pm, less than 85 pm, less than 90 pm, less than 95 pm, less than 100 pm, less than 125 pm, less than 150 pm, less than 175 pm, less than 200 pm, less than 225 pm, less than 250 pm, less than 275 pm, less than 300 pm, less than 325 pm, less than 350 pm, less than 375 pm, less than 400 pm, less than 425 pm, less than 450 pm, less than 475 pm, less than 500 pm, less than 525 pm, less than 550 pm , less than 575 pm
, less than 600 pm , less than 625 pm , less than 650 pm , less than 675 pm , less than 700 pm , less than 725 pm , less than 750 pm , less than 775 pm , less than 800 pm , less than 825 pm , less than 850 pm , less than 875 pm , less than 900 pm , less than 925 um, less than 950 pm , less than 975 pm .
[0435] In another embodiment, nucleic acid vaccine may be delivered using smaller LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 nm to about 50 nm, from about 20 nm to about 50 nm, from about 30 nm to about 50 nm, from about 40 nm to about 50 nm, from about 20 nm to about 60 nm, from about 30 nm to about 60 nm, from about 40 nm to about 60 nm, from about 20 nm to about 70 nm, from about 30 nm to about 70 nm, from about 40 nm to about 70 nm, from about 50 nm to about 70 nm, from about 60 nm to about 70 nm, from about 20 nm to about 80 nm, from about 30 nm to about 80 nm, from about 40 nm to about 80 nm, from about 50 nm to about 80 nm, from about 60 nm to about 80 nm, from about 20 nm to about 90 nm, from about 30 nm to about 90 nm, from about 40 nm to about 90 nm, from about 50 nm to about 90 nm, from about 60 nm to about 90 nm and/or from about 70 nm to about 90 nm. [0436] In some embodiments, the nucleic acid vaccine may be formulated in lipid nanoparticles having a diameter from about 10 nm to about 100 nm such as, but not limited to, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm, about 30 nm to about 100 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 40 nm to about 90 nm, about 40 nm to about 100 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 50 nm to about 100 nm, about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm to about 100 nm, about 80 nm to about 90 nm, about 80 nm to about 100 nm and/or about 90 nm to about 100 nm.
[0437] In some embodiments, the nucleic acid vaccine may be formulated in lipid nanoparticles having a diameter from 10-1000 nm. The nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,
225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,
315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400,
405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490,
495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580,
585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,
675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760,
765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850,
855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940,
945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm.
[0438] In some embodiments, the lipid nanoparticles may have a diameter from about 10 to 500 nm.
[0439] In some embodiments, the lipid nanoparticle may have a diameter 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
[0440] The nucleic acid vaccine compositions of the disclosure can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERX™ polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™ (PHASERX®, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers. RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but not limited to, PHASERX® (Seattle, WA). [0441] A non-limiting example of chitosan-based formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No. US20120258176; the contents of which are herein incorporated by reference in their entirety). Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N- carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.
[0442] In some embodiments, the polymers used in the present disclosure have undergone processing to reduce and/or inhibit the attachment of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer. The polymer may be processed by methods known and/or described in the art and/or described in PCT Patent Application Publication No. WO2012150467; the contents of which are herein incorporated by reference in their entirety.
[0443] A non-limiting example of PLGA based formulations include, but are not limited to, PLGA-based injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space. The PLGA-based injectable depots may be long- acting.
[0444] Many of these polymer approaches have demonstrated efficacy in delivering oligonucleotides in vivo into the cell cytoplasm (reviewed in de Fougerolles Hum Gene Ther. 2008, 19: 125-132; the contents of which are herein incorporated by reference in their entirety). Two polymer approaches that have yielded robust in vivo delivery of nucleic acids, i.e., in the case of small interfering RNA (siRNA), are dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches uses dynamic poly conjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887; the contents of which are herein incorporated by reference in their entirety). This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and A-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (See again, Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887). On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer. Through replacement of the A-acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells. Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycation nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in transferrin receptor-expressing Ewing’s sarcoma tumor cells (Hu-Li eskovan et a!.. Cancer Res. 2005, 65: 8984-8982; herein incorporated by reference in its entirety) and siRNA formulated in these nanoparticles was well tolerated in non-human primates (Heidel et al., Proc Natl Acad Sci USA 2007, 104:5715-21; herein incorporated by reference in its entirety). Both of these delivery strategies incorporate rational approaches using both targeted delivery and endosomal escape mechanisms.
[0445] The polymer formulation can permit the sustained or delayed release of nucleic acid vaccine compositions (e.g., following intramuscular, subcutaneous, intraparenchymal, intrathecal, intracerebroventricular administration). The altered release profile for the nucleic acid vaccine compositions can result in, for example, translation of an encoded protein, or polypeptide or peptide over an extended period of time. Biodegradable polymers have been previously used to protect nucleic acids from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887; Sullivan et al., Expert Opin Drug Deliv. 2010, 7: 1433- 1446; Convertine et al., Biomacromolecules. 2010, Oct 1; Chu et al., Acc Chem Res. 2012, Jan 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 Fougerolles Hum Gene Ther. 2008, 19: 125-132; Schaffert and Wagner, Gene Ther. 2008, 16: 1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011, 8: 1455-1468; Davis, AfoZ Pharm. 2009, 6:659-668; Davis, Nature, 2010, 464: 1067-1070; the contents of each of which are herein incorporated by reference in their entirety).
[0446] In some embodiments, the nucleic acid vaccines of the present disclosure may be formulated for controlled release. One form of controlled-release formulation contains the therapeutic compound or its salt dispersed or encapsulated in a slowly degrading, nontoxic, non-antigenic polymer such as copoly(lactic/glycolic) acid, as described in the pioneering work of Kent et al., US Patent No. 4,675,189, the contents of which are incorporated by reference herein in their entirety. The compounds, or their salts, may also be formulated in cholesterol or other lipid matrix pellets, or silastomer matrix implants. As a non-limiting example, the nucleic acid vaccines of the present disclosure may be dispersed or encapsulated in the polymers disclosed in US Patent No. 4,675,189 for controlled release. An additional form of controlled-release formulation comprises a solution of biodegradable polymer, such as copoly(lactic/gly colic acid) or block copolymers of lactic acid and PEG, which is injected subcutaneously or intramuscularly to achieve a depot formulation for controlled release.
[0447] In some embodiments, the pharmaceutical compositions may be sustained release formulations. In further embodiments, the sustained release formulations may be for subcutaneous delivery. Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EV Ac), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
[0448] As a non-limiting example, nucleic acid vaccine compositions may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the nucleic acid vaccine compositions in the PLGA microspheres while maintaining the integrity of the nucleic acid vaccine compositions during the encapsulation process. EV Ac are non-biodegradable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications. Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxy ethylene-polyoxypropylene-polyoxy ethylene having a low viscosity at temperatures less than 5°C and forms a solid gel at temperatures greater than 15°C. PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.
[0449] Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N- acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011, 12:2708-2714; Rozema et al., Proc Natl Acad Sci USA. 2007, 104: 12982-12887; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature ,2010 464: 1067-1070; the contents of each of which are herein incorporated by reference in their entirety).
[0450] The nucleic acid vaccine compositions of the disclosure may be formulated with or in a polymeric compound. The polymeric compound may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, poly acrylates, polymethacrylates, poly cyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine- containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof.
[0451] As a non-limiting example, the nucleic acid vaccine compositions of the disclosure may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its entirety. The formulation may be used for transfecting cells in vitro or for in vivo delivery of the nucleic acid vaccine compositions. In another example, the nucleic acid vaccine compositions may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. US20090042829 and US20090042825; the contents of each of which are herein incorporated by reference in their entirety.
[0452] As another non-limiting example, the nucleic acid vaccine compositions of the disclosure may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, herein incorporated by reference in their entireties) or PLGA-PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, herein incorporated by reference in its entirety). As a non-limiting example, the nucleic acid vaccine compositions of the disclosure may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety). [0453] In some embodiments, the nucleic acid vaccines compositions may be formulated with branched PEG molecules as described in or made by the methods described in PCT Patent Application Publication No. W020180126084; the contents of which are herein incorporated by reference in their entirety. As a non-limiting example, the branched PEG which may be used in the formulations described herein may have the formula I, formula II, formula III, formula IV, formula V, formula VI of PCT Publication No. WO20180126084, the contents of which are herein incorporated by reference in their entirety.
[0454] A polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. US20100260817; the contents of which are herein incorporated by reference in their entirety). As a non-limiting example, the nucleic acid vaccine compositions of the present disclosure may be formulated using the polyamine derivative described in U.S. Pub. No. US20100260817; the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, the nucleic acid vaccine compositions of the present disclosure may 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 dialkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; the contents of which are herein incorporated by reference in their entirety).
[0455] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be formulated with at least one polymer and/or derivatives thereof described in PCT Patent Application Publication Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No. US20120283427, the contents of each of which are herein incorporated by reference in their entireties. The nucleic acid vaccine compositions of the present disclosure may be formulated with a polymer of formula Z as described in WO2011115862; the contents of which are herein incorporated by reference in their entirety. The nucleic acid vaccine compositions may be formulated with a polymer of formula Z, Z’ or Z” as described in PCT Patent Application Publication Nos. WO2012082574 or WO2012068187 and U.S. Pub. No. US2012028342; the contents of each of which are herein incorporated by reference in their entireties. The polymers formulated with the nucleic acid vaccine compositions of the present disclosure may be synthesized by the methods described in PCT Patent Application Publication Nos. WO2012082574 or WO2012068187, the contents of each of which are herein incorporated by reference in their entireties.
[0456] The nucleic acid vaccine compositions of the disclosure may be formulated with at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), poly cyanoacrylates and combinations thereof.
[0457] Formulations of nucleic acid vaccine compositions of the disclosure may include at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof.
[0458] For example, the nucleic acid vaccine compositions of the disclosure may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof. The biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S. Pat. No. 6,696,038, and U.S. Pub. Nos. US20030073619 and US20040142474; the contents of each of which are herein incorporated by reference in their entireties. The poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. US20100004315, which is herein incorporated by reference in its entirety. The biodegradable polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos. 6,517,869 and 6,267,987, the contents of each of which are each incorporated herein by reference in their entirety. The linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886; the contents of which are each incorporated herein by reference in their entirety. The PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,217,912; the contents of which are herein incorporated by reference in their entirety. The PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co- glycolides). The biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. US2012009145; the contents of each of which are herein incorporated by reference in their entireties. For example, the multi -block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines. Further, the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. US20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912; the contents of each of which are herein incorporated by reference in their entireties.
[0459] The nucleic acid vaccine compositions of the disclosure may be formulated with at least one degradable polyester which may contain polycationic side chains. Degradable 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 degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[0460] The nucleic acid vaccine compositions of the disclosure may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in US Pub. No. US20120269761; the contents of which herein are incorporated by reference in their entirety. [0461] In some embodiments, the polymers described herein may be conjugated to a lipid-terminating PEG. As a non-limiting example, PLGA may be conjugated to a lipid- terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use with the present disclosure include those described in PCT Patent Application Publication No. W02008103276; the contents of which are herein incorporated by reference in their entirety. The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363; the contents of which are herein incorporated by reference in their entirety.
[0462] In some embodiments, the nucleic acid vaccine compositions described herein may be conjugated with another compound. Non-limiting examples of conjugates are described in US Pat. Nos. 7,964,578 and 7,833,992; the contents of each of which are herein incorporated by reference in their entireties. In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be conjugated with conjugates of formula 1-122 as described in US Pat. Nos. 7,964,578 and 7,833,992; the contents of each of which are herein incorporated by reference in their entireties. The nucleic acid vaccine compositions described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Journ. Amer. Chem. Soc. 2009, 131(6): 2072-2073; the contents of which are herein incorporated by reference in their entirety). In some embodiments, the nucleic acid vaccine compositions described herein may be conjugated and/or encapsulated in gold-nanoparticles (PCT Application Publication No.
WO201216269 and U.S. Pub. No. US20120302940; the contents of each of which are herein incorporated by reference in their entirety).
[0463] As described in U.S. Pub. No. US20100004313, a gene delivery composition may include a nucleotide sequence and a poloxamer. As a non-limiting example, the nucleic acid vaccine compositions of the present disclosure may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. US20100004313; the contents of which are each incorporated herein by reference in their entirety.
[0464] In some embodiments, the polymer formulations comprising the nucleic acid vaccines of the present disclosure may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. US20090042829; the contents of which are herein incorporated by reference in their entirety.
[0465] The cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycosidepolyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP), N- [l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 3B-[N — (N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1) diheptadecylamidoglycyl spermidine (DOGS), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N- dimethylammonium chloride DODAC) and combinations thereof.
[0466] In some embodiments, the nucleic acid vaccine compositions of the disclosure may be formulated in a polyplex of one or more polymers (U.S. Pub. Nos. US20120237565 and US20120270927; the contents of each of which are herein incorporated by reference in their entirety). In some embodiments, the polyplex comprises two or more cationic polymers. The cationic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
[0467] The nucleic acid vaccine compositions of the disclosure can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate. Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so delivery of the nucleic acid vaccine compositions may be enhanced (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 which are herein incorporated by reference in their entirety). As a non-limiting example, the nanoparticle 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 herein incorporated by reference in their entirety).
[0468] Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers may be used to deliver nucleic acid vaccine compositions in vivo. In some embodiments, a lipid coated calcium phosphate nanoparticle, which may also contain a targeting ligand such as anisamide, may be used to deliver the nucleic acid vaccine compositions of the present disclosure. For example, to effectively deliver siRNA in a mouse metastatic lung model a lipid coated calcium phosphate nanoparticle was used (Li et al., JContr 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 each of which are herein incorporated by reference in their entirety). This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
[0469] In some embodiments, calcium phosphate with a PEG-polyanion block copolymer may be used to delivery nucleic acid vaccine compositions of the disclosure (Kazikawa et al., J Contr Rel. 2004, 97:345-356; Kazikawa et al., J Contr Rel. 2006, 111 :368-370; the contents of each of which are herein incorporated by reference in their entirety).
[0470] In some embodiments, a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011, 32:3106-3114; the contents of which are herein incorporated by reference in their entirety) may be used to form a nanoparticle to deliver the nucleic acid vaccine compositions of the present disclosure. The PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
[0471] In some embodiments, a core-shell nanoparticle may be used to form a nanoparticle to deliver the nucleic acid vaccine compositions of the present disclosure. The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci U SA. 2011, 108: 12996-13001; the contents of which are herein incorporated by reference in their entirety). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver nucleic acid vaccine compositions to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
[0472] In some embodiments, the nanoparticles described herein may be nanoparticles which include 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 nanomolar binding affinities toward their targets, a peptide aptamer, an antibody, a small molecule ligand such as, but not limited to, folate, anisamide, and galactose. (Leng et al. Journal of Drug Delivery. 2017, 17, Article ID 6971297; the contents of which are herein incorporated by reference in their entirety).
[0473] In some embodiments, a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of the nucleic acid vaccine compositions of the present disclosure. As a non-limiting example, the lipid- polymer-lipid hybrid nanoparticle may be used to deliver the nucleic acid vaccine compositions described herein (Shi et al, Angew Chem Int Ed. 2011, 50:7027-7031; the contents of which are herein incorporated by reference in their entirety).
[0474] Core-shell nanoparticles for use with the nucleic acid vaccine compositions of the present disclosure may be formed by the methods described in U.S. Pat. No.
8,313,777; the contents of which are herein incorporated by reference in their entirety. [0475] In some embodiments, the core-shell nanoparticles may comprise a core of the nucleic acid vaccine compositions described herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the nucleic acid vaccine compositions in the core, (see, e.g., US Publication No. 20120321719; the contents of which are herein incorporated by reference in their entirety).
[0476] In some embodiments, the polymer used with the formulations described herein may be a modified polymer (such as, but not limited to, a modified polyacetal) as described in PCT Application Publication No. WO2011120053; the contents of which are herein incorporated by reference in their entirety.
[0477] In some embodiments, the nucleic acid vaccine compositions may be delivered to the cell or cytosol of a target cell by contacting the cell with a membrane-destabilizing polymer and a conjugate of the nucleic acid vaccine composition, a targeting ligand and an optional linker. Non-limiting examples of membrane-destabilizing polymers are taught in International PCT Application Publication No. W02020093061, the contents of which are herein incorporated by reference in their entirety, such as, but not limited to, the membrane-destabilizing polymers of formula XX therein.
Excipients
[0478] In some embodiments, pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, anti-oxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21" Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
[0479] In some embodiments, a 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, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[0480] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents.
[0481] 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, cornstarch, powdered sugar, etc., and/or combinations thereof.
[0482] Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cationexchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
[0483] Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chon- drux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, 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, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate (TWEEN®20), polyoxyethylene sorbitan (TWEEN®60), polyoxyethylene sorbitan monooleate (TWEEN®80), sorbitan monopalmitate (SPAN®40), sorbitan monostearate (SPAN®60), sorbitan tristearate (SPAN®65), glyceryl monooleate, sorbitan monooleate (SPAN®80)), polyoxyethylene esters (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, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof. [0484] Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch 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. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
[0485] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. In order to prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenyl ethyl 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, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®1 15, GERMAB EN®! !, NEOLONE™, KATHON™, and/or EUXYL®. [0486] In some embodiments, the pH of the pharmaceutical solutions are maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/or sodium malate. In another embodiment, the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions; however, these are not preferred due to complex formation and/or mRNA degradation.
[0487] Exemplary buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, etc., and/or combinations thereof. [0488] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof. [0489] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
[0490] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/ or perfuming agents can be present in the composition, according to the judgment of the formulator.
[0491] Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents can be used. [0492] In some embodiments of the present disclosure, the nucleic acid vaccine compositions described herein may comprise at least one nucleic acid vaccine that is formulated in a lipid nanoparticle (LNP) and at least one excipient. As non-limiting examples, the excipient may be a sugar such as sucrose.
Adjuvants
[0493] Adjuvants may also be administered with or in combination with one or more of the nucleic acid vaccines described herein, e.g., the mRNA vaccine. Adjuvants may be used to enhance the immunogenicity of the nucleic acid vaccine, modify the immune response, reduce the amount of nucleic acid vaccine needed for immunization, reduced the frequency of additional or “booster” immunizations needed or to create an improved immune response in those with weakened or immunocompromised immune systems or the elderly. The adjuvants may be a component of the formulation containing the nucleic acid vaccine or they may be co-administered with the nucleic acid vaccines compositions. Coadministration of the adjuvant may be any method known in the art or described herein such as, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC) or intradermal (ID).
[0494] In some embodiments, the adjuvant is natural or synthetic. The adjuvants may also be organic or inorganic.
[0495] In some embodiments, the adjuvant used with the nucleic acid vaccine is 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 emulsion, surfactant based emulsion, purified saponin, and oil-in water emulsion), inert vehicles, particulate adjuvants (e.g., unilamellar liposomal vehicles such as virosomes or a structured complex of saponions and lipids such as polylactide coglycolide (PLG)), microbial derivatives, endogenous human immunomodulators, and tensoactive compounds. Listings of adjuvants which may be used with the nucleic acid vaccines described herein may be found on the web-based vaccine adjuvant database Vaxjo (see e.g., violinet.org/vaxjo or Sayers et al., Journal of Biomedicine and Biotechnology. 2012; 2012: 831486. PMID: 22505817; the contents of which are herein incorporated by reference in their entirety).
[0496] Adjuvants may be selected for use with the nucleic acid vaccines by one of ordinary skill in the art. Adjuvants may be interferons, TNF-alpha, TNF-beta, chemokines (e.g., CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD40L, CD28 agonists, PD1, soluble PD1, PDL1, PDL2) or interleukins (e.g., IL1, IL2, IL4, IL6, IL7, IL10, IL12, IL13, IL15, IL17, IL18, IL21, and IL23). Nonlimiting examples of adjuvants include Abisco-100 vaccine adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Adjumer™, AF03, Albumin-heparin microparticles vaccine adjuvant, Algal Glucan, Algammulin, alhydrogel, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, Aluminum vaccine adjuvant, amorphous aluminum hydroxyphosphate sulfate adjuvant, Arlacel A, ASO, 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 Liposomal Vaccine Adjuvant, cationic liposome-DNA complex JVRS- 100, Cholera toxin, Cholera toxin B subunit, Corynebacterium-derived P40 Vaccine Adjuvant, CpGDNA Vaccine Adjuvant, CRL1OO5, CTA1-DD gene fusion protein, DDA Adjuvant, DHEA vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide)) vaccine adjuvant, DOC/ Alum Complex, E. coli heat-labile toxin, Etx B subunit Adjuvant, Flagellin, Freund’s Complete Adjuvant, Freund’s Incomplete Adjuvant, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Imiquimod, Immunoliposomes Containing Antibodies to Costimulatory Molecules, ISCOM(s)™, ISCOMA-TRIX®, Killed Cory neb acterium parvum Vaccine Adjuvant, Lipopolysaccharide, Liposomes, Loxoribine, LTK63 Vaccine Mutant Adjuvant, LTK72 vaccine adjuvant, LTR192G Vaccine Adjuvant, Matrix-S, MF59, Montanide Incomplete Seppic Adjuvant, Montanide ISA 51, Montanide ISA 720 Adjuvant, MPL-SE vaccine adjuvant, MPL™ Adjuvant, MTP-PE Liposomes, Murametide, Muramyl Dipeptide Adjuvant, Murapalmitine, D-Murapalmitine, NAGO, nanoemulsion vaccine adjuvant, Non-Ionic Surfactant Vesicles, non-toxic mutant El 12K of Cholera Toxin mCT-El 12K, PMMA, Poly(LC), Polygen Vaccine Adjuvant, Protein Cochleates, QS-21, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, Recombinant hlFN-gamma/Interferon-g, Rehydragel EV, Rehydragel HP A, Resiquimod, Ribi Vaccine Adjuvant, SAF-1, Saponin Vaccine Adjuvant, Sclavo peptide, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Specol, SPT (Antigen Formulation), Squalene-based Adjuvants, Stearyl Tyrosine, Theramide®, Threonyl muramyl dipeptide (TMDP), Titer- Max Gold Adjuvant, Ty Particles vaccine adjuvant, and VSA-3 Adjuvant.
[0497] In some embodiments, the nucleic acid vaccines described herein may be used as a vaccine and may further comprise an adjuvant which may enable the vaccine to elicit a higher immune response. As a non-limiting example, the adjuvant could be a sub-micron oil-in-water emulsion which can elicit a higher immune response in human pediatric populations (see e.g., the adjuvanted vaccines described in US Patent Publication No.
US20120027813 and U.S. Pat. No. 8,506,966, the contents of each of which are herein incorporated by reference in their entirety).
Dosing and Administration
[0498] The present disclosure encompasses the delivery of nucleic acid vaccine compositions including, for example, nucleic acid vaccine for COVID-19 for any therapeutic, prophylactic, pharmaceutical, diagnostic or imaging use by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
[0499] The nucleic acid vaccine compositions of the present disclosure may be delivered to a cell naked. As used herein in, “naked” refers to delivering nucleic acid vaccine compositions free from agents which promote transfection. For example, the nucleic acid vaccine compositions delivered to the cell may contain no modifications. The naked nucleic acid vaccine compositions may be delivered to the cell using routes of administration known in the art and described herein.
[0500] The nucleic acid vaccine compositions of the present disclosure may be formulated, using the formulation components and methods described herein. The formulations may contain nucleic acid vaccine compositions which may be modified and/or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulated nucleic acid vaccine compositions may be delivered to the cell using routes of administration known in the art and described herein.
[0501] The nucleic acid vaccine compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like. The nucleic acid vaccine compositions of the present disclosure may also be cloned into a retroviral replicating vector (RRV) and transduced to cells.
[0502] The pharmaceutical composition may be packaged as a percentage of fill of the capacity of a glass vial. As a non-limiting example, the fill may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% of the capacity of the glass vial. In some embodiments, the pharmaceutical composition may be packaged as a 2.4 mL fill in a 6 mL glass vial. In some embodiments, the pharmaceutical composition may be packaged as a 2 mL fill in a 3 mL glass vial.
Dosing
[0503] Provided herein also include methods comprising administering the nucleic acid vaccines described herein to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, health, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[0504] The present disclosure contemplates dosage levels of between about 0.001 and about 500 mg nucleic acid vaccine (e.g., nucleic acid vaccine for COVID-19, e.g., mRNA vaccine for COVID-19)/kg body weight per day, about 0.001 and about 200 mg/kg, about 0.001 and about 100 mg/kg, 0.01 and about 100 mg/kg, preferably between about 0.005 and about 50 mg/kg, 0.01 and about 50 mg/kg, 0.01 and about 40 mg/kg, 0.01 and about 30 mg/kg, 0.01 and about 10 mg/kg, 0.05 and about 50 mg/kg, 0.05 and about 30 mg/kg, 0.05 and about 10 mg/kg, 0.05 and about 5 mg/kg, 0.1 and about 50 mg/kg, 0.1 and about 30 mg/kg, 0.1 and about 10 mg/kg, 0.1 and about 1 mg/kg, 1.0 and about 50 mg/kg, 1.0 and about 40 mg/kg, 1.0 to about 30 mg/kg, 10 to about 50mg/kg body weight. Other embodiments contemplate a dosage of between 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, 10-100mg/kg body weight. The dosages may be administered about hourly, multiple times per day, daily, every other day, weekly, every other week, monthly, every other month, or on an as-needed basis.
[0505] In some embodiments, compositions of the nucleic acid vaccines may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 1 mg/kg to about 50 mg/kg, from about 10 mg/kg to about 100 mg/kg, from about 10 mg/kg to about 50 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage 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 multiple administrations are employed, split dosing regimens such as those described herein may be used.
[0506] In some embodiments, compositions of the nucleic acid vaccines described herein may be administered at dosage levels sufficient to deliver to a subject, about 1 pg, lOpg. 15pg, 20pg, 25pg, 30pg, 35pg, 40pg, 50pg , 60pg, 70pg, 80pg, 90pg, or lOOpg of the nucleic acid composition.
[0507] In some embodiments, the nucleic acid vaccines may be administered in splitdose regimens. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose. In some embodiments, the nucleic acid vaccines described herein are administered to a subject in split doses. The nucleic acid vaccines may be formulated in buffer only or in a formulation described herein.
[0508] In some embodiments, the nucleic acid vaccine compositions described herein may be administered to a subject in two separate phases, a loading dosing phase and a maintenance dosing phase. The dosing regimen may comprise an initial higher loading dose of the nucleic acid vaccine that is given to the subject first time at the beginning of a course of prevention, alleviation and/or treatment, e.g., first dose for preventing COVID- 19, and a lower maintenance dose following the first loading dose. In some embodiments, the loading dose and the maintenance dose have the same amount of the nucleic acid vaccines of the present disclosure. In some embodiments, more than one maintenance doses are administered to the subject. The multiple maintenance doses may be administered biweekly, every three weeks, every four weeks, monthly, bimonthly, every three months, every four months, every five months, or every six months. In the context of vaccination for prevention of a disorder, e.g., the nucleic acid (e.g., mRNA) vaccine for CO VID-19, the maintenance doses of the nucleic acid vaccines may also be referred to as booster doses. As used herein, a “booster dose” (or “booster shot) is an extra or supplemental dose of a vaccine after an initial primer dose. The booster dose may have the same amount of the nucleic acid vaccine as the initial loading dose. Alternatively, the booster dose has an amount of the nucleic acid vaccine that is smaller than the original amount of the nucleic acid vaccine in the initial dose. In some embodiments, the subject may receive one, two, three, four or more booster doses.
[0509] Such administration can be used as a chronic or acute treatment or prevention of a clinic-concerning condition. The amount of drug that may be combined with the carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80%, 30% to about 70%, 40% to about 60%, or about 50% active compound. In other embodiments, the preparations used in the present disclosure 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.
[0510] Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of the present disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
[0511] As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, gender, diet, time of administration, rate of excretion, drug combination, the severity and course of an infection, the patient's disposition to the infection and the judgment of the treating physician.
Delivery
[0512] In some embodiments, the delivery of the nucleic acid vaccines may be naked or formulated.
[0513] In some embodiments, the nucleic acid vaccines described herein may be delivered to a cell naked. As used herein in, “naked” refers to delivering nucleic acid vaccines free from agents which promote transfection. For example, the nucleic acid vaccines delivered to the cell may contain no modifications. The naked nucleic acid vaccines may be delivered to the cell using routes of administration known in the art and described herein.
[0514] In some embodiments, the nucleic acid vaccines described herein may be formulated, using the methods described herein. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulated nucleic acid vaccines may be delivered to the cell using routes of administration known in the art and described herein.
[0515] The compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
Administration
[0516] In some embodiments, the nucleic acid vaccine compositions of the present disclosure may be administered by any route which results in a prophylactic or therapeutically effective outcome. These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electroosmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracistemal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique, ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. In specific embodiments, compositions may be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
[0517] Delivery of the nucleic acid vaccines described herein to a subject over prolonged periods of time, for example, for periods of one week to one year, may be accomplished by a single administration of a controlled release system containing sufficient active ingredient for the desired release period. Various controlled release systems, such as monolithic or reservoir-type microcapsules, depot implants, polymeric hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms may be utilized for this purpose. Localization at the site to which delivery of the active ingredient is desired is an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders. [0518] In some embodiments, the nucleic acid vaccines described herein may be administered intranasally similar to the administration of live vaccines. In another aspect the polynucleotide may be administered intramuscularly or intradermally similarly to the administration of inactivated vaccines known in the art.
[0519] In certain embodiments for transdermal administration, delivery across the barrier of the skin would be enhanced using electrodes (e.g., iontophoresis), electroporation, or the application of short, high-voltage electrical pulses to the skin, radiofrequencies, ultrasound (e.g. sonophoresis), microprojections (e.g. microneedles), jet injectors, thermal ablation, magnetophoresis, lasers, velocity, or photomechanical waves. The drug can be included in single-layer drug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, or vapor style patches, or could utilize patchless technology. Delivery across the barrier of the skin could also be enhanced using encapsulation, a skin lipid fluidizer, or a hollow or solid microstructured transdermal system (MTS, such as that manufactured by 3M), jet injectors. Additives to the formulation to aid in the passage of therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell penetrating peptides, permeation enhancers, encapsulation technologies, enzymes, enzyme inhibitors, gels, nanoparticles and peptide or protein chaperones.
[0520] Additional slow release, depot implant or injectable formulations will be apparent to the skilled artisan. 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 are incorporated by reference in their entirety.
[0521] Mixing of the nucleic acid vaccines described herein with a polymeric formulation comprising biodegradable polymers that can form a depot formulation upon administration, is suitable to achieve very long duration of action formulations.
[0522] When formulated for nasal administration, the absorption across the nasal mucous membrane may be further enhanced by surfactants, such as, for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cycledextrins and the like in an amount in the range of between about 0.1 and 15 weight percent, between about 0.5 and 4 weight percent, or about 2 weight percent. An additional class of absorption enhancers reported to exhibit greater efficacy with decreased irritation is the class of alkyl maltosides, such as tetradecylmaltoside (Arnold, JJ et al., J Pharm Set, 2004, 93: 2205-13; Ahsan, F et al., Pharm Res, 2001,18: 1742-46) and references therein, all of which are hereby incorporated by reference in their entirety.
[0523] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol. [0524] The pharmaceutical compositions of the present disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. [0525] The pharmaceutical compositions of present disclosure may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the active ingredient the present disclosure with a suitable nonirritating excipient that is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
[0526] Topical administration of the pharmaceutical compositions of the present disclosure is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the present disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the present disclosure may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included in the present disclosure.
[0527] The pharmaceutical compositions of the present disclosure may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
[0528] When formulated for delivery by inhalation, a number of formulations offer advantages. Adsorption of the therapeutic agents to readily dispersed solids such as diketopiperazines (for example, Technosphere particles (Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv 2: 1097-1106) or similar structures gives a formulation that results in rapid initial uptake of the therapeutic compound. Lyophilized powders, especially glassy particles, containing the therapeutic compound and an excipient are useful for delivery to the lung with good bioavailability, for example, see Exubera® (inhaled insulin, Pfizer, Inc. and Aventis Pharmaceuticals Inc.) and Afrezza® (inhaled insulin, Mannkind, Corp.).
Dosage Forms
[0529] A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
Liquid Dosage Forms
[0530] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMO- PHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Injectable forms
[0531] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
[0532] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0533] In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the nucleic acid vaccine then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered nucleic acid vaccine may be accomplished by dissolving or suspending the nucleic acid vaccine in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the nucleic acid vaccine in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of nucleic acid vaccine to polymer and the nature of the particular polymer employed, the rate of polynucleotides release can be controlled.
Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the nucleic acid vaccine in liposomes or microemulsions which are compatible with body tissues.
Pulmonary
[0534] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, e.g., by rapid inhalation through the nasal passage from a container of the powder held close to the nose. [0535] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
[0536] General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.
Solid Dosage Forms: Coatings or Shells
[0537] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
Properties of the Pharmaceutical Compositions
[0538] The nucleic acid vaccine pharmaceutical compositions described herein may be characterized using one or more of bioavailability, therapeutic window, volume of distribution, biological effect and detection of polynucleotides by mass spectrometry. Bioavailability
[0539] The nucleic acid vaccines, when formulated into a composition with a delivery agent as described herein, can exhibit an increase in bioavailability as compared to a composition lacking a delivery agent as described herein. As used herein, the term “bioavailability” refers to the systemic availability of a given amount of nucleic acid vaccines administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration of the unchanged form of a compound following administration of the compound to a mammal. AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis). Generally, the AUC for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modem Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, N.Y, Inc., 1996, herein incorporated by reference in its entirety.
[0540] The Cmax value is the maximum concentration of the compound achieved in the serum or plasma of a mammal following administration of the compound to the mammal. The Cmax value of a particular compound can be measured using methods known to those of ordinary skill in the art. The phrases “increasing bioavailability” or “improving the pharmacokinetics,” as used herein mean that the systemic availability of a first nucleic acid vaccine, measured as AUC, Cmax, or Cmin, in a mammal is greater, when co-administered with a delivery agent as described herein, than when such coadministration does not take place. In some embodiments, the bioavailability of the nucleic acid vaccines can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
[0541] In some embodiments, liquid formulations of nucleic acid vaccines may have various in vivo half-life, requiring modulation of doses to yield a prophylactic or therapeutic effect. To address this, in some embodiments, nucleic acid vaccine formulations may be designed to improve bioavailability and/or prophylactic or therapeutic effect during repeat administrations. Such formulations may enable sustained release of nucleic acid vaccines and/or reduce nucleic acid vaccine degradation rates by nucleases. In some embodiments, suspension formulations are provided comprising nucleic acid vaccines, water immiscible oil depots, surfactants and/or co-surfactants and/or co-solvents. Combinations of oils and surfactants may enable suspension formulation with nucleic acid vaccines. Delivery of nucleic acid vaccines in a water immiscible depot may be used to improve bioavailability through sustained release of polynucleotides from the depot to the surrounding physiologic environment and/or prevent polynucleotide degradation by nucleases.
[0542] In some embodiments, cationic nanoparticles comprising combinations of divalent and monovalent cations may be formulated with nucleic acid vaccines. Such nanoparticles may form spontaneously in solution over a given period (e.g. hours, days, etc.). Such nanoparticles do not form in the presence of divalent cations alone or in the presence of monovalent cations alone. The delivery of nucleic acid vaccines in cationic nanoparticles or in one or more depot comprising cationic nanoparticles may improve nucleic acid vaccine bioavailability by acting as a long-acting depot and/or reducing the rate of degradation by nucleases.
Therapeutic Window
[0543] The nucleic acid vaccines, when formulated into a composition with a delivery agent as described herein, can exhibit an increase in the therapeutic window of the administered nucleic acid vaccine composition as compared to the therapeutic window of the administered nucleic acid vaccine composition lacking a delivery agent as described herein. As used herein “therapeutic window” refers to the range of plasma concentrations, or the range of levels of therapeutically active substance at the site of action, with a high probability of eliciting a prophylactic or therapeutic effect. In some embodiments, the therapeutic window of the nucleic acid vaccines when co-administered with a delivery agent as described herein can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
Volume of Distribution
[0544] The nucleic acid vaccines, when formulated into a composition with a delivery agent as described herein, can exhibit an improved volume of distribution (Vdist), e.g., reduced or targeted, relative to a composition lacking a delivery agent as described herein. The volume of distribution (Vdist) relates the amount of the drug (e.g., nucleic acid vaccine of the present disclosure) in the body to the concentration of the drug in the blood or plasma. As used herein, the term “volume of distribution” refers to the fluid volume that would be required to contain the total amount of the drug in the body at the same concentration as in the blood or plasma: Vdist equals the amount of drug in the body/concentration of drug in blood or plasma. For example, for a 10 mg dose and a plasma concentration of 10 mg/L, the volume of distribution would be 1 liter. The volume of distribution reflects the extent to which the drug is present in the extravascular tissue. A large volume of distribution reflects the tendency of a compound to bind to the tissue components compared with plasma protein binding. In a clinical setting, Vdist can be used to determine a loading dose to achieve a steady state concentration. In some embodiments, the volume of distribution of the nucleic acid vaccines when co-administered with a delivery agent as described herein can decrease at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
Biological Effect
[0545] In some embodiments, the biological effect of the nucleic acid vaccine delivered to the animals may be categorized by analyzing the protein expression in the animals. The protein expression may be determined from analyzing a biological sample collected from a mammal administered the nucleic acid vaccine described herein.
Detection of Polynucleotides by Mass Spectrometry
[0546] Mass spectrometry (MS) is an analytical technique that can provide structural and molecular mass/concentration information on molecules after their conversion to ions. The molecules are first ionized to acquire positive or negative charges and then they travel through the mass analyzer to arrive at different areas of the detector according to their mass/charge (m/z) ratio.
[0547] Mass spectrometry is performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example, ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. The skilled artisan will understand that the choice of ionization method can be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.
[0548] After the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., m/z). Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers. The ions may be detected using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).
[0549] Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupled with stable isotope labeled dilution of peptide standards has been shown to be an effective method for protein verification (e.g., Keshishian et al., Mol Cell Proteomics, 2009, 8: 2339-2349; Kuhn et al., Clin Chem 2009, 55: 1108-1117; Lopez et al., Clin Chem, 2010, 56:281- 290; the contents of each of which are herein incorporated by reference in their entirety). Unlike untargeted mass spectrometry frequently used in biomarker discovery studies, targeted MS methods are peptide sequence-based modes of MS that focus the full analytical capacity of the instrument on tens to hundreds of selected peptides in a complex mixture. By restricting detection and fragmentation to only those peptides derived from proteins of interest, sensitivity and reproducibility are improved dramatically compared to discovery -mode MS methods. This method of mass spectrometry based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples.
[0550] In some embodiments, the biological sample, once obtained from the subject, may be subjected to enzyme digestion. As used herein, the term “digest” means to break apart into shorter peptides. As used herein, the phrase “treating a sample to digest proteins” means manipulating a sample in such a way as to break down proteins in a sample. These enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin.
[0551] In some embodiments, a biological sample may be analyzed for protein using electrospray ionization. Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to aid in the transfer of ions from the solution to the gaseous phase before they are analyzed by mass spectrometry. Samples may be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003, 24(1):3-12; herein incorporated by reference in its entirety). The ionic species contained in solution may be transferred into the gas phase by dispersing a fine spray of charge droplets, evaporating the solvent and ejecting the ions from the charged droplets to generate a mist of highly charged droplets. The mist of highly charged droplets may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers such as, but not limited to, a quadropole mass analyzer. Further, the mass spectrometry method may include a purification step. As a non-limiting example, the first quadrapole may be set to select a single m/z ratio so it may filter out other molecular ions having a different m/z ratio which may eliminate complicated and time-consuming sample purification procedures prior to MS analysis.
[0552] In some embodiments, a biological sample may be analyzed for protein in a tandem ESIMS system (e.g., MS/MS). As non-limiting examples, the droplets may be analyzed using a product scan (or daughter scan) a precursor scan (parent scan) a neutral loss or a multiple reaction monitoring.
[0553] In some embodiments, a biological sample may be analyzed using matrix- assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides for the nondestructive vaporization and ionization of both large and small molecules, such as proteins. In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, an ultraviolet absorbing weak organic acid. Non-limiting examples of matrices used in MALDI are a-cyano-4-hy- droxy cinnamic acid, 3, 5 -dimethoxy -4-hydroxy cinnamic acid and 2,5-dihydroxybenzoic acid. Laser radiation of the analyte-matrix mixture may result in the vaporization of the matrix and the analyte. The laser induced desorption provides high ion yields of the intact analyte and allows for measurement of compounds with high accuracy. Samples may be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; the contents of which are herein incorporated by reference in their entirety). As non-limiting examples, mass analyzers used in the MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or a Fourier transform mass analyzer.
Expression Systems [0554] In some embodiments, nucleic acid vaccines described herein may be operably linked to one or more regulatory nucleotide sequences and encoded in an expression construct. Regulatory nucleotide sequences will generally be appropriate for a host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, transcriptional start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow the selection of transformed host cells. Certain embodiments include an expression vector encoding a nucleic acid vaccine for COVID-19 sequence operably linked to at least one regulatory sequence. Regulatory sequences for use herein include promoters, enhancers, and other expression control elements. In certain embodiments, an expression vector is designed considering the choice of the host cell to be transformed, the particular nucleic acid vaccine sequence to be expressed, the vector's copy number, the ability to control that copy number, or the expression of other proteins encoded by the vector, such as antibiotic markers.
[0555] In some embodiments, the nucleic acids described herein may be expressed in microorganisms. As a non-limiting example, the nucleic acid may be expressed in a bacterial system, for example, in Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Caulobacter crescentus, Escherichia coli and their derivatives. Exemplary promoters include the 1-arabinose inducible araBAD promoter (PBAD), the lac promoter, the 1- rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter Pl, and the anhydrotetracycline-inducible tetA promoter/operator. [0556] In some embodiments, the nucleic acids described herein may be expressed in a yeast expression system. Non-limiting examples of promoters which may be used in yeast vectors include the promoters for 3 -phosphoglycerate kinase (Hitzeman et al., J. Biol.
Chem. 255:2073 (1980)); other glycolytic enzymes (Hess et al., J. Adv. Enzyme Res. 7: 149 (1968); Holland et al., Biochemistry 17:4900 (1978). Others promoters are from, e.g., enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase alcohol oxidase I (A0X1), alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter and termination sequences, with or without an origin of replication, is suitable. Certain yeast expression systems are commercially available, for example, from Clontech Laboratories, Inc. (Palo Alto, Calif., e.g., Pyex 4T family of vectors for S. cerevisiae), Invitrogen (Carlsbad, Calif., e.g. Ppicz series Easy Select Pichia Expression Kit) and Stratagene (La Jolla, Calif., e.g. ESP.TM. Yeast Protein Expression and Purification System for S. pombe and Pesc vectors for S. cerevisiae).
[0557] In some embodiments, the nucleic acids described herein may be expressed in mammalian expression systems. Non-limiting examples of mammalian promoters include, for example, promoters from the following genes: ubiquitin/S27a promoter of the hamster (WO 97/15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are the actin, immunoglobulin or heat shock promoter(s). In a specific embodiment, a yeast alcohol oxidase promoter is used. [0558] In some embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers et al., Nature 273: 113-120 (1978). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. Greenaway, P. J. et al., Gene 18: 355-360 (1982). The foregoing references are incorporated by reference in their entirety.
[0559] In some embodiments, the nucleic acids described herein may be expressed in insect cell expression systems. Eukaryotic expression systems employing insect cell hosts may rely on either plasmid or baculoviral expression systems. Typical insect host cells are derived from the fall army worm (Spodoptera frugiperdct). For expression of a foreign protein these cells are infected with a recombinant form of the baculovirus Autographa californica nuclear polyhedrosis virus which has the gene of interest expressed under the control of the viral polyhedron promoter. Other insects infected by this virus include a cell line known commercially as "High 5" (Invitrogen) which is derived from the cabbage looper (Trichoplusia ni). Another baculovirus sometimes used is the Bombyx mori nuclear polyhedorsis virus which infect the silkworm (Bombyx mori). Numerous baculovirus expression systems are commercially available, for example, from Thermo Fisher (Bac-N- Blue™k or BAC-TO-BAC™ Systems), Clontech (BacPAK™ Baculovirus Expression System), Novagen (Bac Vector System™), or others from Pharmingen or Quantum Biotechnologies. Another insect cell host is the common fruit fly, Drosophda melanogaster, for which a transient or stable plasmid-based transfection kit is offered commercially by Thermo Fisher (The DES™ System).
[0560] In some embodiments, cells are transformed with vectors that express a nucleic acid described herein. Transformation techniques for inserting new genetic material into eukaryotic cells, including animal and plant cells, are well known. Viral vectors may be used for inserting expression cassettes into host cell genomes. Alternatively, the vectors may be transfected into the host cells. Transfection may be accomplished by methods as described in the art such as, but not limited to, calcium phosphate precipitation, electroporation, optical transfection, protoplast fusion, impalefection, and hydrodynamic delivery.
IV. METHODS OF USE
[0561] One aspect of the present disclosure provides methods of using nucleic acid vaccines of the present disclosure and pharmaceutical compositions and formulations comprising the nucleic acid vaccines and at least one pharmaceutically acceptable carrier. Provided herein are compositions, methods, kits, and reagents for diagnosis, treatment, alleviation or prevention of a disease or condition in humans or other mammals where the active agent is the nucleic acid vaccine, cells containing the nucleic acid vaccine or polypeptides translated from nucleic acid vaccine polynucleotides.
[0562] In some embodiments, the methods of use can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of disease progression, including stabilization, slowing down and complete arrest;
(2) reduction in the number of disease episodes and/or symptoms; (3) inhibition (z.e., reduction, slowing down or complete stopping) of a disease cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e. reduction, slowing down or complete stopping) of disease spread; (5) decrease of an autoimmune condition; (6) favorable change in the expression of a biomarker associated with the disorder; (7) relief, to some extent, of one or more symptoms associated with a disorder; (8) increase in the length of disease-free presentation following treatment; or (9) decreased mortality at a given point of time following treatment.
Therapeutic or Prophylactic Uses
[0563] The nucleic acid vaccines described herein may be used to protect, treat or cure infection arising from contact with an infectious agent such as, but not limited to, viruses, bacteria, fungi, parasites and protozoa. As a non-limiting example, the infectious agent is a virus and the virus is SARS-CoV-2 and/or a variant thereof. In some embodiments, the variants of SARS-CoV-2 are VOI, VOC and VOHC variants.
[0564] The nucleic acid vaccines described herein may be used as prophylactic agents where the nucleic acid vaccines are administered to a subject, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the prevention of COVID-19.
[0565] The nucleic acid vaccines described herein may be used as therapeutic agents where the nucleic acid vaccines are administered to a subject, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the alleviation of one or more symptoms of COVID-19.
[0566] In some embodiments, provided are methods for treating or preventing a viral infection and/or a disease, disorder, or condition associate with a viral infection or a symptom thereof, in a subject, by administering a nucleic acid vaccine comprising one or more polynucleotides encoding a viral polypeptide. The administration may be in combination with an anti-viral or anti-bacterial agent or a small molecule compound described herein or known in the art.
[0567] In some embodiments, the nucleic acid vaccines described herein may be used to protect against and/or prevent the transmission of an emerging or engineered threat which may be known or unknown.
[0568] In some embodiments, provided herein are methods of inducing translation of a polypeptide (e.g., one or more proteins, peptides, fragments or variants thereof of SARS- CoV-2 in a cell, tissue or organism using the nucleic acid polynucleotides described herein. The translated polypeptide may be used for the prevention, alleviation and/or treatment of COVID-19. Such translation can be in vitro, in vivo, ex vivo, or in culture. The cell, tissue or organism may be contacted with an effective amount of a composition or pharmaceutical composition containing the nucleic acid vaccine which includes a polynucleotide with at least one region encoding the polypeptide of interest (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or prevention of CO VID-19.
[0569] In some embodiments, the effective amount of the nucleic acid vaccine described herein provided to a cell, a tissue or a subject may be enough for immune prophylaxis.
[0570] An “effective amount” of the composition of the nucleic acid vaccine is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and the number of unmodified and modified nucleosides) and other components of the nucleic acid vaccine. An effective amount of the composition containing the nucleic acid vaccine described herein is one that provides an induced or boosted immune response as a function of production in the cell of one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 as compared to an untreated cell. Increased production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid vaccine), increased protein translation from the polynucleotide or altered innate immune response of the host cell.
[0571] Provided herein are directed to methods of inducing in vivo translation of one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 in a mammalian subject in need thereof. An effective amount of a nucleic acid vaccine composition containing a polynucleotide that has at least one translatable region encoding the polypeptide (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2) is administered to the subject using the delivery methods described herein. The polynucleotide is provided in an amount and under other conditions such that the polynucleotide is translated in the cell. The cell in which the polynucleotide is localized, or the tissue in which the cell is present, may be targeted with one or more rounds of nucleic acid vaccine administration.
[0572] In certain embodiments, the administered nucleic acid vaccine comprising polynucleotides directs production of one or more polypeptides that provide a functional immune system-related activity which is substantially absent in the cell, tissue or organism n in which the polypeptide is translated. For example, the missing functional activity may be enzymatic, structural, or gene regulatory in nature. In related embodiments, the administered polynucleotides direct production of one or more polypeptides that increases a functional activity related to the immune system which is present but substantially deficient in the cell in which the polypeptide is translated.
[0573] Additionally, the polypeptide translated from the nucleic acid vaccine may antagonize, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell. Non-limiting examples of biological moi eties that may be antagonized include a nucleic acid, a carbohydrate, a protein toxin such as shiga and tetanus toxins, lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), or a small molecule toxin (e.g., cholera, botulinum, and diphtheria toxins). In some embodiments, the biological molecule which may be antagonized may be an endogenous protein that may have an undesirable activity such as, but not limited to, cytotoxic or cytostatic activity. The proteins described herein may be engineered for localization within the cell, potentially within a specific compartment such as the cytoplasm or nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
[0574] In some embodiments, the polynucleotides of the nucleic acid vaccines and their encoded polypeptides may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to viral infections (e.g., infections caused by SARS-CoV-2 and/or a variant thereof).
[0575] The subject to whom the nucleic acid vaccine may be administered suffers from or may be at risk of developing a disease, disorder, or deleterious condition. Provided are methods of identifying, diagnosing, and classifying subjects on these bases, which may include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.
[0576] The agents (e.g., compositions of nucleic acid vaccines and any additional moieties) can be administered simultaneously, for example in a combined unit dose (e.g., providing simultaneous delivery of both agents). The agents can also be administered at a specified time interval, such as, but not limited to, an interval of minutes, hours, days or weeks. Generally, the agents may be concurrently bioavailable, e.g., detectable, in the subject. In some embodiments, the agents may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In other embodiments, the agents may be delivered in separate unit dosages. The agents may be administered in any order, or as one or more preparations that includes two or more agents. In a preferred embodiment, at least one administration of one of the agents, e.g., the first agent, may be made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent. In some embodiments, combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than additive results. [0577] In some embodiments, the nucleic acid vaccine described herein may be administrated with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 21 days, 28 days , 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years.
[0578] In some embodiments, administration of a booster (or booster vaccine) may result in an increase in antibody titer at least 3 weeks after the administration of the booster as compared to antibody titer levels prior to the administration of the booster. The increase may be seen, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or greater than 8 weeks after administration of the booster. The difference in titer level after administration of the booster as compared to before the booster may be, but is not limited to at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11- fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80- fold, 85-fold, 90-fold, 95-fold, or 100-fold, or about 1 to 5-fold, about 5 to 8-fold, about 5 to 10-fold, about 5 to 20-fold, about 5 to 30-fold, about 5 to 40-fold, about 5 to 50-fold, about 5 to 60-fold, about 10 to 20-fold, about 10 to 30-fold, about 10 to 40-fold, about 10 to 50-fold, about 10 to 60-fold, about 20 to 30-fold, about 20 to 40-fold, about 20 to 50- fold, about 20 to 60-fold, about 30 to 40-fold, about 30 to 50-fold, about 30 to 60-fold, about 40 to 50-fold, about 40 to 60-fold, about 50 to 60-fold, about 50 to 75-fold, about 50 to 100-fold, about 50 to 125-fold, about 50 to 150-fold, about 50 to 175-fold, about 50 to
200-fold, about 50 to 225-fold, about 50 to 300-fold, about 50 to 375-fold, about 75 to
100-fold, about 75 to 125-fold, about 75 to 150-fold, about 75 to 175-fold, about 75 to
200-fold, about 75 to 225-fold, about 75 to 300-fold, about 75 to 375-fold, about 100 to
125-fold, about 100 to 150-fold, about 100 to 175-fold, about 100 to 200-fold, about 100 to 225-fold, about 100 to 300-fold, about 100 to 375-fold, about 125 to 150-fold, about 125 to 175-fold, about 125 to 200-fold, about 125 to 225-fold, about 125 to 300-fold, about 125 to 375-fold, about 150 to 175-fold, about 150 to 200-fold, about 150 to 225- fold, about 150 to 300-fold, about 150 to 375-fold, about 175 to 200-fold, about 175 to 225-fold, about 175 to 300-fold, about 175 to 375-fold, about 200 to 225-fold, about 200 to 300-fold, about 200 to 375-fold, about 225 to 300-fold, about 225 to 375-fold, about 300 to 375-fold, about 100 to 350-fold, about 125 to 350-fold, about 150 to 350-fold, about 175 to 350-fold, about 200 to 350-fold, about 225 to 350-fold, about 250 to 350- fold, about 275 to 350-fold, about 300 to 350-fold, about 325 to 350-fold, about 350 to 375-fold, or more than 375-fold increase.
[0579] In some embodiments, the nucleic acid vaccines may be formulated by the methods described herein. In one aspect, the formulation may comprise a nucleic acid vaccine or polynucleotide which can have a therapeutic and/or prophylactic effect on more than one disease, disorder or condition. As a non-limiting example, the formulation may comprise polynucleotides encoding one or more proteins, polypeptide, peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-19.
[0580] In some embodiments, the nucleic acid vaccines described herein may be used for research in many applications, such as, but not limited to, identifying and locating intracellular and extracellular proteins, protein interaction, signal pathways and cell biology.
Modulation of the Immune Response
[0581] In some embodiments, the nucleic acid vaccines comprising the polynucleotides described herein may act as a single composition as a vaccine. As used herein, a “vaccine” refers to a composition, a substance or preparation that stimulates, induces, causes or improves immunity in an organism, e.g., an animal organism, for example, a mammalian organism (e.g., a human). Preferably, a vaccine provides immunity against one or more diseases or disorders in the organism, including prophylactic and/or therapeutic immunity. Exemplary vaccines include one or more agents that resembles an infectious agent, e.g., a disease-causing microorganism, and can be made, for example, from live, attenuated, modified, weakened or killed forms of disease-causing microorganisms, or antigens derived therefrom, including combinations of antigenic components. In exemplary embodiments, a vaccine stimulates, induces causes or improves immunity in an organism or causes or mimics infection in the organism without inducing any disease or disorder. A vaccine introduces an antigen into the tissues, extracellular space or cells of a subject and elicits an immune response, thereby protecting the subject from a particular disease or pathogen infection. The nucleic acid vaccines described herein may encode an antigen and when the polynucleotides are expressed in cells, a desired immune response is achieved. As a non-limiting example, the nucleic acid vaccines described herein may encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 and when the polynucleotides are expressed in cells, a desired immune response against SARS-CoV-2 is achieved to treat and/or prevent COVID-19.
[0582] Nucleic acid vaccines may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms.
[0583] The nucleic acid vaccines described herein may also be administered as a second line treatment after the standard first line treatments such as antibiotics and antivirals have failed to induce passive immunity. In this regard, the nucleic acid vaccines described herein are useful in settings where resistance to first line treatments has developed and disease persists and induces chronic disease.
[0584] Nucleic acid vaccines may be administered as part of a treatment regimen for latent viral infections, such as SARS-CoV-2 infections. In this embodiment, one or more polynucleotides are administered which ultimately produce proteins which result a desired immune response against SARS-CoV-2 is achieved to treat and/or prevent COVID-19. [0585] The use of RNA in or as a vaccine overcomes the disadvantages of conventional genetic vaccination involving incorporating DNA into cells in terms of safeness, feasibility, applicability, and effectiveness to generate immune responses. RNA molecules are considered to be significantly safer than DNA vaccines, as RNAs are more easily degraded. They are cleared quickly out of the organism and cannot integrate into the genome and influence the cell’s gene expression in an uncontrollable manner. It is also less likely for RNA vaccines to cause severe side effects like the generation of autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), vol. 2010, article ID623687). Transfection with RNA requires only insertion into the cell’s cytoplasm, which is easier to achieve than into the nucleus. However, RNA is susceptible to RNase degradation and other natural decomposition in the cytoplasm of cells. [0586] Various attempts to increase the stability and shelf life of RNA vaccines. US Pub. No. US 20050032730 to Von Der Mulbe et al. discloses improving the stability of mRNA vaccine compositions by increasing G(guanosine)/C(cytosine) content of the mRNA molecules. U.S. Pat. No. 5,580,859 to Feigner et al. teaches incorporating polynucleotide sequences coding for regulatory proteins that binds to and regulates the stabilities of mRNA. While not wishing to be bound by theory, it is believed that the nucleic acid vaccines described herein may result in improved stability and therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs. Additionally, modified nucleosides, or combinations thereof, may be introduced into the nucleic acid vaccines described herein to activate the innate immune response. Such activating molecules are useful as adjuvants when combined with polypeptides and/or other vaccines. In certain embodiments, the activating molecules contain a translatable region which encodes for a polypeptide sequence useful as a vaccine, thus providing the ability to be a self-adjuvant.
[0587] In some embodiments, the nucleic acid vaccines described herein may be used in the prevention, treatment and diagnosis of diseases and physical disturbances caused by infectious agents such as, but not limited to, SARS-CoV-2, or a VOC, VOI or VOHC of SARS-CovV-2. The nucleic acid vaccines described herein may encode at least one polypeptide of interest (e.g., one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2) and may be provided to an individual in order to stimulate the immune system to protect against the disease-causing agents. As a nonlimiting example, the biological activity and/or effect from an infectious agent may be inhibited and/or abolished by providing neutralizing antibodies which have the ability to bind and neutralize the infectious agent; the neutralizing antibodies produced from the immune system stimulated by the polypeptides translated from the nucleic acid vaccine. [0588] As a non-limiting example, the polynucleotides encoding an immunogen may be delivered to cells to trigger multiple innate response pathways (see PCT Patent Application Publication Nos. W02012006377 and US Patent Publication No. US20130177639; the contents of each of which are herein incorporated by reference in their entirety). As another non-limiting example, the nucleic acid vaccines described herein may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see PCT Patent Application Publication Nos. W02012006372 and WO20 12006369 and US Publication Nos. US20130149375 and US20130177640; the contents of each of which are herein incorporated by reference in their entirety).
[0589] In some embodiments, the nucleic acid vaccines described herein may be delivered to a mammal (e.g., human) in a dose amount large enough to be immunogenic for stimulating an immune response in the mammal. The immune response can defend a viral infection, thereby, prevent and/or treat a disease. As a non-limiting example, the nucleic acid vaccines described herein may treat and/or prevent infectious diseases including viral infectious diseases such as COVID-19 caused by SARS-CoV-2, or a VOC, VOI or VOHC of SARS-CoV-2.
[0590] Nucleic acid vaccines described herein may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the nucleic acid vaccines described herein may be utilized to treat and/or prevent COVID-19 infection, including the diseases and conditions related to COVID-19 infection (including infection by the original and mutated versions of SARS- CoV-2).
[0591] Symptoms of COVID-19 infection are changing as more is learned about the disease but the current symptoms include fever or chills, cough, shortness of breath or difficulty breathing, fatigue, body aches, muscle aches, headaches, sore throat, congestion or runny nose, nausea and/or vomiting, diarrhea, and a new loss of taste or smell.
[0592] In some embodiments, the nucleic acid vaccines described herein may be better designed, as compared to current anti-viral treatments, to produce the appropriate protein conformation on translation as the nucleic acid vaccines co-opt natural cellular machinery. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the nucleic acid vaccines are presented to the cellular system in a more native fashion. In some embodiments, the nucleic acid vaccines described herein are a tailored active vaccine for COVID-19 that not only can prevent infection by SARS-CoV-2 but can limit transmission of SARS-CoV-2.
[0593] In some embodiments, the nucleic acid vaccines described herein may be used to prevent pandemic COVID-19 by reacting to emerging new strains with the very rapid nucleic acid based vaccine production process.
[0594] In some embodiments, a single injection of a nucleic acid vaccine may provide protection for an entire season.
[0595] In some embodiments, the nucleic acid vaccines described herein may be immunostimulatory. The polynucleotide sequence of the nucleic acid vaccine may further comprise a sequence region encoding a cytokine that promotes the immune response, such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-y, GM-CFS, LT-a, or growth factors such as hGH.
Treatment and/or Prevention of CO VID- 19
[0596] In some embodiments, the nucleic acid vaccines described herein encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 and may be used for the treatment and/or prevention of COVID-19
[0597] In some embodiments, the nucleic acid vaccines described herein can produce much higher neutralizing antibody titers and they may produce responses early than commercially available anti-virals. As a non-limiting example, the nucleic acid vaccines described herein can produce 10 times, or 9X, or 8X, or 7X, or 6X, or 5X, or 4X, or 3X more neutralizing antibody titers than other vaccines.
[0598] In some embodiments, administration of the nucleic acid vaccine may result in an increase in neutralizing antibody titer above a previous level (e.g., a pre-administration baseline level). The difference in titer level between the post nucleic acid administration and the pre-administration levels may be, but is not limited to, at least 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14- fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95- fold, or 100-fold, or about 1 to 5-fold, about 5 to 10-fold, about 5 to 20-fold, about 5 to 30- fold, about 5 to 40-fold, about 5 to 50-fold, about 5 to 60-fold, about 10 to 20-fold, about 10 to 30-fold, about 10 to 40-fold, about 10 to 50-fold, about 10 to 60-fold, about 20 to 30- fold, about 20 to 40-fold, about 20 to 50-fold, about 20 to 60-fold, about 30 to 40-fold, about 30 to 50-fold, about 30 to 60-fold, about 40 to 50-fold, about 40 to 60-fold, about 50 to 60-fold, about 50 to 75-fold, about 50 to 100-fold, about 50 to 125-fold, about 50 to 150-fold, about 50 to 175-fold, about 50 to 200-fold, about 50 to 225-fold, about 50 to 300-fold, about 50 to 375-fold, about 75 to 100-fold, about 75 to 125-fold, about 75 to 150-fold, about 75 to 175-fold, about 75 to 200-fold, about 75 to 225-fold, about 75 to 300-fold, about 75 to 375-fold, about 100 to 125-fold, about 100 to 150-fold, about 100 to 175-fold, about 100 to 200-fold, about 100 to 225-fold, about 100 to 300-fold, about 100 to 375-fold, about 125 to 150-fold, about 125 to 175-fold, about 125 to 200-fold, about 125 to 225-fold, about 125 to 300-fold, about 125 to 375-fold, about 150 to 175-fold, about 150 to 200-fold, about 150 to 225-fold, about 150 to 300-fold, about 150 to 375- fold, about 175 to 200-fold, about 175 to 225-fold, about 175 to 300-fold, about 175 to 375-fold, about 200 to 225-fold, about 200 to 300-fold, about 200 to 375-fold, about 225 to 300-fold, about 225 to 375-fold, about 300 to 375-fold, about 100 to 350-fold, about 125 to 350-fold, about 150 to 350-fold, about 175 to 350-fold, about 200 to 350-fold, about 225 to 350-fold, about 250 to 350-fold, about 275 to 350-fold, about 300 to 350- fold, about 325 to 350-fold, about 350 to 375-fold, or more than 375-fold increase.
[0599] In some embodiments, administration of the nucleic acid vaccine results in neutralizing antibodies being present in a subject for at least 1 day post administration. Antibodies may be present in a subject for, but not limited to, about 1 day, about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days, about 1 to 5 weeks, about 1 to 10 weeks, about 1 to 15 weeks, about 1 to 20 weeks, about 1 to 25 weeks, about 1 to 30 weeks, about 5 to 10 weeks, about 5 to 15 weeks, about 5 to 20 weeks, about 5 to 25 weeks, about 5 to 30 weeks, about 10 to 15 weeks, about 10 to 20 weeks, about 10 to 25 weeks, about 10 to 30 weeks, about 15 to 20 weeks, about 15 to 25 weeks, about 15 to 30 weeks, about 20 to 25 weeks, about 20 to 30 weeks, about 25 to 30 weeks, about 30 to 40 weeks, about 30 to 50 weeks, about 30 to 60 weeks, about 40 to 50 weeks, about 40 to 60 weeks, about 50 to 60 weeks, and more than 1 year post administration.
[0600] In some embodiments, administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at least 1 day post administration. Antibodies may be detectable in a subject for, but not limited to, about 1 day, about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days, about 1 to 5 weeks, about 1 to 10 weeks, about 1 to 15 weeks, about 1 to 20 weeks, about 1 to 25 weeks, about 1 to 30 weeks, about 5 to 10 weeks, about 5 to 15 weeks, about 5 to 20 weeks, about 5 to 25 weeks, about 5 to 30 weeks, about 10 to 15 weeks, about 10 to 20 weeks, about 10 to 25 weeks, about 10 to 30 weeks, about 15 to 20 weeks, about 15 to 25 weeks, about 15 to 30 weeks, about 20 to 25 weeks, about 20 to 30 weeks, about 25 to 30 weeks, about 30 to 40 weeks, about 30 to 50 weeks, about 30 to 60 weeks, about 40 to 50 weeks, about 40 to 60 weeks, about 50 to 60 weeks, and more than 1 year post administration.
[0601] In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 1 day post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 14 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 21 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 28 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 35 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 42 days post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about 6 months (180 days) post administration. In some embodiments administration of the nucleic acid vaccine results in neutralizing antibodies being detectable in a subject for at about one year post administration.
[0602] In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing for at least 1 day post administration. Antibodies may be produced by a subject for, but not limited to, about 1 day, about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days, about 1 to 5 weeks, about 1 to 10 weeks, about 1 to 15 weeks, about 1 to 20 weeks, about 1 to 25 weeks, about 1 to 30 weeks, about 5 to 10 weeks, about 5 to 15 weeks, about 5 to 20 weeks, about 5 to 25 weeks, about 5 to 30 weeks, about 10 to 15 weeks, about 10 to 20 weeks, about 10 to 25 weeks, about 10 to 30 weeks, about 15 to 20 weeks, about 15 to 25 weeks, about 15 to 30 weeks, about 20 to 25 weeks, about 20 to 30 weeks, about 25 to 30 weeks, about 30 to 40 weeks, about 30 to 50 weeks, about 30 to 60 weeks, about 40 to 50 weeks, about 40 to 60 weeks, about 50 to 60 weeks, and more than 1 year post administration.
[0603] In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at least about 1 day post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 14 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 21 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 28 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 35 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 42 days post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 6 months (180 days or 26 weeks) post administration. In some embodiments, administration of the nucleic acid vaccine results in a subject producing neutralizing antibodies for at about 1 year post administration.
[0604] In some embodiments, the nucleic acid vaccines described herein co-opt the natural cellular machinery to produce the appropriate protein conformation on translation. Unlike traditional vaccines which are manufactured ex vivo and may trigger unwanted cellular responses, the nucleic acid vaccines described herein are introduced to the cellular system in a way that is closer to the native way or the way normal cellular processing occurs. Additionally, formulations may be used to shield or target delivery of the nucleic acid vaccines to specific cells or tissues in the subject.
[0605] In some embodiments, nucleic acid vaccines described herein represent a targeted active vaccine that not only can prevent infection but can limit transmission of COVID-19.
[0606] In some embodiments, the nucleic acid vaccines may be used to prevent pandemic SARS-CoV-2 infection or COVID-19 by reacting to emerging new strains with the very rapid vaccine production process.
[0607] In some embodiments a single injection of nucleic acid vaccines encoding one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 may provide protection for at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, at least 13 years, at least 15 years or more than 15 years.
[0608] The nucleic acid vaccines described herein may also be used to maintain or restore antigenic memory in a subject or population as part of a vaccination plan for COVID-19 or other diseases caused by SARS-CoV-2.
[0609] In some embodiments, nucleic acid vaccines compositions may be created which include polynucleotides that encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 which are showing prevalence increased infection rates for the year. The protein sequences of SARS-CoV-2 have been shown to change or mutate over time, wherein some of the mutations have shown increased infection rates. As a non-limiting example, the nucleic acid vaccines compositions may be created which include polynucleotides that encode one or more proteins, polypeptides, peptides, fragments or variants thereof of SARS-CoV-2 which are showing prevalence increased infection rates for the year such as, but not limited to the D614G mutation in the spike protein.
[0610] In some embodiments, the nucleic acid vaccines may be used to induce neutralizing antibodies in a subject. The neutralization activity of the neutralizing antibodies induced by the present nucleic acid vaccines may correlate to the resulting effectiveness (e.g., immune protection) of the vaccines described herein. In some aspects, the immune protection provided by the present nucleic acid vaccines may be greater than the immune protection provided in convalescent subjects. The induced neutralization activity from the present nucleic acid vaccines may increase the recovery rate of those exposed to SARS-CoV-2 or whom are in convalescence. As compared to the other mRNA vaccines available such as BNT162b2 and mRNA- 1273, the present nucleic acid vaccines induce higher neutralizing antibody titers in treated subjects. In some embodiments, the nucleic acid vaccines include at least 10 times, 9 times, 8 times, 7 times, 6 times, 5 times or 4 times more neutralizing antibody titers than other vaccines for COVID-19.
[0611] In some embodiments, a vaccination scheme or plan is developed which allows for not only ongoing vaccination in the current year but memory booster vaccinations across years, strains, or groups thereof to establish and maintain memory in a population. In this manner, a population is less likely to succumb to any pandemic or outbreak involving recurrence of older strains. Any combination of a prior vaccine component strain can be utilized to create or design a memory booster vaccine.
[0612] In some embodiments, nucleic acid vaccines which are memory booster vaccines are administered to boost memory across a time period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 years.
[0613] In some embodiments, nucleic acid vaccines which are memory booster vaccines are administered to boost memory for alternating historic years including every other year from the past vaccine component strains relative to a current year. In some embodiments the selection of the vaccine components can be from every 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th or more years.
[0614] In some embodiments, nucleic acid vaccines which are memory booster vaccines are administered to boost memory over ten-year periods.
[0615] In some embodiments, the nucleic acid booster vaccine may be used in a population either once or periodically to create herd immunity which means greater than 30% of a population is protected.
[0616] In some embodiments, the nucleic acid booster vaccine may be used in a population either once or periodically to create herd immunity against COVID-19 which means greater than 30% of a population is protected.
[0617] In some embodiments, the nucleic acid vaccines are used to target at risk populations for COVID-19 such as those having pre-existing conditions including, but not limited to, cancer, chronic kidney disease, chronic obstructive pulmonary disease (COPD), immunocompromised state (weakened immune system) from solid organ transplant, blood or bone marrow transplant, immune deficiencies, HIV, and use of corticosteroids or other immune weaking medicines, obesity (body mass index (BMI) of 30 or higher), heart conditions such as heart failure, coronary artery disease, or cardiomyopathies, sickle cell disease, type 1 or type 2 diabetes mellitus, asthma (moderate-to-severe), cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, neurological conditions such as dementia, liver disease, pregnancy, pulmonary fibrosis, smoking, and thalassemia.
[0618] In some embodiments, the nucleic acid vaccines are used to protect healthcare workers who are at risk of being exposed to SARS-CoV-2.
[0619] As a non-limiting example, the nucleic acid vaccine of the present disclosure comprises a LNP formulated polynucleotide encoding the full-length S protein with D614G (SEQ ID NO: 27) (as referred to as “PTX-B”). Methods for use of PTX-B vaccine to induce a protective immune response in a subject is provided. The protective immune response can protect a subject against a viral infection, such as infection by SAR-CoV-2 original strain and its variant thereof. The SARS-CoV-2 variant can be any VOC, VOI and/or VOHC strain. As non-limiting examples, the variant is an Alpha variant, a Beta variant, or a Delta variant.
[0620] In some embodiments, the nucleic acid vaccine PTX-B is used to protect against an Alpha variant. In some embodiments, the Alpha variant is Alpha (B.1.1.7).
[0621] In some embodiments, the nucleic acid vaccine PTX-B is used to protect against a Beta variant. In some embodiments, the Beta variant is beta (B.1.351).
[0622] In some embodiments, the nucleic acid vaccine PTX-B is used to protect against a Delta variant. In some embodiments, the Delta variant is delta (B.1.617.2).
[0623] In some embodiments, the nucleic acid vaccine PTX-B is used to induce neutralizing antibodies in a subject. The nucleic acid vaccine PTX-B induces antibodies with high neutralization titers. For example, the neutralization titers of induced antibodies are as least 100 fold, 90 fold, 80 fold, 70 fold, 60 fold, 50 fold, 40 fold, 30 fold, 25 fold, 20 fold, 15 fold, 10 fold or 5 fold of the titers in sera from infected subjects or convalescent subjects.
[0624] In some embodiments, a dosing regimen of the nucleic acid vaccine PTX-B is provided. The dose of PTX-B ranges from 1 pg to 500 pg, from 1 pg to Img, from Img to lOmg, from Img to lOOmg, or from lOmg to lOOmg/kg of a subject body weight. In some examples, a dose of PTX-B can achieve a dose level of about 10 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 60pg, 70 pg, 80 pg, 90 pg, or 100 pg of the nucleic acid vaccine. At least one dose of the nucleic acid vaccine PTX-B is administered. In some embodiments, the at least one dose administered is 16 ug. In other embodiments, the at least one dose administered is 40 ug. In further embodiments, the at least one dose administered is 100 ug. In some embodiments, at least one booster dose of PTX-B is administered. The booster dose may be administered to the subject, one month, two months, three months, four months, six months, or one year or greater after the subj ect receives the first dose of PTX-B. In some instances, more than one booster dose, e.g., two, three, four or more, is administered to the subject. In some embodiments, the booster dose administered is 16 ug. In other embodiments, the booster dose administered is 40 ug. In further embodiments, the booster dose administered is 100 ug.
[0625] In another embodiment, the nucleic acid vaccine of the present disclosure comprises a LNP formulated polynucleotide encoding the full-length S protein of SARS- CoV-2 variant B.1.351 (South African variant (SEQ ID NO: 43).
[0626] In some embodiments, the nucleic acid vaccine may be administered to a black/ African American population. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) black/ African American subjects. In some embodiments the nucleic acid vaccine may be administered to a population comprised of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% black/ African American subjects. In some embodiments, the nucleic acid vaccine may be administered to a population comprised about 88% black/ African American subjects.
[0627] In some embodiments, the nucleic acid vaccine may be administered to subjects from any country in the world. In some embodiments, the nucleic acid vaccine may be administered to subjects from Afghanistan, Albania, Algeria, Andorra, Angola, Antigua and Barbuda, Argentina, Armenia, Australia, Austria, Azerbaijan, Bahamas, Bahrain, Bangladesh, Barbados, Belarus, Belgium, Belize, Benin, Bhutan, Bolivia, Bosnia and Herzegovina, Botswana, Brazil, Brunei, Bulgaria, Burkina Faso, Burundi, Cabo Verde, Cambodia, Cameroon, Canada, Central African Republic (CAR), Chad, Chile, China, Colombia, Comoros, Congo, Democratic Republic of the, Congo, Republic of the, Costa Rica, Cote d'Ivoire, Croatia, Cuba, Cyprus, Czechia, Denmark, Djibouti, Dominica, Dominican Republic, Ecuador, Egypt, El Salvador, Equatorial Guinea, Eritrea, Estonia, Eswatini, Ethiopia, Fiji, Finland, France, Gabon, Gambia, Georgia, Germany, Ghana, Greece, Grenada, Guatemala, Guinea, Guinea-Bissau, Guyana, Haiti, Honduras, Hungary, Iceland, India, Indonesia, Iran, Iraq, Ireland, Israel, Italy, Jamaica, Japan, Jordan, Kazakhstan, Kenya, Kiribati, Kosovo, Kuwait, Kyrgyzstan, Laos, Latvia, Lebanon, Lesotho, Liberia, Libya, Liechtenstein, Lithuania, Luxembourg, Madagascar, Malawi, Malaysia, Maldives, Mali, Malta, Marshall Islands, Mauritania, Mauritius, Mexico, Micronesia, Moldova, Monaco, Mongolia, Montenegro, Morocco, Mozambique, Myanmar, Namibia, Nauru, Nepal, Netherlands, New Zealand, Nicaragua, Niger, Nigeria, North Korea, North Macedonia, Norway, Oman, Pakistan, Palau, Palestine, Panama, Papua New Guinea, Paraguay, Peru, Philippines, Poland, Portugal, Qatar, Romania, Russia, Rwanda, Saint Kitts and Nevis, Saint Lucia, Saint Vincent and the Grenadines, Samoa, San Marino, Sao Tome and Principe, Saudi Arabia, Senegal, Serbia, Seychelles, Sierra Leone, Singapore, Slovakia, Slovenia, Solomon Islands, Somalia, South Africa, South Korea, South Sudan, Spain, Sri Lanka, Sudan, Suriname, Sweden, Switzerland, Syria, Taiwan, Tajikistan, Tanzania, Thailand, Timor-Leste, Togo, Tonga, Trinidad and Tobago, Tunisia, Turkey, Turkmenistan, Tuvalu, Uganda, Ukraine, United Arab Emirates (UAE), United Kingdom (UK), United States of America (USA), Uruguay, Uzbekistan, Vanuatu, Vatican City (Holy See), Venezuela, Vietnam, Yemen, Zambia, Zimbabwe, or any combination thereof. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of subjects from one country. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of subjects from South Africa.
[0628] In some embodiments, the nucleic acid vaccine may be administered to a population comprised of both males and females. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of males or a majority (i.e., at least about 50.1%) of females. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of males. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of a majority (i.e., at least about 50.1%) of females. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of about 52% of males. In some embodiments, he nucleic acid vaccine may be administered to a population comprised of about 48% of females.
[0629] In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of any age. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages from about 1 year old to about 120 years old or more. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages from about birth to about 18 years old inclusive (i.e., minors). In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages from greater than about 18 years old to about 64 years old inclusive (i.e., adults). In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects of ages greater than 64 years old to death (i.e., seniors or the elderly).
[0630] In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects whose mean age is about 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 years old. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects whose mean age is about 31 years old. In some embodiments, the nucleic acid vaccine may be administered to a population comprised of subjects whose mean age is about 32 years old. [0631] In some embodiments, subjects may be administered more than one dose of the nucleic acid vaccine. In certain embodiments, subjects may be administered at least 2 doses of the nucleic acid vaccine. In some embodiments, subjects may be administered at least one first dose and at least one second dose of the nucleic acid vaccine. In some embodiments, the at least one second dose may be administered between about 1 and 30 days after the at least one first dose. In some embodiments, the at least one second dose may be administered between about 1 to 5 days, about 1 to 10 days, about 1 to 15 days, about 1 to 20 days, about 1 to 25 days, about 1 to 30 days, about 5 to 10 days, about 5 to 15 days, about 5 to 20 days, about 5 to 25 days, about 5 to 30 days, about 10 to 15 days, about 10 to 20 days, about 10 to 25 days, about 10 to 30 days, about 15 to 20 days, about 15 to 25 days, about 15 to 30 days, about 20 to 25 days, about 20 to 30 days, about 25 to 30 days after the at least one first dose. In some embodiments, the at least one second dose may be administered between about 20 after the at least one first dose. In some embodiments, the at least one second dose may be administered between about 27 after the at least one first dose.
[0632] In some embodiments, the safety and/or tolerability of the nucleic acid vaccine may be assayed. Various methods of assaying unintended or unanticipated effects , also known as “side-effects,” of a vaccine, including nucleic acid vaccines, are known in the art and may be suitable for use in assaying safety and/or tolerability. Example methods for assaying safety and/or tolerability include but are not limited to monitoring for and recording incidences of: injection site reactions (e.g., arm check evaluations including pain, tenderness, erythema/redness, induration/swelling), solicited adverse events (SAEs; e.g., fever, chills, nausea/vomiting, diarrhea, headache, fatigue, myalgia), unsolicited adverse events (AEs), treatment emergent adverse events (TEAEs) including medically attendant adverse events (MAAEs new onset chronic diseases (NOCDs), adverse event of special interests (AESIs), potentially immune-mediated medical conditions (PIMMCs) and any combinations thereof.
[0633] In some embodiments, the safety and/or tolerability of the nucleic acid vaccine may be assayed for a period of time after administration of at least one dose of the nucleic acid vaccine. For example, safety and/or tolerability may be assayed for about 1 week, about 1 to 4 weeks, about 2 to 6 weeks, about 1 to 8 weeks, about 1 to 12 weeks, about 1 to 24 weeks, about 1 to 36 weeks, about 1 to 54 weeks, more than 54 weeks, or any combination thereof post administration of the first dose of the nucleic acid vaccine.
V. KITS AND DEVICES
Kits [0634] The disclosure provides a variety of kits for conveniently and/or effectively carrying out methods of the present disclosure. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
[0635] In some embodiments, the present disclosure provides kits for modulating the expression of genes in vitro or in vivo, comprising nucleic acid vaccine compositions of the present disclosure or a combination of nucleic acid vaccine compositions of the present disclosure, nucleic acid vaccine compositions modulating other genes, siRNAs, miRNAs or other oligonucleotide molecules.
[0636] The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation, e.g., for administration to a subject in need of treatment using the nucleic acid vaccine compositions described herein. The delivery agent may comprise a saline, a buffered solution, a lipidoid, a dendrimer or any suitable delivery agent.
[0637] In one non-limiting example, the buffer solution may include sodium chloride, calcium chloride, phosphate and/or EDTA. In another non-limiting example, the buffer solution may include, but is not limited to, saline, saline with 2mM calcium, 5% sucrose, 5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium, Ringer’s lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See U.S. Pub. No. 20120258046; herein incorporated by reference in its entirety). In yet another nonlimiting example, the buffer solutions may be precipitated or it may be lyophilized. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of nucleic acid vaccine compositions in the buffer solution over a period of time and/or under a variety of conditions.
Devices
[0638] The present disclosure provides for devices which may incorporate nucleic acid vaccine compositions of the present disclosure. These devices can contain a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient. [0639] Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, electroporation devices, iontophoresis devices, multi-layered microfluidic devices. The devices may be employed to deliver nucleic acid vaccine compositions of the present disclosure according to single, multi- or split-dosing regiments. The devices may be employed to deliver nucleic acid vaccine compositions of the present disclosure across biological tissue, intradermal, subcutaneously, or intramuscularly. More examples of devices suitable for delivering oligonucleotides are disclosed in International Publication WO 2013/090648, the contents of which are incorporated herein by reference in their entirety.
VI. DEFINITIONS
[0640] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges.
[0641] About: As used herein, the term “about” means +/-10% of the recited value. [0642] Administered in combination: As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
[0643] Adjuvant: As used herein, the term “adjuvant” means a substance that enhances a subject’s immune response to an antigen. The nucleic acid vaccines described herein may optionally comprise one or more adjuvants.
[0644] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
[0645] Antigen: As defined herein, the term “antigen” refers to a composition, for example, a substance or agent which causes an immune response in an organism, e.g., causes the immune response of the organism to produce antibodies against the substance or agent in particular, which provokes an adaptive immune response in an organism. Antigens can be any immunogenic substance including, in particular, proteins, polypeptides, polysaccharides, nucleic acids, lipids and the like. Exemplary antigens are derived from infectious agents. Such agents can include parts or subunits of infectious agents, for example, coats, coat components, e.g., coat protein or polypeptides, surface components, e.g., surface proteins or polypeptides, capsule components, cell wall components, flagella, fimbrae, and/or toxins or toxoids) of infectious agents, for example, bacteria, viruses, and other microorganisms. Certain antigens, for example, lipids and/or nucleic acids are antigenic, preferably, when combined with proteins and/or polysaccharides.
[0646] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0647] Associated with: As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.
[0648] Bifunctional: As used herein, the term “bifunctional” refers to any substance, molecule or moiety which is capable of or maintains at least two functions. The functions may affect the same outcome or a different outcome. The structure that produces the function may be the same or different.
[0649] Biocompatible: As used herein, the term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.
[0650] Biodegradable: As used herein, the term “biodegradable” means capable of being broken down into innocuous products by the action of living things.
[0651] Biologically active: As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, a polynucleotide described herein may be considered biologically active if even a portion of the polynucleotides is biologically active or mimics an activity considered biologically relevant.
[0652] Chimera: As used herein, “chimera” is an entity having two or more incongruous or heterogeneous parts or regions.
[0653] Compound: As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. [0654] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.
[0655] Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
[0656] Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. [0657] The compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
[0658] Conserved: As used herein, the term “conserved” refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
[0659] In some embodiments, two or more sequences are said to be “completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an polynucleotide or polypeptide or may apply to a portion, region or feature thereof.
[0660] Controlled Release: As used herein, the term “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
[0661] Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing, suppressing the growth, division, or multiplication of a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
[0662] Cytotoxic: As used herein, “cytotoxic” refers to killing or causing injurious, toxic, or deadly effect on a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a combination thereof.
[0663] Delivery: As used herein, “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.
[0664] Delivery Agent: As used herein, “delivery agent” refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide to targeted cells.
[0665] Destabilized: As used herein, the term “destable,” “destabilize,” or “destabilizing region” means a region or molecule that is less stable than a starting, wildtype or native form of the same region or molecule.
[0666] Detectable label: As used herein, “detectable label” refers to one or more markers, signals, or moieties which are attached, incorporated or associated with another entity that is readily detected by methods known in the art including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable labels may be located at any position in the peptides or proteins disclosed herein. They may be within the amino acids, the peptides, or proteins, or located at the N- or C-termini.
[0667] Digest: As used herein, the term “digest” means to break apart into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.
[0668] Dosing regimen: As used herein, a “dosing regimen” is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.
[0669] Encapsulate: As used herein, the term “encapsulate” means to enclose, surround or encase.
[0670] Encoded protein cleavage signal: As used herein, “encoded protein cleavage signal” refers to the nucleotide sequence which encodes a protein cleavage signal.
[0671] Engineered: As used herein, embodiments of the nucleic acid vaccines are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.
[0672] Effective Amount: As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.
[0673] Exosome: As used herein, “exosome” is a vesicle secreted by mammalian cells or a complex involved in RNA degradation. [0674] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. [0675] Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.
[0676] Formulation: As used herein, a “formulation” includes at least a polynucleotide of a nucleic acid vaccine and a delivery agent.
[0677] Fragment: A “fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
[0678] Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
[0679] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
[0680] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
[0681] Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and nonidentical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M, ed., Oxford University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, N.Y, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M, and Griffin, H. G., eds., Humana Press, N.J., 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, N.Y, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al, J. Molec. Biol., 215, 403 (1990)).
[0682] Infectious Agent: As used herein, the phrase “infectious agent” means an agent capable of producing an infection in an organism, for example, in an animal. An infectious agent may refer to any microorganism, virus, infectious substance, or biological product that may be engineered as a result of biotechnology, or any naturally occurring or bioengineered component of any such microorganism, virus, infectious substance, or biological product, can cause emerging and contagious disease, death or other biological malfunction in a human, an animal, a plant or another living organism.
[0683] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
[0684] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
[0685] Isolated: As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. Substantially isolated: By “substantially isolated” is meant that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
[0686] Linker: As used herein, a “linker” refers to a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end. The linker may be of sufficient length as to not interfere with incorporation into a nucleic acid sequence.
[0687] Modified: As used herein “modified” refers to a changed state or structure of a molecule described herein. Molecules may be modified in many ways including chemically, structurally, and functionally.
[0688] Mucus: As used herein, “mucus” refers to the natural substance that is viscous and comprises mucin glycoproteins.
[0689] Naturally occurring: As used herein, “naturally occurring” means existing in nature without artificial aid.
[0690] Neutralizing antibody: As used herein, a “neutralizing antibody” refers to an antibody which binds to its antigen and defends a cell from an antigen or infectious agent by neutralizing or abolishing any biological activity it has. [0691] Non-human vertebrate: As used herein, a “non-human vertebrate” includes all vertebrates except Homo sapiens, including wild and domesticated species. Examples of non- human vertebrates include, but are not limited to, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep water buffalo, and yak.
[0692] Nucleic Acid Vaccine: As used herein, “nucleic acid vaccine” refers to a vaccine or vaccine composition which includes a nucleic acid or nucleic acid molecule (e.g., a polynucleotide) encoding an antigen (e.g., an antigenic protein or polypeptide.) In exemplary embodiments, a nucleic acid vaccine includes a ribonucleic (“RNA”) polynucleotide, ribonucleic acid (“RNA”) or ribonucleic acid (“RNA”) molecule. Such embodiments can be referred to as ribonucleic acid (“RNA”) vaccines.
[0693] Off-target: As used herein, “off target” refers to any unintended effect on any one or more target, gene, or cellular transcript.
[0694] Open reading frame: As used herein, the term “open reading frame” or “ORF” refers to a continuous polynucleotide sequence, for example, a DNA sequence or RNA sequence (e.g., an mRNA sequence), comprising a start codon, a subsequent region comprising a plurality of amino acid-encoding codons, and a terminal stop codon, wherein the region comprising the plurality of amino acid-encoding codons contains no stop codons.
[0695] Operably linked: As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
[0696] Part: As used herein, a “part” or “region” of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide.
[0697] Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
[0698] Paratope: As used herein, a “paratope” refers to the antigen-binding site of an antibody. [0699] Patient: As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
[0700] Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0701] Pharmaceutically acceptable excipients: The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and noninflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BEIT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (com), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
[0702] Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropi onate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Beige et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
[0703] Pharmaceutically acceptable solvate: The term “pharmaceutically acceptable solvate,” as used herein, means a compound described herein wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidi- none (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethyl- formamide (DMF), N,N' -dimethylacetamide (DMAC), 1,3- dimethyl-2-imidazolidinone (DMEU), l,3-dimethyl-3,4,5, 6-tetrahydro-2-(lH)- pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”
[0704] Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME where: (A) Absorption is the process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.
[0705] Physicochemical: As used herein, “physicochemical” means of or relating to a physical and/or chemical property. [0706] Polypeptide per unit drug (PUD): As used herein, a PUD or product per unit drug, is defined as a subdivided portion of total daily dose, usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) as measured in body fluid or tissue, usually defined in concentration such as pmol/mL, mmol/ mL, etc. divided by the measure in the body fluid. [0707] Preventing: As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition. [0708] Proliferate: As used herein, the term “proliferate” means to grow, expand or increase or cause to grow, expand or increase rapidly. “Proliferative” means having the ability to proliferate. “Anti-proliferative” means having properties counter to or inapposite to proliferative properties.
[0709] Prophylactic: As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.
[0710] Prophylaxis: As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease. An “immune prophylaxis” refers to a measure to produce active or passive immunity to prevent the spread of disease.
[0711] Protein cleavage site: As used herein, “protein cleavage site” refers to a site where controlled cleavage of the amino acid chain can be accomplished by chemical, enzymatic or photochemical means.
[0712] Protein cleavage signal: As used herein “protein cleavage signal” refers to at least one amino acid that flags or marks a polypeptide for cleavage.
[0713] Protein of interest: As used herein, the terms “proteins of interest” or “desired proteins” include those provided herein and fragments, mutants, variants, and alterations thereof. [0714] Purified: As used herein, “purify,” “purified,” “purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.
[0715] Repeated transfection: As used herein, the term “repeated transfection” refers to transfection of the same cell culture with a polynucleotide a plurality of times. The cell culture can be transfected at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more.
[0716] Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.
[0717] Signal Sequences: As used herein, the phrase “signal sequences” refers to a sequence which can direct the transport or localization of a protein.
[0718] Single unit dose: As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.
[0719] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
[0720] Split dose: As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses.
[0721] Stable: As used herein “stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
[0722] Stabilized: As used herein, the term “stabilize”, “stabilized,” “stabilized region” means to make or become stable.
[0723] Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).
[0724] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
[0725] Substantially equal: As used herein as it relates to time differences between doses, the term means plus/minus 2%.
[0726] Substantially simultaneously: As used herein and as it relates to plurality of doses, the term means within 2 seconds.
[0727] Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition. [0728] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
[0729] Sustained release: As used herein, the term “sustained release” refers to a pharmaceutical composition or compound release profile that conforms to a release rate over a specific period of time.
[0730] Synthetic: The term “synthetic” means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules described herein may be chemical or enzymatic.
[0731] Vaccine: As used herein, a vaccine is a compound or composition which comprises at least one polynucleotide encoding at least one antigen.
[0732] Targeted Cells: As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient. [0733] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
[0734] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
[0735] Therapeutically effective outcome: As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
[0736] Total daily dose: As used herein, a “total daily dose” is an amount given or prescribed in 24 hr. period. It may be administered as a single unit dose.
[0737] Transfection: As used herein, the term “transfection” refers to methods to introduce exogenous nucleic acids into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments and cationic lipids or mixtures.
[0738] Translation: As used herein “translation” is the process by which a polynucleotide molecule is processed by a ribosome or ribosomal-like machinery, e.g., cellular or artificial, to produce a peptide or polypeptide.
[0739] Transcription: As used herein “transcription” is the process by which a polynucleotide molecule is processed by a polymerase or other enzyme to produce a polynucleotide, e.g., an RNA polynucleotide.
[0740] Treating: As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, infection, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, infection, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, infection, disorder, and/or condition.
[0741] Unmodified: As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.
[0742] Vaccine: As used herein, the phrase “vaccine” refers to a biological preparation that improves immunity in the context of a particular disease, disorder or condition.
[0743] Viral protein: As used herein, the phrase “viral protein” means any protein originating from a virus.
VII. ENUMERATED EMBODIMENTS
[0744] Embodiment 1. A method of inducing an immune response in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32.
[0745] Embodiment 2. The method of Embodiment 1, where the first sequence region is at least 95% identical to, at least 99% identical to or comprising SEQ ID NO: 7.
[0746] Embodiment 3. The method of Embodiment 2, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2.
[0747] Embodiment 4. The method of Embodiment 2, wherein at least 50% of the polynucleotide sequence is codon optimized. [0748] Embodiment 5. The method of Embodiment 4, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine.
[0749] Embodiment 6. The method of Embodiment 5, wherein all uracil nucleosides are modified to be N1 -methylpseudouridine.
[0750] Embodiment 7. The method of any of Embodiments 1-6, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
[0751] Embodiment 8. The method of Embodiment 7, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a noncationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid; (iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid; (v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid; (vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15- 50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid; (vii) the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a noncationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid; (viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5- 50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid; (xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid; (xii) the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid; (xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA; or (xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA.
[0752] Embodiment 9. The method of any of Embodiment 1-8, wherein the pharmaceutical composition comprises about 0.2 mg/mL of nucleic acid vaccine.
[0753] Embodiment 10. The method of Embodiment 9, wherein the pharmaceutical composition is suitable for intramuscular (IM) injection.
[0754] Embodiment 11. The method of Embodiment 10, wherein the pharmaceutical composition is packaged as a 2.4 mL fill in a 6 mL glass vial or a 2 mL fill in a 3 mL glass vial.
[0755] Embodiment 12. The method of any of Embodiments 1-11, wherein the immune response comprises a T-cell response.
[0756] Embodiment 13. The method of any of Embodiments 1-11, wherein the immune response comprises a B cell response.
[0757] Embodiment 14. The method of any of Embodiments 1-13, further comprising administering a booster subsequent to the first administration.
[0758] Embodiment 15. The method of Embodiment 14, wherein the subject is negative prior to the booster administration.
[0759] Embodiment 16. The method of any of Embodiments 1-15, wherein the subject is seronegative.
[0760] Embodiment 17. A method of treating COVID-19 in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32.
[0761] Embodiment 18. A method of preventing COVID-19 in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32.
[0762] Embodiment 19. A method of mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32.
[0763] Embodiment 20. The method of any of Embodiments 17-19, where the first sequence region is at least 95% identical to, at least 99% identical to or comprising SEQ ID NO: 7.
[0764] Embodiment 21. The method of Embodiment 20, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2.
[0765] Embodiment 22. The method of Embodiment 20, wherein at least 50% of the polynucleotide sequence is codon optimized. [0766] Embodiment 23. The method of Embodiment 22, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine.
[0767] Embodiment 24. The method of Embodiment 23, wherein all uracil nucleosides are modified to be N1 -methylpseudouridine.
[0768] Embodiment 25. The method of any of Embodiments 17-24, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
[0769] Embodiment 26. The method of Embodiment 25, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid; (iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid; (v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35- 45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid; (vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid; (vii) the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid; (viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5- 50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid; (xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid, (xii) the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid; (xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA; or (xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA.
[0770] Embodiment 27. The method of any of Embodiments 17-26, wherein the dose of nucleic acid vaccine is administered to the subject is (i) about 5 pg to about 100 pg, (ii) about 16 pg, (iii) about 40 pg, or (iv) about 100 pg.
[0771] Embodiment 28. The method of any of Embodiments 17-27, wherein the volume of pharmaceutical composition administered to the subject is about 0.025 mL to about 0.5 mL.
[0772] Embodiment 29. The method of Embodiment 28, wherein the volume of pharmaceutical composition administered to the subject is about 0.025 mL, 0.05mL, 0.08mL, 0.2mL, or 0.5mL.
[0773] Embodiment 30. The method of any of Embodiments 17-29, comprising administering a second dose of the nucleic acid vaccine or pharmaceutical composition after between about 1 and about 5 weeks of a first dose.
[0774] Embodiment 31. The method of Embodiment 30, wherein the second dose is administered about 3 to 4 weeks after said first dose.
[0775] Embodiment 32. The method of Embodiment 31, wherein the subject is negative for COVID-19 when administered the second dose.
[0776] Embodiment 33. The method of any of Embodiments 17-32, wherein the pharmaceutical composition is suitable for intramuscular (IM) injection.
[0777] Embodiment 34. The method of any of Embodiments 17-33, wherein the subject is seronegative for COVID-19. [0778] Embodiment 35. A method of inducing SARS-CoV-2 neutralizing antibody production in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32.
[0779] Embodiment 36. The method of Embodiment 35, where the first sequence region is at least 95% identical to, at least 99% identical to or comprising SEQ ID NO: 7. [0780] Embodiment 37. The method of Embodiment 36, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2.
[0781] Embodiment 38. The method of Embodiment 36, wherein at least 50% of the polynucleotide sequence is codon optimized.
[0782] Embodiment 39. The method of Embodiment 38, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine.
[0783] Embodiment 40. The method of Embodiment 39, wherein all uracil nucleosides are modified to be N1 -methylpseudouridine.
[0784] Embodiment 41. The method of any of Embodiments 35-40, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
[0785] Embodiment 42. The method of Embodiment 41, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid; (iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid; (v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35- 45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid; (vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid; (vii) the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid; (viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5- 50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid; (xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid; (xii) the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid; (xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA; or (xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA.
[0786] Embodiment 43. The method of any of Embodiments 35-42, wherein the SARS-CoV-2 neutralizing antibodies can be detected in the subject by day 28 after administration. [0787] Embodiment 44. The method of Embodiment 43, wherein the SARS-CoV-2 neutralizing antibody production is enhanced in the subject at day 42 after receiving a second dose of the nucleic acid vaccine or pharmaceutical composition.
[0788] Embodiment 45. The method of Embodiment 43, wherein the level of neutralizing antibodies to SARS-CoV-2 is increased ten-fold by day 42.
[0789] Embodiment 46. The method of any of Embodiments 35-42, wherein the subject is seronegative for SARS-CoV-2.
[0790] Embodiment 47. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 1 days post initial administration.
[0791] Embodiment 48. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 21 days post initial administration.
[0792] Embodiment 49. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 28 days post initial administration.
[0793] Embodiment 50. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 42 days post initial administration.
[0794] Embodiment 51. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 42 days post initial administration.
[0795] Embodiment 52. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 6 months (180 days) post initial administration.
[0796] Embodiment 53. The method of any of Embodiments 35-42, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 1 year post initial administration. [0797] Embodiment 54. The method of any of Embodiments 35-53, wherein the amount of neutralizing antibody in a subject increased by at least about 4-fold relative to pre administration levels.
[0798] Embodiment 55. The method of any of Embodiments 35-54, wherein the amount of neutralizing antibody in a subject increased by about 100-fold relative to pre administration levels.
[0799] Embodiment 56. The method of any of Embodiments 35-55, wherein the amount of neutralizing antibody in a subject increased by about 200-fold relative to pre administration levels.
[0800] Embodiment 57. The method of any of Embodiments 35-42, wherein the nucleic acid vaccine is administered as a booster vaccine.
[0801] Embodiment 58. The method of Embodiment 57, wherein the subject is seropositive for SARS-CoV-2 antibodies.
[0802] Embodiment 59. The method of Embodiment 57, wherein the subject is has previously received at least one SARS-CoV-2 vaccine.
[0803] Embodiment 60. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 1 day post administration of the nucleic acid vaccine.
[0804] Embodiment 61. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 21 days post administration of the nucleic acid vaccine.
[0805] Embodiment 62. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 28 days post administration of the nucleic acid vaccine.
[0806] Embodiment 63. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 42 days post administration of the nucleic acid vaccine. [0807] Embodiment 64. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 42 days post administration of the nucleic acid vaccine.
[0808] Embodiment 65. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 6 months (180 days) days post administration of the nucleic acid vaccine.
[0809] Embodiment 66. The method of any of Embodiments 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 1 year post administration of the nucleic acid vaccine.
[0810] Embodiment 67. The method of any of Embodiments 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 4-fold relative to levels prior to the administration of the nucleic acid vaccine.
[0811] Embodiment 68. The method of any of Embodiments 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 5-fold relative to levels prior to the administration of the nucleic acid vaccine.
[0812] Embodiment 69. The method of any of Embodiments 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 6-fold relative to levels prior to the administration of the nucleic acid vaccine.
[0813] Embodiment 70. The method of any of Embodiments 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 7-fold relative to levels prior to the administration of the nucleic acid vaccine.
[0814] Embodiment 71. The method of any of Embodiments 57-66, wherein the amount of neutralizing antibody in a subject increased by about 100-fold relative to levels prior to the administration of the nucleic acid vaccine.
[0815] Embodiment 72. The method of any of Embodiments 57-66, wherein the amount of neutralizing antibody in a subject increased by about 200-fold relative to levels prior to the administration of the nucleic acid vaccine.
[0816] Embodiment 73. The pharmaceutical composition as defined in any one of Embodiment 72. 1-72 for use in therapy. [0817] Embodiment 74. The pharmaceutical composition as defined in any one of Embodiment 1-72 for use inducing an immune response in a subject, for treating COVID- 19 in a subject, for preventing COVID-19 in a subject, for mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject, or for inducing SARS-CoV-2 neutralizing antibody production in a subject.
[0818] Embodiment 75. The pharmaceutical composition as defined in any one of Embodiment 1-72 for the manufacture of a medicament for inducing an immune response in a subject, for treating COVID-19 in a subject, for preventing COVID-19 in a subject, for mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject, or for inducing SARS-CoV-2 neutralizing antibody production in a subject.
[0819] Embodiment 76. A method of inducing SARS-CoV-2 neutralizing antibody production in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7 and 27 and wherein the SARS-CoV-2 neutralizing antibody is to alpha (B. l.1.7), beta (B.1.351) or delta (B.1.617.2) strains of SARS-CoV-2.
[0820] Embodiment 77. The method of Embodiment 76, where the first sequence region is at least 95% identical to, at least 99% identical to or comprising SEQ ID NO: 7. [0821] Embodiment 78. The method of Embodiment 77, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2.
[0822] Embodiment 79. The method of Embodiment 77, wherein at least 50% of the polynucleotide sequence is codon optimized.
[0823] Embodiment 80. The method of Embodiment 79, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine. [0824] Embodiment 81. The method of Embodiment 80, wherein all uracil nucleosides are modified to be N1 -methylpseudouridine.
[0825] Embodiment 82. The method of any of Embodiments 76-81, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
[0826] Embodiment 83. The method of Embodiment 82, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid; (iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid; (v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35- 45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid; (vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid; (vii) the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid; (viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5- 50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid; (xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid; (xii) the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid; (xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA; or (xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA.
[0827] Embodiment 84. The method of any of Embodiments 76-83, wherein the SARS-CoV-2 neutralizing antibodies can be detected in the subject by day 28 after administration.
[0828] Embodiment 85. The method of any of claims Embodiments 76-83, wherein the SARS-CoV-2 neutralizing antibody production is enhanced in the subject at day 42 after receiving a second dose of the nucleic acid vaccine or pharmaceutical composition.
[0829] Embodiment 86. The method of any of Embodiments 76-85, wherein the dose administered is 16 ug.
[0830] Embodiment 87. The method of any of Embodiments 76-85, wherein the dose administered is 40 ug.
[0831] Embodiment 88. The method of any of Embodiments 76-85, wherein the dose administered is 100 ug.
[0832] Embodiment 89. The method of any of Embodiments 76-88, wherein the SARS-CoV-2 neutralizing antibody is to alpha (B.l.1.7).
[0833] Embodiment 90. The method of any of Embodiments 76-88, wherein the SARS-CoV-2 neutralizing antibody is to beta (B.1.351).
[0834] Embodiment 91. The method of any of Embodiments 76-88, wherein the SARS-CoV-2 neutralizing antibody is to delta (B.1.617.2).
EXAMPLES
Example 1. Comparison of PTX-B to COMIRNATY® in Humans
Study Overview [0835] A Phase II, In-Human, Randomized, Double-Dummy, Observer-Blind, study to evaluate the safety, tolerability, and immunogenicity Study of PTX-B compared to COMIRNATY® vaccine was conducted in healthy adults (male and non-pregnant females) aged 18-64 who are negative to SARS-CoV-2 (COVID-19 disease).
Inclusion Criteria for Study
[0836] Subjects were required to meet all inclusion criteria (number 1-8 below) to be eligible for study participation.
[0837] 1) Willing and able to provide written informed consent to participate prior to commencing any study-related activities.
[0838] 2) Must be able to attend all visits (scheduled and unscheduled, as applicable) for the duration of the study and comply with all study procedures.
[0839] 3) Healthy adult males and females 18 to 64 years of age, inclusive, at
Screening Visit (Visit 1).
[0840] 4) Negative to SARS-CoV-2 (COVID-19 disease) at screening and at each dosing, without evidence of recent of exposure or viral respiratory disease not identified as influenza or respiratory syncytial virus (RSV) (febrile or lower respiratory tract infection). [0841] 5) Must be in general good health before study participation with no clinically relevant abnormalities that could interfere with study assessments at investigator’s discretion. Subjects may have stable comorbidities (no change in medications, exacerbations, or hospitalizations in past three (3) months).
[0842] 6) Women of childbearing potential (WOCBP) and men whose sexual partners are WOCBP must be able and willing to use at least one (1) highly effective method of contraception (i.e., including hysterectomy, bilateral salpingectomy, and bilateral oophorectomy, hormonal oral [in combination with male condoms with spermicide], transdermal, implant, or injection, barrier [i.e., condom, diaphragm with spermicide]; intrauterine device; vasectomized partner [six months minimum], clinically sterile partner; or abstinence) during the study. A female subject is considered a WOCBP after menarche and until she is in a postmenopausal state for 12 consecutive months (without an alternative medical cause) or otherwise permanently sterile. Subjects not of childbearing potential are not required to use any other forms of contraception during the study. Non- childbearing potential is defined as subject confirmed: Surgical sterilization (e.g., bilateral oophorectomy, bilateral salpingectomy, bilateral occlusion by cautery, hysterectomy, or tubal ligation). Postmenopausal (defined as permanent cessation of menstruation for at least 12 consecutive months prior to screening); if postmenopausal status is unclear, pregnancy tests will be performed prior to vaccinations.
[0843] 7) WOCBP must have a negative pregnancy test before each vaccination. If menopausal status is unclear, a pregnancy test is required.
[0844] 8) For inclusion into the open label safety extension the subject must have received at least one (1) vaccination prior to Day 42. However, subjects who have had any procedures required for Visit 1 (Screening Visit) performed under a separate approved Pre-screening Protocol PRO-CL-PS, may be enrolled in this study, and be deemed to have completed those procedures for the screening visit if performed within 30 days of Visit 1 (Screening Visit). Following review and completion of any remaining procedures, subjects may be eligible for Visit 2 providing a COVID-19 is negative within seven (7) days of Visit 2. Records of all procedures performed (including results of laboratory tests) must be available from the Screening Protocol.
Exclusion Criteria for Study
[0845] Subjects will not be eligible for study participation if they meet any of the exclusion criteria:
[0846] 1) History of an acute or chronic medical condition that, in the opinion of the investigator, would render vaccination unsafe or would interfere with the evaluation of responses.
[0847] 2) History of ongoing clinical condition or medication or treatments that may adversely affect the immune system.
[0848] 3) Subjects with pulse oximetry <90% at screening and prior to each vaccination. [0849] 4) Individuals who are antigen positive, seropositive or reverse transcriptase polymerase chain reaction (RT-PCR) positive for SARS-CoV-2, including prior to a second dose of PTX-B.
[0850] 5) Individuals who are at increased risk of exposure to SARS-CoV-2 (e.g., healthcare workers, emergency responders).
[0851] 6) Close contact of anyone known to have SARS-CoV-2 infection within 30 days prior to vaccine administration.
[0852] 7) Individuals with any elevated (Grade 1 or higher) laboratory test assessed as clinically significant for age/sex by the investigator at screening.
[0853] 8) Individuals known to have anti-platelet antibodies.
[0854] 9) Individuals with myorcadtitis and/or arrythmias.
[0855] 10) Active neoplastic disease (excluding non-melanoma skin cancer that was successfully treated) or a history of any hematological malignancy. (“Active” is defined as having received treatment within the past five years).
[0856] 11) Long-term (> two (2) weeks) use of oral or parenteral steroids or high-dose inhaled steroids (> 800 pg/day of beclomethasone dipropionate or equivalent) within six months before screening (nasal and topical steroids are allowed).
[0857] 12) History of autoimmune, inflammatory disease, or potential immune- mediated medical conditions (PIMMCs).
[0858] 13) Women currently pregnant, lactating, or planning a pregnancy between enrollment and Day 42.
[0859] 14) History of immune thrombocytopenic purpura (ITP).
[0860] 15) History of Guillain-Barre Syndrome or any degenerative neurology disorder.
[0861] 16) History of anaphylactic-type reaction to any injected vaccines.
[0862] 17) Known or suspected hypersensitivity to one or more of the components of the vaccine.
[0863] 18) Known history of alcohol abuse, illicit drug use, physical dependence to any opioid, or any history of drug abuse or addiction within 12 months of screening. [0864] 19) Acute illness or fever (temperature >37.5C) within three (3) days before study enrollment (enrollment may be delayed for full recovery if acceptable to the investigator and within Protocol-defined windows).
[0865] 20) Individuals currently participating or planning to participate in a study that involves an experimental agent (vaccine, drug, biologic, device, or medication); or who have received an experimental agent within one (1) month (three (3) months for immunoglobulins) before enrollment in this study; or who expect to receive another experimental agent up to Day 90 in this study.
[0866] 21) Receipt of immunoglobulin or another blood product within the three (3) months before enrollment in this study or those who expect to receive immunoglobulin or another blood product during this study.
[0867] 22) Individuals using prescription medications for prophylaxis of SARS-CoV-2.
[0868] 23) Individuals who plan to receive another vaccine within the first three (3) months of the study (except influenza vaccine which should not be given within two (2) weeks of vaccine).
[0869] 24) Receipt of any other SARS-CoV-2 or other experimental coronavirus
(MERS, SARS etc.) vaccine at any time prior to or during the study.
[0870] 25) Receipt of any investigational vaccine or investigational drug within one (1) month of enrollment and through the end of the study (one (1) year after the last vaccination).
[0871] 26) Planning international travel from enrollment through Day 43.
[0872] 27) History of surgery or major trauma within 12 weeks of screening, or surgery planned during the study.
[0873] 28) Significant blood loss (> 400 mL) or has donated one or more units of blood
(within 56 days for males and 84 days for females) or plasma (within two (2) weeks) prior to study participation.
[0874] 29) Positive urine drugs of abuse screen test result.
[0875] 30) Positive screen for HIV-1 and HIV-2 antibodies, HbsAg, or HCV antibody.
[0876] 31) Involved in the planning or conduct of this study. [0877] 32) Unwilling or unlikely to comply with the requirements of the study.
[0878] 33) Subjects would be excluded from the open label safety extension for participation in a study that would affect potential immunogenicity evaluation during ongoing participation in this study. Participation in any other study is allowed after Day 90.
Study Compositions
[0879] The vaccine product was presented as a 0.2 mg/mL, 2 mL fill in a 3 mL United States Pharmacopeia / European Pharmacopeia Type I borosilicate glass vial with a fluororesin laminated bromobutyl rubber stopper and an aluminum cover seal with a red, plastic, flip-off cap. PTX-B (SEQ ID NO: 27) messenger ribonucleic acid Humoral Vaccine was an injectable IM 0.2 mg/mL 0.5 mL solution administered at a dose of 40 pg. COMIRNATY® messenger ribonucleic acid Humoral Vaccine was an injectable IM 0.2 mg/mL 0.3 mL solution administered at a dose of 30 pg.
[0880] The placebo was commercially available sodium chloride 0.9% injectable IM solution 0.0 mg/mL 0.5 mL.
Overall Study Design
[0881] Enrollment started with 15 subjects assigned in an open-label manner to 40 pg PTX-B to obtain an early assessment of cell-mediated immunity at 2 weeks post second vaccination (Day 42) and continued in the randomized fashion with subjects assigned to one of the study groups in a 2: 1 ratio respectively for a total of 350 individuals assigned to the PTX-B vaccine group and 175 assigned to the COMIRNATY® vaccine group.
[0882] There were no clinically meaningful differences in demographic and baseline characteristics between the PTX-B and COMIRNATY® groups. The majority of the overall population was Black/ African American (88.7%) primarily from South Africa and 52.4% of subjects were male. The mean age was similar in the PTX-B and COMIRNATY® groups; 31.4 years and 32.8 years, respectively. Demographics and baseline characteristics for the subjects who received at least one dose of the study vaccine are provided in Table 6.
Table 6. Demographics and Baseline Characteristics
Figure imgf000283_0001
[0883] Subjects in both groups received their first dose of vaccine at Day 1. Subjects assigned to the COMIRNATY® vaccine treatment arm received their second dose at Day 21 and subjects assigned to the PTX-B treatment arm received their second dose at Day 28. In order to preserve the observer-blind (including treatment assignment), placebo was administered to the relevant treatment arm at each of the dosing visits. Blood draws for antibody titer assays were taken prior to administration on Days 1, 8, 21, 28, and 2-weeks post 2nd vaccine dose.
[0884] Subjects visit the clinical site for screening in a separate visit up to 30 days prior to receiving a first dose (Day 1). Study visits were performed on Days 1, 8, 21, 28, 35 and 42 and additional study visits will be conducted on Days, 90, 180 and 365. Safety phone calls to subjects were performed on Days 2 and 29. Screening procedures included informed consent, evaluation of entry criteria, demographics, height, weight, body mass index, medical and surgical histories, safety evaluations (including SAE evaluations, recording prior medications and procedures, physical examinations, vital sign assessments, clinical blood and urine samples, point-of-care analysis by rapid antigen tests were performed at each visit, with the option to confirm results by nasopharyngeal (NP) swab if the rapid antigen test were positive), and blood samples for immunogenicity analysis (antibodies). On Day 1, randomization was performed. On Day 1, 21 and 28, eligibility was confirmed and safety evaluations, paper diary training and distribution, and vaccine or placebo administration was performed. Safety evaluations, blood and urine samples for safety evaluations were performed through the end of study visit. Assessments of unsolicited AEs were performed on Day 1 through Day 42 and for subjects that continued to the next visit at Day 90, a safety data cut was performed at Day 57 for a 4 weeks post 2nd dose evaluation. Medically attended AE (MAAE) assessments were performed when subjects visited medical or other healthcare providers outside of routine and/or study clinic visits. Assessments of NOCDs, AESIs (including COVID-19 cases), and PIMMCs were performed from Day 1 through the end of study. The SAEs were assessed throughout the study.
[0885] Vaccine and placebo were prepared by unblinded site pharmacists and administered by unblinded site pharmacist or unblinded administrator in a blinded fashion , i.e., subjects could not see the vials or preparation materials or identify contents of the syringe. The IM injection was made in the upper arm deltoid muscle. Administration to the non-dominant side was preferred but not required, and injection location was recorded by the EDC. Assessments were performed by blinded personnel.
[0886] Subjects were observed for immediate AEs and/or reactogenicity for approximately 15 minutes after administration of vaccine. Subjects were provided with a Diary Card and trained to record specifically elicited systemic and local symptoms daily, as well as any additional AEs, during the follow-up period after each vaccination.
[0887] The duration of ongoing monitoring of subjects is approximately 12 months for each subj ect.
Safety and Tolerability
[0888] The primary safety analysis of results consisted of a comparison of PTX-B vaccine to COMIRNATY® vaccine. The safety analysis time period corresponded to the data collected until 28 days following second vaccine (or placebo). Safety data were analyzed and reported using descriptive summary statistics.
[0889] Primary safety and tolerability endpoints were collected until 28 days following second administration (vaccine or placebo) and included: injection site reactions (e.g., arm check evaluations including pain, tenderness, erythema/redness, induration/swelling) through the seventh day post each administration; incidence of solicited adverse events (SAEs; e.g., fever, chills, nausea/vomiting, diarrhea, headache, fatigue, myalgia) through the seventh day post each administration; incidence of unsolicited adverse events (AEs) from Day 1 through 4 weeks following second administration; Treatment Emergent Adverse Events (TEAEs) including medically attendant adverse events (MAAEs), new onset chronic diseases (NOCDs), SAEs, adverse event of special interests (AESIs), and potentially immune-mediated medical conditions (PIMMCs) from Day 1 through 4 weeks following second vaccination (or Day 57 for subjects who miss their second vaccination). For longer-term follow-up, AEs and SAEs continue to be reported until end of the study (approximately 1 year post first administration).
[0890] The Safety Analysis Population included a total of 565 subjects who received at least 1 dose of a study vaccine (i.e., either PTX-B or COMIRNATY®). An overall 475 subjects (84.1%) who were randomized received both doses of vaccine; 15 subjects (100%) in the Open label part of the study and 285 subjects (79.4%) in the randomized part for the PTX-B group and 175 subjects (91.6%) in the COMIRNATY® group. Of the 71 (19.8%) randomized subjects in the PTX-B group that did not receive the second vaccine, 57.7% did not meet eligibility criteria for second injection due to being positive for SARS-CoV-2 during the Omicron wave in South Africa and 26.8% were not dosed due to non-compliance. The rate of subjects who did not meet eligibility criteria for the second injection due to being positive for SARS-CoV-2 infection was comparable to the 66.7% in the COMIRNATY® group. No subjects in either treatment group were discontinued from treatment due to an AE. Of the 565 subjects, 38 (6.7%) discontinued the study before Day 35/42 (2 weeks post second dose), 28 (7.8%) in the PTX-B group and 10 (5.2%) in the COMIRNATY® vaccine group. The primary reason for study discontinuation was non- compliance in both groups. No subject permanently discontinued the study due to an AE. A complete set of subject enrollment and disposition information was recorded in Table 7.
Table 7. Subject Enrollment and Disposition
Figure imgf000286_0001
[0891] PTX-B was found to have an acceptable safety profile characterized by transient injection site pain (61.2%), headache (30.1%), fatigue (28.1%), muscle pain (25.0%) and joint pain (13.2%). Reactogenicity was mostly mild to moderate and short-lived after dosing, and the AE profile did not suggest new safety concerns.
[0892] The overall incidence of all-causality solicited TEAEs was similar between both treatment groups after the first and second vaccine, 71.6% and 59.0% for PTX-B and 74.2% and 62.4% for COMIRNATY®, respectively. The incidence of local reactions was similar between the groups. Treatment emergent solicited systemic events were generally higher among subjects who received COMIRNATY® in comparison to those who received PTX-B after each dose. The overall incidence of solicited local and systemic AEs was higher after the first dose for both treatment groups. Local site reactions for PTX-B were mostly mild and comparable to COMIRNATY®, occurring after each dose in 62.6% vs 65.1% and 53.8% vs 57.1% of the Safety Population. Respectively. Solicited systemic reactions were generally lower among participants who received PTX-B vs COMIRNATY® after each dose occurred in 49.7% vs 58.6% and 36.9% vs 45.3%, respectively. Fig. 1 A shows the solicited adverse events by dose and severity. Solicited AEs reported as Grade 3 for PTX-B vs COMIRNATY® after each dose occurred in 3.7% vs 2.7% and 3.8% vs 1.2%, respectively. No Grade 4 (potentially life threatening) reactions were reported. The most commonly reported solicited AEs following any dose of PTX-B were pain at the injection site (61.2%), headache (30.1%), fatigue (28.1%), muscle pain (25.0%) and joint pain (13.2%). The majority were Grade 1 and resolved within several days of onset. Pain at the injection site was similar between PTX-B and COMIRNATY®, 61.2% vs 64% after Dosel and 53.4% vs 54.7% after Dose 2, respectively. Erythema, induration, and swelling were reported in <10% subjects and were numerically higher in the PTX-B. Fig. 1C shows the solicited systemic events within 7 days after each dose. In general, the incidence of systemic events was lower in the PTX-B group compared to the COMIRNATY® group. Fig. ID shows the solicited local reactions within 7 days after each dose. Most reactions were Grade 1 to 2 and Grade 3 reactions were low (2.0% overall).
[0893] The overall proportions of PTX-B subjects who reported an unsolicited adverse event were generally similar to COMIRNATY® subjects. Unsolicited AEs were mostly mild (PTX-B: 36.9% and COMIRNATY®: 36.6%) and moderate (PTX-B: 14.7% and COMIRNATY®: 14.1%) in severity. There were no other notable patterns or numerical imbalances between treatment groups for specific categories of unsolicited adverse events. An overview of unsolicited AEs reported for all enrolled subjects was recorded in Table 8 A and Table 8B. The percentage of PTX-B participants with unsolicited adverse events was similar to COMIRNATY® participants. There were no reports of serious adverse events (SAE) or specific categories of adverse events of special interests (AESI) including allergic, neurologic, inflammatory and thrombotic events.
Table 8A. Overall Summary of Unsolicited Treatment Emergent Adverse Events (TEAE)
Figure imgf000288_0001
Table 8B. Summary of Unsolicited Treatment Emergent Adverse Events (TEAE)
Figure imgf000288_0002
Figure imgf000289_0001
[0894] The frequency and severity of reported solicited local and systemic reactions, respectively, within 7 days following each dose of PTX-B and COMIRNATY® was recorded in Table 9 and Table 10, respectively.
Table 9. Subjects with Solicited Local Reactions by Maximum Severity
Figure imgf000290_0001
Figure imgf000290_0003
Table 10. Subjects with Solicited Systemic by Maximum Severity
Figure imgf000290_0002
Figure imgf000291_0001
Figure imgf000292_0001
[0895] Table 11 A shows rates of unsolicited AEs that occurred within 28 days post second vaccination and at rates of >1%. Severe AEs (> Grade 3 in severity) were reported in 2.1% of PTX-B (in 8 subjects) and 1.6% of COMIRNATY® (3 subjects). No subjects had AEs of myocarditis, pericarditis, or anaphylaxis. Palpitations ware reported in 3 PTX- B subjects. All cases were considered not related to study drug, short in duration and 1 case was linked to concomitant medications. Lymphadenopathy was reported in 3 subjects (1 PTX-B and 2 COMIRNATY®) (0.5%). All cases were considered not related to study drug. No AEs of lymphadenopathy in the arm and neck region were reported within 7 days after vaccination. 3 SAEs were reported in the PTX-B group (0.8%); all 3 SAEs (acute hepatitis, limb injury and hyperglycemia) were unrelated. One (1) pregnancy was reported in the PTX-B group following the first vaccine. The subject gave birth to a healthy term baby. No AESIs were prespecified in the study protocol. Table 1 IB shows rates of unsolicited AEs that occurred from 0 to 2 months post dose or 3 to 12 months post dose. The pre-specified medically significant events that had the potential to be causally associated with vaccine products including immune mediated events (autoimmune and neuroinflammatory disorders) were not reported by subjects.
Table 11A. Unsolicited Treatment-Emergent Adverse Events by System Organ Class and Preferred Term in >1% of Subjects in Any Treatment Group
Figure imgf000293_0001
Figure imgf000294_0001
Table 11B. Unsolicited Treatment-Emergent Adverse Events (>2%) by System
Organ Class (SOC) and Preferred Term (PT) in
Figure imgf000294_0002
Figure imgf000295_0001
[0896] There is no overall indication of decreased safety or tolerance for PTX-B as compared to COMIRNATY®. The overall proportions of unsolicited AEs over 12 months post vaccination were generally similar between PTX-B and COMIRNATY®. The long term safety profile of PTX-B is consistent with the previously authorized mRNA vaccines. Immunogenicity
[0897] Immune response endpoints evaluated included immunogenicity analysis using both the evaluable immunogenicity population (EIP) ant the modified intention-to-treat (mITT) populations. The EIP included subjects in the randomized arm of the Safety Population (above) without evidence of SARS-CoV-2 infection before or during vaccination assessment period, who received both doses of vaccine and have post-baseline immunogenicity (204 (15+189) subjects for PTX-B and 140 subjects for COMIRNATY® groups, 344 total subjects). The seronegative mITT population included subjects (222 subjects for PTX-B and 127 subjects for COMIRNATY® groups, 369 total subjects) in the randomized arm of the Safety Population (above) for who had no evidence of SARS- CoV-2 infection throughout the assessment period. The mITT population included subjects in the randomized arm of the Safety Population (above) who received both doses of vaccine regardless of SARS-CoV-2 status (301 subjects for PTX-B and 174 subjects for COMIRNATY® groups, 475 total subjects). The full analysis set included 565 total subjects with 374 subjects for PTX-B and 191 subjects for COMIRNATY®.
[0898] As seen in FIG. IB, subjects with no COVID infection (i.e., the EIP, right side histogram) had similar profile of local and systemic AEs to the total Safety Population (left side histogram) both for subjects receiving PTX-B (left bar at each time point) and COMIRNATY® (right bar at each time point).
[0899] Immune response endpoints evaluated included: Immunogenicity analysis using both the EIP and mITT populations. The geometric mean titer (GMT) was determined for SARS-CoV-2 pseudo virus neutralizing antibody titers (NT50) at Day 1 (pre- administration), Day 21 or 28 (COMIRNATY® or PTX-B respectively) preadministration of dose 2, and 14-days post dose 2 (Day 35 or 42 for COMIRNATY® or PTX-B respectively). Comparison of overall immunogenicity was summarized as the geometric mean ratio (GMR) at 2 weeks post second vaccination. Neutralizing antibody titers were determined by ELISA. A SARS-CoV-2 neutralizing antibody titers with a > 4- fold increase over pre-vaccination (baseline) was designated as a positive seroresponse, and the seroresponse rate determined at each time point.
[0900] As seen in FIG. 2 the Nab titer levels were similar at all time points between PTX-B (left box-plot at each time point) and COMIRNATY® (right box-plot at each time point) hand with a final GMT ratio of 0.81. This may have indicated a slightly increased response in the COMIRNATY® but the distribution of the 2-week post injection data suggests this ratio likely indicates no significant difference. The seroconversion Nab rate was likewise similar between both vaccine groups. At Day 21/28 the seroconversion rates were 74.0% and 75.2% for PTX-B and COMIRNATY® respectively. At Day 35/42 the rates were 89.1% and 92.8% for PTX-B and COMIRNATY® respectively. The uexpected high Nab titer at Day 1 in this population likely indicates some members of the population were previously exposed to SARS-CoV-2 prior to the study, despite the lack of any other evidence of previous infection. Additional post-hoc analysis was conducted to control for these potential outliers.
[0901] In order to control for unexpectedly high baseline, Nab titer levels, a post-hoc analysis was conducted on only those EIP subjects whose baseline Nab titer < 30 (~ LLQ). As seen in FIG. 3, the baseline (day 1) Nab titer levels are as expected and again about the same Nab titer levels were observed at all time points. Here, the GMT ratio between PTX- B (left box-plot at each time point) and COMIRNATY® (right box-plot at each time point) was 1.00 on Day 1, 1.61 on Day 21, 1.02 post dose 1 and 1.22 post dose 2, which indicated a slightly greater response in the PTX-B group. Again, the distribution of the data within the groups may indicate the differences are minor, but it is clear that the PTX- B vaccine caused at least as high an immune response as the COMIRNATY® vaccine. [0902] The ability of the PTX-B vaccine to cause an equal or better response compared to the COMIRNATY® as also observed when analyzing the fold increase in Nab (FIG. 4). Here the fold increase in Nab titer was higher in the PTX-B (194.0-fold increase, dark grey left bar at each time point) group at 2 weeks post 2nd dose compared to the COMIRNATY® (186.8-fold increase, light grey bar at each time point) and was generally consistent with levels seen with primary immunization.
[0903] In the mITT population, when high baseline Nab titers (Nab titer > 30) were eliminated in post-hock analysis, (FIG. 5) PTX-B (left box-plot at each time point) caused a similar or greater increase in Nab titer levels as compared to COMIRNATY® (right boxplot at each time point) with a GMT ratio of 1.01 on Day 1, 1.20 on Day 21, 0.92 post dose 1, and 1.29 post dose 2. Covid infections were balanced between groups. In order to examine the overall immunogenicity, the seroresponse rates of both vaccines in both groups was compared (Table 12). As was evident, both vaccines resulted in a 100% seroconversion rate after dose 2.
Table 12. Seroconversion in mITT and EIP (<30 titer at baseline)
Figure imgf000297_0001
[0904] When the analysis was expanded to include all subjects in the mITT population
(FIG. 6A), it was again observed that PTX-B (left box-plot at each time point) caused at least an equal increase in Nab titer levels as COMIRNATY® (right box-plot at each time point), with a GMR of 0.86 on Day 1, 0.89 on Day 21, 0.65 post dose 1, and 0.80 post dose 2.
[0905] The evaluable immunogenicity set were participants who received two doses of vaccine within the prespecified vaccination window and remained SARS-CoV-2 infection- naive throughout the immunogenicity assessment period. As shown in Fig. 6B, in the evaluable immunogenicity set PTX-B demonstrated non-inferiority as compared to COMIRNATY® with respect to GMT ratio of neutralizing antibodies. In the figure, GMT = Geometric Mean Titer; CI = Confidence Interval; Pre-specified non-inferiority margin of GMTs = 0.67; GMT ratio based on pre-specified ANCOVA model adjusting for age and baseline titer. Additionally, as shown in Table 13 PTX-B demonstrated non-inferiority as compared to COMIRNATY® with respect to seroresponse rate.
Table 13. Seroresponse Rate for EIP
Figure imgf000298_0001
[0906] Additional analysis was conducted on the subjects with no serological evidence of neutralizing antibody titers (defined as NT50 less than or equal to 30) at baseline (day 1). The mITT population with no baseline antibody titers included 219 total subjects with 149 subjects for PTX-B and 70 subjects for COMIRNATY®, the seronegative mITT population with no baseline antibody titers included 172 total subjects with 109 subjects for PTX-B and 63 subjects for COMIRNATY®, and the EIP with no baseline antibody titers included 159 total subjects with 99 subjects for PTX-B and 60 subjects for COMIRNATY®. As shown in Fig. 6C, the EIP showed that the GMT ratio was noninferior and numerically higher in favor of PTX-B versus COMIRNATY® (GMT = Geometric Mean Titer; CI = Confidence Interval; Pre-specified non-inferiority margin of GMTs = 0.67; GMT ratio based on pre-specified ANCOVA model adjusting for age and baseline titer). Additionally, as shown in Table 14 the seroresponse rates for PTX-B and COMIRNATY® were both 100%. For the mITT (participants who received both vaccinations and had at least one immunogenicity assessment) and seronegative mITT populations (participants who had no evidence of SARS-CoV-2 infection throughout the immunogenicity assessment period), the GMT ratio was non-inferior and numerically higher in favor of PTX-B compared to COMIRNATY® and all seroresponse rates were 100% for all groups (see Table 15A and Fig. 6D).
Table 14. GMT and Seroresponse Rate for Seronegative EIP at Baseline
Figure imgf000299_0001
Table 15A. GMT and Seroresponse Rate for mITT
Figure imgf000299_0002
[0907] For the EIP group, cell mediated immunity was evaluated as shown in Fig. 6E. Cellular immunity in PTX -B group shows a predominant IFN-y production by Day 42, which indicates a favorable Thl response (tested by ELISpot after stimulation with Spike peptide pool using peripheral blood mononuclear cells (PBMCs)) that is comparable to COMIRNATY®.
[0908] Primary immunization with PTX-B demonstrated non-inferiority to COMIRNATY® (saliva antibody responses for anti-RBD and anti-spike shown in FIG. 6F) and comparable durability of the long-term response (Tables 15B and 15C). Examination of the durability of neutralizing antibody titers for ancestral SARS-CoV-2 showed that 2 vaccinations elicited durable neutralizing antibody responses up to 12 months in both groups. A continuous decline of binding antibody was observed until Day 365. At the peak the levels, binding antibodies were approximately 1.5-fold higher in COMIRNATY® participants. There was approximately a 2-fold decline in PTX-B group and 2.5 decline in Comirnaty group at Day 90 resulting in similar levels of binding antibody titers. An additional approximately 2-fold decline was observed at the 6 month timepoint in both groups. During the 12-month follow-up, subjects with breakthrough infections had higher titers with a less steep rate of decay.
Table 15B. Neutralizing Titers for Ancestral SARS-CoV-2 (mlTT Population)
Figure imgf000300_0001
Table 15C. Durability of S-Binding IgG for Ancestral SARS-CoV-2 (mlTT
Population)
Figure imgf000300_0002
after vaccination) representing approximately 50-fold increase from baseline. A continuous and comparable decrease was then observed up to Day 365. At 6 months, neutralizing antibodies were comparable between 2 treatment groups and were in a similar range at Day 21 levels showing an approximately 2-fold decline between 2 weeks and month 6 after vaccination. The rate of decline of binding antibody was higher in both treatment groups over the course of the 12-month follow up. Two doses of PTX-B vaccine elicited a rapid induction of humoral response followed by relatively slow waning of the immune response with approximately 58% and 75% lower neutralizing antibody and IgG levels (Table 15D), respectively, 6 months after the second dose. The results of neutralizing antibody waning are consistent with data published for primary vaccination with COMIRNATY®.
Table 15D. Increase and Waning of Neutralizing and S-Binding IgG Antibodies Over
12 Months Post-Vaccination
Figure imgf000301_0002
The fold-rise is calculated relative to baseline and decrease is calculated relative to Day 35/42.
Figure imgf000301_0001
[0910] The subjects with a breakthrough omicron SARS-CoV-2 infection during the 12-month follow-up had a somewhat less steep rate of decay in neutralizing-antibody titers. Elevated T-cell responses (predominantly Thl) were maintained at 6-months (FIG. 6G). Analysis of time to COVID-19 infection showed a lower incidence of disease with
PTX-B versus COMIRNATY® (Table 15E).
Table 15E. Time to COVID-19 Infection (Seronegative mITT Population; p=0.00019)
Figure imgf000301_0003
[0911] The hazard ratio for SARS-CoV-2 infection was not significant between PTX-B and COMIRNATY® for the mITT population (Table 16) and for the full analysis population (Table 17). The analysis period for SARS-CoV-2 infection was from 2 weeks post first dose to 4 weeks post second dose or, for Table 16, Day 57 for participants who did not receive the second dose. Participants with SARS-CoV-2 infection earlier than 2 weeks post first dose were not included in the analysis; Percentages are based on the total number of participants included in the analysis (Total column); CI = confidence interval.
Table 16. Hazard Ratio for mITT Population
Figure imgf000302_0001
Table 17. Hazard Ratio for Full Analysis Population
Figure imgf000302_0002
[0912] In summary, PTX-B demonstrated non-inferiority to COMIRNATY® in terms of GMT ratio (0.84, 95% confidence interval 0.69-1.02) as well as non-inferiority in terms of seroresponse rates at two weeks after the second vaccination in adults aged 18 to 64 years. In the analysis of participants with no detectable neutralizing antibody titers at baseline showed numerically higher GMT ratio of 1.23 (95% CI 0.96, 1.57) for PTX-B vs. COMIRNATY® and 100% seroresponse rate. Non-inferiority of GMT ratios was observed with the primary analysis population was confirmed in the mITT populations, the T-cell responses analyzed in a sub-set of participants showed predominant Thl response, and the hazard ratios for COVID-19 were similar between PTX-B and COMIRNATY®. [0913] These results showed that the PTX-B vaccine was at least as effective in increasing the Nab titer level and inducing seroconversion in humans as the COMIRNATY® vaccine. They also showed that the PTX-B vaccine was at least as safe and well tolerated as the COMIRNATY® vaccine. Example 2. Variant Efficacy
[0914] In order to analyze the efficacy of PTX-B and COMIRNATY® against the
D614G variant pseudovirus, the geometric mean titer (GMT) was determined for SARS- CoV-2 pseudo virus neutralizing antibody titers 2 weeks after the second vaccine dose as described in Example 1. The GMR was calculated for comparison of PTX-B to COMIRNATY®. This analysis utilized samples collected from individuals participating in the study described in Example 1. Specifically, 285 samples were chosen from individuals receiving the PTX-B vaccine and 175 from those receiving COMIRNATY®. The samples were then sorted into EIP or mITT populations as described previously. A summary of the neutralizing antibody titer results was recoded in Table 18.
Table 18. Nab Titers for Two Variants
Figure imgf000303_0001
antibody titer and 0.79 for the D614G neutralizing antibody titer. Post hoc analysis of the mITT population with a baseline Nab titer of <30 showed a GMR of 1.35 for the Ori neutralizing antibody titer and 1.27 for the D614G neutralizing antibody titer.
[0916] For the total EIP population, the GMR was 0.85 for the Ori neutralizing antibody titer and 0.83 for the D614G neutralizing antibody titer. Post hoc analysis of the mITT population with a baseline Nab titer of <30 showed a GMR of 1.29 for the Ori neutralizing antibody titer and 1.29 for the D614G neutralizing antibody titer.
[0917] These results showed that the PTX-B vaccine was effective against the D614G variant, and that PTX-B was overall as, or more, effective than COMIRNATY®.
Example 3. Booster Efficacy
[0918] In order to evaluate the efficacy of PTX-B to be used as a booster to increase antibody levels in subjects already possessing neutralizing antibodies, the geometric mean titer (GMT) was determined for SARS-CoV-2 pseudo virus neutralizing antibody titers (NT50) for the mITT and EIB populations in Example 1 with baseline Nab titers >30.
[0919] A summary of the GMT results was recorded in Table 19 and overall seroconversion was recorded in Table 20.
Table 19. Nab Titers for Variable Baseline Nab Levels
Figure imgf000304_0001
Figure imgf000305_0001
Table 20. Seroconversion in mlTT and EIP
Figure imgf000305_0002
[0920] As seen here and in Figure 7, individuals with pre-existing antibody levels who received PTX-B when PTX-B showed an increased level of titers. For example, the antibody levels at day 21 (for those tested at 3 weeks post administration) or day 28 (for those tested 4 weeks post administration) were higher than those seen in individuals with less than 30 Nab at baseline. Also, individuals who had Nab titers greater than 100 who produced the maximum amount of antibodies after a single dose of PTX-B as the antibody levels were consistent after the second dose received (either day 21 or day 28) and measured at day 42.
Example 4. Long-Term Efficacy of PTX-B
[0921] In order to evaluate the long-term efficacy of PTX-B, the antibody titer levels in subjects were evaluated at day 1 (prior to the first administration of PTX-B), day 8, day 28 (prior to second administration of PTX-B), day 90, day 180, day 365 and day 395 (12 months after second administration of PTX-B). PTX-B was evaluated in a study including 60 healthy volunteers 18 to 64 years of age who were SARS-CoV-2 seronegative and RT- PCR negative. Subjects were treated with two doses of PTX-B at dose levels of 16 pg, 40 pg, and 100 pg, administered at a 4 week interval. Each cohort included 20 subjects (15 receiving PTX-B and 5 receiving placebo). All subjects active in the study completed a 12-month follow-up after second immunization (Day 395).
I. Anti-Spike Protein and Anti-RBD Immunoglobin G and Immunoglobin A Analysis Analytical Methods
[0922] The antibody response induced by PTX-B was analyzed in serum samples by two methods, MSD (a multiplex immunoassay sandwich-based method with an electrochemiluminescent (ECL) readout on the Mesoscale Discovery platform which is referred to as the MSD method) and ELISA.
[0923] For MSD, anti-Spike (S) and anti RBD immunoglobulin G (IgG) and immunoglobulin A (IgA) were measured for all doses from days 8, 28, 42, 90, 180 and 365 using MSD. Individual values are reported in Binding Antibody Units (BAU/mL) since the assay was calibrated with the NIB SC international standard. Cohort values are reported as Geometric Mean Titers (GMT) and 95% CI. Additionally, 21 samples from COVID-19 RNA diagnosed convalescence serum were also tested and used as a benchmark to assess the strength of antibody induction by PTX-B vaccination.
[0924] For ELISA, anti-S IgG was determined on the same serum samples for all doses by ELISA on day 0, 28 and 42. IgG Antibody titers values are reported in BAU/mL since the assay was calibrated with the NIBSC international standard. Sera from 29 SARS-CoV- 2 convalescence patients was also tested by ELISA and IgG titers were used to evaluate the strength of antibody induction by PTX-B vaccination.
Results
[0925] For the modified intent-to-treat (mITT) population, small to no increases were seen in anti-RBD or anti-S (IgG) GeoMeans by Day 8, and the small decrease noted (PTX-B 40 pg dose group) was not statistically significant. However, by Day 28, the increases in anti-RBD and anti-S titers for all 3 dose levels, compared to pooled placebo, were statistically significant (p < 0.0001), with titers more than 20-times the base line indicating seroconversion in all subjects. Anti-RBD and anti-S levels continued to increase and by Day 42 the PTX-B 100 pg dose group had anti-RBD and anti-S levels that were -1959 and -1293 fold greater than baseline respectively, followed by the PTX-B 40 pg dose group with -823 and -568-fold greater than baseline, and PTX-B 16 pg dose group with -647 and -175-fold greater than baseline respectively. There were no changes in anti-RBD or anti-S levels over time in the pooled placebo group (Fig. 8).
[0926] Similar results were observed by ELISA (Fig. 9). Small increases were seen in total anti-S protein (IgG) Geometric Means as early as Day 8; however, the increases were not statistically significant. By Day 28, the increases in total anti-S protein levels for all 3 dose levels were statistically significant when compared to placebo (p = 0.0001). Total anti-S-protein levels continued to increase and by Day 42 the PTX-B 100 pg dose group had anti-S protein levels that were -622-fold greater than baseline, followed by the PTX-B 40 pg dose group with -503-fold greater than baseline, and PTX-B 16 pg dose group with -372-fold greater than baseline. There were no changes in total anti-S protein levels over time in the pooled placebo group. The level of antibodies produced in subjects by PTX-B compare favorably to the levels of antibodies produced by other mRNA vaccines that have been approved for use against COVID-19.
[0927] IgG titers after 6 months (Day 180, week 26) post vaccination stayed elevated were higher than the levels observed after the first immunization and reflected the dose dependency (Fig. 9). The titers levels were consistent within each cohort and the levels of antibodies induced by vaccination with 40 and 100 pg of PTX-B at six months post vaccination were higher than COVID-19 convalescence serum antibodies. The 16 pg vaccination anti-RBD/anti-S antibody titers at six months were similar to COVID-19 convalescence serum titers. These results indicate that vaccination with PTX-B can induce a long-lasting antibody response against SARS-CoV-2 for at least 6 months.
[0928] PTX-B vaccination also induced production of anti-RBD and anti-S IgA antibodies that were detected in serum of all three cohorts by Day 28. IgA titers were the highest by Day 42. Anti-RBD and anti-Spike for the 16 ug dose group was more than 6- fold and 4-fold the base line respectively, for the 40 pg dose group was more than 7-fold and 14 fold respectively and for the 100 pg dose was more than 26-fold and 36-fold respectively (Fig. 10 for anti-spike). The duration of the IgA antibody titers in sera was shorter than the one observed for IgG. At 3 months post vaccination (Day 90, 12 weeks), anti-RBD IgA titers were 2 to 5-fold higher than base line for 40 pg and 100 pg dose cohorts respectively, and by Day 180 only the 100 pg cohort had a 4-fold titer higher than base line (Fig. 11). These results indicate that vaccination with PTX-B can induce antibody isotype switching to produce an IgA response against SARS-CoV-2 and doses of 40 and 100 pg induce long lasting titers for at least 6 months post vaccination.
II. CO VID- 19 Neutralizing Antibody Titer
Analytical Methods
[0929] The neutralizing antibody response induced by PTX-B against SARS-CoV-2 was analyzed by two methods. The first is a MSD S-ACE2:S blocking binding assay which is a competition binding assay that measures antibodies that block the binding of ACE-2, labeled with MSD-Sulfotag, to the SARS-CoV-2 S and RBD antigens. Here results are reported in Unit/mL, which corresponds to a neutralizing activity of 1000 ng/mL of antibody that can bind to S protein. Serum samples from all three cohorts were tested at Days 1, 8, 28, 42, 90, 180 and 365. The assay was performed and validated at CIRION BioPharma Research Inc.
[0930] The second is a pseudovirus neutralization assay where the pseodtyped virus is an rVSV that encodes the Spike protein of SARS-CoV-2 Wuhan strain and A G-luciferase. The virus can infect Vero E6 cells which results in cellular Luciferase expression. The neutralizing activity of the sera is measured by its capacity to reduce Luciferase levels during infection. Results of NT50 titers are further reported as lU/mL based on the calibration of NIB SC international standard. Serum samples from all three cohorts were tested at Days 1, 28, and 42. The assay was validated and performed at Nexelis (a CEPI network contract research laboratory that uses standardized methods to evaluate COVID- 19 vaccines around the world).
Results
[0931] The blocking antibody levels in U/mL indicated that all subjects in the Phase 1 study produced neutralizing antibodies by Day 28 with the first immunization. The anti- COVID-19 neutralization titers GeoMeans titers for all 3 dose levels, compared to pooled placebo, were statistically significant (p < 0.0028). Anti-COVID-19 neutralization titers continued to increase and by Day 42 the PTX-B 100 pg dose group had anti-COVID-19 neutralization titers that were ~112-fold greater than baseline, followed by the PTX-B 40 pg dose group with ~47-fold greater than baseline, and PTX-B 16 pg dose group with -31 fold greater than baseline. There were no changes in anti-COVID-19 neutralization titers over time in the pooled placebo group. The antibody levels decreased after Day 42 but remained higher than the titers after the first immunization until Day 180, indicating strong and lasting blocking response. These results indicate that PTX-B induced a strong neutralizing antibody response against Spike of SARS-CoV-2.
[0932] The blocking antibody titers were analyzed as percentage of inhibition based on the total binding of the ACE-2 receptor in the absence of antibodies. All subjects from the 3 dose levels cohorts showed >50% average blocking activity by Day 28 and all groups reached 100% blocking activity by Day 42 with samples diluted at 1 : 100 or greater.
[0933] When samples were tested for neutralizing antibodies with the pseudovirus assay the Geometric Mean increases in anti-COVID-19 50% serum neutralization titers (NT50) for all 3 dose levels were statistically significant by Day 28 when compared to pooled placebo (p < 0.0001). Anti-COVID-19 neutralization titers continued to increase and by Day 42 the PTX-B 100 pg dose group had anti-COVID-19 neutralization titers that were -423 -fold greater than baseline, followed by the PTX-B 40 pg dose group with titers ~ 161 -fold greater than baseline, and PTX-B 16 pg dose group with titers ~87 fold greater than baseline. There were no changes in anti-COVID-19 neutralization titers over time in the pooled placebo group (FIG. 12). Since the assay was calibrated with the WHO international standard for anti-SARS-CoV-2 immunoglobulin (NIBSC code 20/136), the NT50 titers were converted to international units using the conversion factor: NT50/ 1.871=IU/mL. Based upon these results, it can be concluded that PTX-B induced high neutralizing antibody levels in a dose dependent manner for all vaccinated subjects. Neutralization titers evident after the first dose were substantially boosted after the second dose, supporting a 2-dose vaccination series.
Example 5. COVID-19 Neutralization Antibodies Against Variants of Concern
[0934] To determine if vaccination with PTX-B could induce neutralizing antibodies against variants of concern (VOC) (i.e., Alpha (B. l.1.7), Beta (B.1.351), and Delta (B.1.617.2) strains of SARS-CoV-2), serum samples were tested in a neutralization assay using pseudoviruses expressing Spike proteins from each of the VOC. The assay utilizes Spike-pseudotyped lentivirus particles expressing either wild-type, D614G or Spike VOCs to infect HEK293T-cells expressing ACE2/TMPRSS2 and evaluates luciferase reduction as the neutralization readout. The neutralization ID50 from the assay using lentivirus correlated very well with the neutralization NT50 from the assay performed at Nexelis using rVSV virus (FIG. 13). These results demonstrated a reliable comparison of both assays and evaluation of PTX-COVID19-B induction of neutralizing antibodies.
[0935] The levels of neutralization against G614 / VOC and ID50, from PTX-B Phase 1 subject sera are shown in FIG. 14. The results indicate that the sera neutralization capacity is reduced for VOC when compared to the G614 strain, with the most reduced being against Beta followed by Delta and then Alpha. However, as the vaccine dose increases from 40 pg to 100 pg, the geometric mean ID50 against the VOC also increases to levels considered protective. Moreover, PTX-B serum neutralization activity compares favorably to the data reported for LTCH workers vaccinated with approved COVID19 mRNA vaccines. The fold decrease of neutralization titers compared to the response to D614G for the Alpha variant is ~2 for all doses, and ~3 for Delta, whereas for Beta it is 6 times lower for the 16 pg dose and ~ 3 for the 40 pg and 100 pg doses (Table 21).
Table 21. Geometric Mean Titer with 95% Confidence Intervals for Serum 50% Neutralization by Vaccination Group on Day 42 against VOC
Figure imgf000311_0001
Figure imgf000311_0002
Equivalents and Scope
[0936] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
[0937] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
[0938] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.
[0939] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0940] In addition, it is to be understood that any particular embodiment that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions described herein (e.g., any therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
[0941] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.
[0942] While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

Claims

1. A method of inducing an immune response in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32.
2. The method of claim 1, where the first sequence region is at least 81%, 82%, 83%, 84%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to or comprising SEQ ID NO: 7.
3. The method of claim 1 or 2, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2.
4. The method of any one of claims 1-3, wherein at least 50% of the polynucleotide sequence is codon optimized.
5. The method of any one of claims 1-4, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine.
6. The method of claim 5, wherein all uracil nucleosides are modified to be Nl- methylpseudouridine.
7. The method of any of claims 1-6, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid.
8. The method of claim 7, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid,
(ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid,
(iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0- 10 mol% of a PEGylated lipid,
(iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid,
(v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid,
(vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid,
(vii)the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid,
(viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid,
(ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid, (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5-50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid,
(xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid,
(xii)the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid,
(xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA, or
(xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA. The method of any of claims 1-8, wherein the pharmaceutical composition comprises about 0.2 mg/mL of nucleic acid vaccine. The method of claim 9, wherein the pharmaceutical composition is suitable for intramuscular (IM) injection. The method of claim 10, wherein the pharmaceutical composition is packaged as a 2.4 mL fill in a 6 mL glass vial. The method of any of claims 1-11, wherein the immune response comprises a T- cell response. The method of any of claims 1-11, wherein the immune response comprises a B cell response. The method of any of claims 1-13, further comprising administering a booster subsequent to the first administration. The method of claim 14, wherein the subject is negative prior to the booster administration. The method of any of claims 1-15, wherein the subject is seronegative. A method of treating COVID-19 in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32. A method of preventing COVID-19 in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32. A method of mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV-2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32. The method of any one of claims 17-19, where the first sequence region is at least 81%, 82%, 83%, 84%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to or comprising SEQ ID NO: 7. The method of any one of claims 17-20, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2. The method of any one of claims 17-21 wherein at least 50% of the polynucleotide sequence is codon optimized. The method of claim any one of claims 17-22, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine. The method of claim 23, wherein all uracil nucleosides are modified to be Nl- methylpseudouridine. The method of any of claims 17-24, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid. The method of claim 25, wherein
(i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid,
(ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid, (iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0- 10 mol% of a PEGylated lipid,
(iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid,
(v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid,
(vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid,
(vii)the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid,
(viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid,
(ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid,
(x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5-50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid,
(xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid, (xii)the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid,
(xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA, or
(xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA. The method of any of claims 17-26, wherein the dose of nucleic acid vaccine is administered to the subject is (i) about 5 pg to about 100 pg, (ii) about 16 pg, (iii) about 40 pg, or (iv) about 100 pg. The method of any of claims 17-27, wherein the volume of pharmaceutical composition administered to the subject is about 0.025 mL to about 0.5 mL. The method of claim 28, wherein the volume of pharmaceutical composition administered to the subject is about 0.025 mL, 0.05mL, 0.08mL, 0.2mL, or 0.5mL. The method of any of claims 17-29, comprising administering a second dose of the nucleic acid vaccine or pharmaceutical composition after between about 1 and about 5 weeks of a first dose. The method of claim 30, wherein the second dose is administered about 3 to 4 weeks after said first dose. The method of claim 31, wherein the subject is negative for COVID-19 when administered the second dose. The method of any of claims 17-32, wherein the pharmaceutical composition is suitable for intramuscular (IM) injection. The method of any of claims 17-33, wherein the subject is seronegative for COVID-19. A method of inducing SARS-CoV-2 neutralizing antibody production in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV- 2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7, 20, 26, 27, and 32. The method of claim 35, where the first sequence region is at least 81%, 82%, 83%, 84%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to or comprising SEQ ID NO: 7. The method of claim 35 or 36, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2. The method of any one of claims 35-37, wherein at least 50% of the polynucleotide sequence is codon optimized. The method of any one of claims 35-38, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine. The method of claim 39, wherein all uracil nucleosides are modified to be Nl- methylpseudouridine. The method of any one of claims 35-40, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid. The method of claim 41, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid,
(ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid,
(iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0- 10 mol% of a PEGylated lipid,
(iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid,
(v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a non-cationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid,
(vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid,
(vii)the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid,
(viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid,
(ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid, (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5-50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid,
(xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid,
(xii)the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25-55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid,
(xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA, or
(xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA. The method of any of claims 35-42, wherein the SARS-CoV-2 neutralizing antibodies can be detected in the subject by day 28 after administration. The method of claim 43, wherein the SARS-CoV-2 neutralizing antibody production is enhanced in the subject at day 42 after receiving a second dose of the nucleic acid vaccine or pharmaceutical composition. The method of claim 43, wherein the level of neutralizing antibodies to SARS- CoV-2 is increased ten-fold by day 42. The method of any of claims 35-45, wherein the subject is seronegative for SARS- CoV-2. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 1 days post initial administration. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 21 days post initial administration. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 28 days post initial administration. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 42 days post initial administration. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 42 days post initial administration. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 6 months post initial administration. The method of any of claims 35-46, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 1 year post initial administration. The method of any of claims 35-53, wherein the amount of neutralizing antibody in a subject increased by at least about 4-fold relative to pre administration levels. The method of any of claims 35-54, wherein the amount of neutralizing antibody in a subject increased by about 100-fold relative to pre administration levels. The method of any of claims 35-55, wherein the amount of neutralizing antibody in a subject increased by about 200-fold relative to pre administration levels. The method of any of claims 35-42, wherein the nucleic acid vaccine is administered as a booster vaccine. The method of claim 57, wherein the subject is seropositive for SARS-CoV-2 antibodies. The method of claim 57, wherein the subject has previously received at least one SARS-CoV-2 vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 1 day post administration of the nucleic acid vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 21 days post administration of the nucleic acid vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 28 days post administration of the nucleic acid vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 42 days post administration of the nucleic acid vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for at least about 42 days post administration of the nucleic acid vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 6 months (180 days) post administration of the nucleic acid vaccine. The method of any of claims 57-59, wherein the subject produces neutralizing antibodies to SARS-CoV-2 for about 1 year post administration of the nucleic acid vaccine. The method of any of claims 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 4-fold relative to levels prior to the administration of the nucleic acid vaccine. The method of any of claims 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 5-fold relative to levels prior to the administration of the nucleic acid vaccine. The method of any of claims 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 6-fold relative to levels prior to the administration of the nucleic acid vaccine. The method of any of claims 57-66, wherein the amount of neutralizing antibody in a subject increased by at least about 7-fold relative to levels prior to the administration of the nucleic acid vaccine. The method of any of claims 57-66, wherein the amount of neutralizing antibody in a subject increased by about 100-fold relative to levels prior to the administration of the nucleic acid vaccine. The method of any of claims 57-66, wherein the amount of neutralizing antibody in a subject increased by about 200-fold relative to levels prior to the administration of the nucleic acid vaccine. The pharmaceutical composition as defined in any one of claims 1-72 for use in therapy. The pharmaceutical composition as defined in any one of claims 1-72 for use inducing an immune response in a subject, for treating COVID-19 in a subject, for preventing COVID-19 in a subject, for mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject, or for inducing SARS-CoV-2 neutralizing antibody production in a subject. The pharmaceutical composition as defined in any one of claims 1-72 for the manufacture of a medicament for inducing an immune response in a subject, for treating COVID-19 in a subject, for preventing COVID-19 in a subject, for mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject, or for inducing SARS-CoV-2 neutralizing antibody production in a subject. A method of inducing SARS-CoV-2 neutralizing antibody production in a subject comprising administering a pharmaceutical composition comprising a pharmaceutical acceptable excipient and a nucleic acid vaccine formulated in a lipid nanoparticle (LNP) to said subject, wherein the nucleic acid vaccine comprises a polynucleotide encoding at least one structural protein of SARS-CoV- 2 or a variant thereof, wherein the at least one structural protein is the spike (S) protein and wherein the polynucleotide comprises a first sequence region, said first sequence region comprising a nucleic acid sequence having at least 80% identity to a member of the group consisting of SEQ ID NO: 7 and 27 and wherein the SARS- CoV-2 neutralizing antibody is to alpha (B.l.1.7), beta (B.1.351) or delta
(B.1.617.2) strains of SARS-CoV-2. The method of claim 76, where the first sequence region is at least 95% identical to, at least 99% identical to or comprising SEQ ID NO: 7. The method of claim 77, wherein the encoded spike protein has a protein sequence of SEQ ID NO: 2. The method of claim 77, wherein at least 50% of the polynucleotide sequence is codon optimized. The method of claim 79, wherein at least one uracil nucleoside is modified to be N1 -methylpseudouridine. The method of claim 80, wherein all uracil nucleosides are modified to be Nl- methylpseudouridine. The method of any of claims 76-81, wherein the LNP comprises (a) 50-85 mol% of a cationic lipid, (b) 13-49.5 mol% of a non-cationic lipid, and (c) 0.5-2 mol% of a conjugated lipid. The method of claim 82, wherein (i) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid (c) about 18.5-48.5 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (ii) the LNP comprises (a) about 35-55 mol% of a cationic lipid, (b) about 5-25 mol% of a noncationic lipid, (c) about 30-40 mol% of a sterol, and (d) about 0-10 mol% of a PEGylated lipid; (iii) the LNP comprises (a) about 35-45 mol% of a cationic lipid, (b) about 25-35 mol% of a non-cationic lipid, (c) about 20-30 mol% of a sterol and (d) about 0-10 mol% of a PEGylated lipid; (iv) the LNP comprises (a) about 45-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25- 40 mol% of a sterol, and (d) about 0.5-10 mol% of a PEGylated lipid; (v) the LNP comprises (a) about 40-60 mol% of a cationic lipid, (b) about 5-15 mol% of a noncationic lipid, (c) about 35-45 mol% of a sterol, and (d) about 0.5-3 mol% of a PEGylated lipid; (vi) the LNP comprises (a) about 30-60 mol% of a cationic lipid, (b) about 0-30 mol% of a non-cationic lipid, (c) about 15-50 mol% of a sterol, and (d) about 0.01-10 mol% of a PEGylated lipid; (vii) the LNP comprises (a) about 10-75 mol% of a cationic lipid, (b) about 0.5-50 mol% of a non-cationic lipid, (c) about 5-60 mol% of a sterol, and (d) about 0.1-20 mol% of a PEGylated lipid; (viii) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30-40 mol% of at least one sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (ix) the LNP comprises (a) about 50-85 mol% of a cationic lipid, (b) about 3-15 mol% of a non-cationic lipid, (c) about 30- 40 mol% of a sterol, and (d) about 0.5-2 mol% of a PEGylated lipid; (x) the LNP comprises (a) about 25-75 mol% of a cationic lipid, (b) about 0.1-15 mol% of a non-cationic lipid, (c) about 5-50 mol% of a sterol, and (d) about 0.5-20 mol% of a PEGylated lipid; (xi) the LNP comprises (a) about 50-65 mol% of a cationic lipid, (b) about 5-10 mol% of a non-cationic lipid, (c) about 25-35 mol% of a sterol, and (d) about 5-10 mol% of a PEGylated lipid; (xii) the LNP comprises (a) about 20-60 mol% of a cationic lipid, (b) about 5-25 mol% of a non-cationic lipid, (c) about 25- 55 mol% of a sterol, and (d) about 0.5-15 mol% of a PEGylated lipid; (xiii) the LNP comprises (a) 45-55 mol% 3D-P-DMA, (b) 8-10 mol% DSPC, (c) 36-42 mol% cholesterol and (d) 1.4-1.8 mol% PEG-DMA; or (xiv) the LNP comprises (a) 49.96 mol% 3D-P-DMA, (b) 9.97 mol% DSPC, (c) 38.44 mol% cholesterol and (d) 1.61% PEG-DMA. The method of any of claims 76-83, wherein the SARS-CoV-2 neutralizing antibodies can be detected in the subject by day 28 after administration. The method of any of claims 76-83, wherein the SARS-CoV-2 neutralizing antibody production is enhanced in the subject at day 42 after receiving a second dose of the nucleic acid vaccine or pharmaceutical composition. The method of any of claims 76-85, wherein the dose administered is 16 ug. The method of any of claims 76-85, wherein the dose administered is 40 ug. The method of any of claims 76-85, wherein the dose administered is 100 ug. The method of any of claims 76-88, wherein the SARS-CoV-2 neutralizing antibody is to alpha (B.1.1.7). The method of any of claims 76-88, wherein the SARS-CoV-2 neutralizing antibody is to beta (B.1.351). The method of any of claims 76-88, wherein the SARS-CoV-2 neutralizing antibody is to delta (B.1.617.2). The pharmaceutical composition as defined in any one of claims 76-91 for use in therapy. The pharmaceutical composition as defined in any one of claims 76-91 for use inducing an immune response in a subject, for treating COVID-19 in a subject, for preventing COVID-19 in a subject, for mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject, or for inducing SARS-CoV-2 neutralizing antibody production in a subject. The pharmaceutical composition as defined in any one of claims 76-91 for the manufacture of a medicament for inducing an immune response in a subject, for treating COVID-19 in a subject, for preventing COVID-19 in a subject, for mitigating or ameliorating the physiologic effects or symptoms of COVID-19 in a subject, or for inducing SARS-CoV-2 neutralizing antibody production in a subject.
PCT/CA2023/051314 2022-10-04 2023-10-04 Compositions and methods for the prevention and/or treatment of covid-19 WO2024073848A1 (en)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US202263378263P 2022-10-04 2022-10-04
US202263378264P 2022-10-04 2022-10-04
US63/378,263 2022-10-04
US63/378,264 2022-10-04
US202263384902P 2022-11-23 2022-11-23
US63/384,902 2022-11-23
US202363514636P 2023-07-20 2023-07-20
US63/514,636 2023-07-20

Publications (1)

Publication Number Publication Date
WO2024073848A1 true WO2024073848A1 (en) 2024-04-11

Family

ID=90607454

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2023/051314 WO2024073848A1 (en) 2022-10-04 2023-10-04 Compositions and methods for the prevention and/or treatment of covid-19

Country Status (1)

Country Link
WO (1) WO2024073848A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021156267A1 (en) * 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
CA3132188A1 (en) * 2020-10-09 2022-04-09 Providence Therapeutics Holdings Inc. Compositions and methods for the prevention and/or treatment of covid-19

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021156267A1 (en) * 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
CA3132188A1 (en) * 2020-10-09 2022-04-09 Providence Therapeutics Holdings Inc. Compositions and methods for the prevention and/or treatment of covid-19

Similar Documents

Publication Publication Date Title
JP7504464B2 (en) Nucleic Acid Vaccines
US12090235B2 (en) Preparation of lipid nanoparticles and methods of administration thereof
US11510977B2 (en) Nucleic acid vaccines for coronavirus
US20220062175A1 (en) Methods of preparing lipid nanoparticles
KR20220144416A (en) coronavirus vaccine
ES2896755T3 (en) Compositions Comprising Synthetic Polynucleotides Encoding CRISPR-Related Proteins and Synthetic sgRNAs and Methods of Use
CN115175698A (en) Coronavirus RNA vaccine
CN109562192B (en) Treatment of primary ciliated dyskinesia with synthetic messenger RNA
AU2017245384A1 (en) Compositions and methods for tolerizing cellular systems
AU2018270111A1 (en) Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
KR20230164648A (en) RNA vaccines against SARS-CoV-2 variants
US20240307522A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
WO2023137550A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3118329A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
WO2024073848A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
WO2024125597A1 (en) Compositions and methods for infectious diseases
WO2023050003A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3146411A1 (en) Compositions and methods for prevention and/or treatment of covid-19
WO2024156053A1 (en) Nucleic acid vaccines for varicella-zoster virus (vzv)
CA3154578A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3113094A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3116932A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3116284A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3128078A1 (en) Compositions and methods for the prevention and/or treatment of covid-19
CA3107232A1 (en) Compositions and methods for the prevention and/or treatment of covid-19

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23874142

Country of ref document: EP

Kind code of ref document: A1