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WO2025074292A2 - Compositions immunogènes - Google Patents

Compositions immunogènes Download PDF

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Publication number
WO2025074292A2
WO2025074292A2 PCT/IB2024/059673 IB2024059673W WO2025074292A2 WO 2025074292 A2 WO2025074292 A2 WO 2025074292A2 IB 2024059673 W IB2024059673 W IB 2024059673W WO 2025074292 A2 WO2025074292 A2 WO 2025074292A2
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WO
WIPO (PCT)
Prior art keywords
lnp
immunogenic composition
mol
rna
lipid
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Pending
Application number
PCT/IB2024/059673
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English (en)
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WO2025074292A3 (fr
Inventor
Bo Chen
Liyang CUI
Qianjing HE
Shuai SHI
Yajing ZHOU
Emily Alexandra CHOU
Ivna Padron DESOUZA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Corp Belgium
Pfizer Corp SRL
Original Assignee
Pfizer Corp Belgium
Pfizer Corp SRL
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Publication of WO2025074292A2 publication Critical patent/WO2025074292A2/fr
Publication of WO2025074292A3 publication Critical patent/WO2025074292A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Therapeutic nucleic acids include, but are not limited to, e.g., messenger RNA (mRNA), self-amplifying RNA (saRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers.
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • antisense oligonucleotides e.g., RNA
  • ribozymes e.g., DNAzymes, DNAzymes, plasmids
  • immune stimulating nucleic acids e.g., antagomir, antimir, mimic, supermir, and aptamers.
  • Some nucleic acids, such as mRNA or plasmids can be used to effect expression of specific cellular products as would be useful in the treatment of, for example, diseases related to a deficiency of a protein or enzyme, or as
  • Lipid nanoparticles formed from cholesterol with other lipid components such as ionizable lipids, neutral lipids, polymer conjugated lipids (e.g., PEGylated lipids), and nucleic acids have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides. Accordingly, there remains a need for improved lipid compounds and lipid nanoparticles for the delivery of nucleic acids.
  • these lipid nanoparticles would provide optimal drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid.
  • these lipid-nucleic acid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient.
  • compositions preferably immunogenic compositions.
  • the lipid nanoparticle (LNP) comprises i. an ionizable cationic lipid; ii. a neutral lipid; iii. a mixture of cholesterol and a cholesterol analog, wherein the molar ratio between the cholesterol and the cholesterol analog in the mixture is 6:4, 1:1 or 4:6; and iv. a polymer-conjugated lipid.
  • embodiment E2 The LNP of embodiment E1, wherein the cholesterol analog is selected from sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, ⁇ -sitosterol, stigmastanol, ⁇ -sitostanol, ergosterol, fecosterol, lupeol, cycloartol, ⁇ 5-avenaserol, ⁇ 7- avenaserol or a ⁇ 7-stigmasterol, tomatidine, ursolic acid or alpha-tocopherol, including analogs, salts or esters thereof.
  • the cholesterol analog is selected from sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, ⁇ -sitosterol, stigmastanol, ⁇ -sitostanol, ergosterol, fecosterol, lupeol
  • the LNP of embodiment E2 wherein the cholesterol analog is ⁇ -sitosterol, stigmasterol or campesterol.
  • the LNP according to any one of embodiments E1-E5, wherein the lipid nanoparticle comprises about 30 mol % to about 60 mol % ionizable cationic lipids, about 0 mol % to about 30 mol % neutral lipids, about 18.5 mol % to about 48.5 mol % mixture of cholesterol and a cholesterol analog, and about 0 mol % to about 10 mol % polymer-conjugated lipid.
  • E7 The LNP according to any of embodiments E1-E6, wherein the LNP comprises 0.9-1.85 mg/mL ALC-0315; 0.11-0.24 mg/mL ALC-0159; 0.18 – 0.41 mg/mL neutral lipid such as DSPC; and 0.36 – 0.78 mg/mL mixture of cholesterol and a cholesterol analog.
  • LNP of any of embodiments E5-E7, wherein the LNP comprises a mol% ratio of ALC- 0315:cholesterol and cholesterol analog mixture:DSPC:ALC-0159 from the group consisting of: a) 47.5:40.7:10:1.8; b) 30:58.2:10:1.8; c) 30:48.2:20:1.8; and d) 50:33.2:15:1.8.
  • the LNP comprises a mol% ratio of ALC- 0315:cholesterol and cholesterol analog mixture:DSPC:ALC-0159 from the group consisting of: a) 47.5:40.7:10:1.8; b) 30:58.2:10:1.8; c) 30:48.2:20:1.8; and d) 50:33.2:15:1.8.
  • An immunogenic composition comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame (ORF) encoding at least one antigenic polypeptide or an immunogenic fragment thereof, formulated in a lipid nanoparticle (LNP) according to any of the above listed embodiments.
  • RNA ribonucleic acid
  • ORF open reading frame
  • LNP lipid nanoparticle
  • FIG.1A-1C depicts 50% neutralization titers 3 weeks post dose 1 against B/Austria; FIG.1B depicts 50% neutralization titers 2 weeks post dose 2 (0.2 ug dose).
  • FIG.7A depicts a comparison of LNPs having an N:P ratio of 10, LNPs having an N:P ratio of 6, and their respective fold change (EC50 cholesterol /EC50 cholesterol analog ) compared to benchmark LNPs
  • FIG.7B depicts a comparison of LNPs having an N:P ratio of 10, LNPs having an N:P ratio of 6, and their respective fold change (MFIcholesterol analog/MFIcholesterol).
  • FIG.8A-8B depicts a comparison of LNPs having an N:P ratio of 10, LNPs having an N:P ratio of 6, and their respective fold change (MFIcholesterol analog/MFIcholesterol).
  • FIG.9A-9F depict the key attributes of various sterol-containing LNPs encapsulating RNA over a 6-month storage period at 5 oC;
  • FIG.9A depicts the LNP size;
  • FIG.9B depicts polydispersity (PDI);
  • FIG.9C depicts percent encapsulation of the encapsulated RNA;
  • FIG.9D depicts percent integrity of the encapsulated RNA;
  • FIG.9E depicts the EC50 for total RSV F protein expression;
  • FIG.9F depicts the EC50 for RSV prefusion F protein expression measured in HEK293T cells.
  • DETAILED DESCRIPTION The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein.
  • Embodiment 1 (E1) is identical to the embodiment of Formula (I) provided above.
  • Exemplary embodiments (E) of the invention provided herein include: E1.
  • a lipid nanoparticle (LNP) comprising i. an ionizable cationic lipid; ii. a neutral lipid; iii. a mixture of cholesterol and a cholesterol analog, wherein the molar ratio between the cholesterol and the cholesterol analog in the mixture is 6:4, 1:1 or 4:6; and iv.
  • the lipid nanoparticle comprises i. an ionizable cationic lipid; ii. a neutral lipid; iii. a mixture of cholesterol and a cholesterol analog, wherein the molar ratio between the cholesterol and the cholesterol analog in the mixture is 6:4, 1:1 or 4:6; and iv. a polymer-conjugated lipid.
  • embodiment E2 The LNP of embodiment E1, wherein the cholesterol analog is selected from sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, ⁇ -sitosterol, stigmastanol, ⁇ -sitostanol, ergosterol, fecosterol, lupeol, cycloartol, ⁇ 5-avenaserol, ⁇ 7- avenaserol or a ⁇ 7-stigmasterol, tomatidine, ursolic acid or alpha-tocopherol, including analogs, salts or esters thereof.
  • the cholesterol analog is selected from sitosterol, stigmasterol, campesterol, sitostanol, campestanol, brassicasterol, fucosterol, ⁇ -sitosterol, stigmastanol, ⁇ -sitostanol, ergosterol, fecosterol, lupeol
  • the LNP of embodiment E2 wherein the cholesterol analog is ⁇ -sitosterol, stigmasterol or campesterol.
  • the ionizable cationic lipid is selected from N,N- dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N- dimethylamino
  • the LNP of embodiment E10, wherein the pegylated lipid is 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide (ALC-0159) or a PEG dialkyoxypropylcarba PEGylated diacylglycerol (PEG-DAG), 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-((O- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG-ceramide, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159
  • the lipid nanoparticle comprises about 30 mol % to about 60 mol % ionizable cationic lipids, about 0 mol % to about 30 mol % neutral lipids, about 18.5 mol % to about 48.5 mol % mixture of cholesterol and a cholesterol analog, and about 0 mol % to about 10 mol % polymer-conjugated lipid.
  • the LNP according to any of embodiments E1-E14, wherein the LNP comprises 0.9- 1.85 mg/mL ALC-0315; 0.11-0.24 mg/mL ALC-0159; 0.18 – 0.41 mg/mL neutral lipid such as DSPC; and 0.36 – 0.78 mg/mL mixture of cholesterol and a cholesterol analog.
  • the LNP comprises 0.9- 1.85 mg/mL ALC-0315; 0.11-0.24 mg/mL ALC-0159; 0.18 – 0.41 mg/mL neutral lipid such as DSPC; and 0.36 – 0.78 mg/mL mixture of cholesterol and a cholesterol analog.
  • LNP of any of embodiments E13-E15, wherein the LNP comprises a mol% ratio of ALC-0315:cholesterol and cholesterol analog mixture:DSPC:ALC-0159 from the group consisting of: a) 47.5:40.7:10:1.8; b) 30:58.2:10:1.8; c) 30:48.2:20:1.8; and d) 50:33.2:15:1.8.
  • the LNP comprises a mol% ratio of ALC-0315:cholesterol and cholesterol analog mixture:DSPC:ALC-0159 from the group consisting of: a) 47.5:40.7:10:1.8; b) 30:58.2:10:1.8; c) 30:48.2:20:1.8; and d) 50:33.2:15:1.8.
  • the LNP of embodiment E16 wherein the LNP comprises a mol% ratio of ALC- 0315:cholesterol:cholesterol analog:DSPC:ALC-0159 from the group consisting of: a) 47.5:16.3:24.4:10:1.8; b) 47.5:20.4:20.4:10:1.8; c) 47.5:24.4:16.3:10:1.8; d) 30:29.1:29.1:10:1.8; e) 30:24.1:24.1:20:1.8; and f) 50:16.6:16.6:15:1.8.
  • E18 The LNP of any one of embodiments E13-E17, wherein the cholesterol analog is beta- sitosterol.
  • E21. An immunogenic composition comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame (ORF) encoding at least one antigenic polypeptide or an immunogenic fragment thereof, formulated in a lipid nanoparticle (LNP) according to any of embodiments E1-E20.
  • RNA polynucleotide comprises a 5’ cap, 5’ UTR, 3’ UTR, and poly-A tail.
  • E24. The immunogenic composition of embodiment E23, wherein the 5’ cap is a 5’ cap analog.
  • E25 The immunogenic composition of embodiment E24, wherein the 5’ cap analog comprises m2 7,3’-O Gppp(m1 2’-O )ApG or . to any of embodiments E21 to E25, wherein the RNA further comprises a modified nucleotide.
  • the immunogenic composition of embodiment E26, wherein the modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methoxyuridine, 2′-O-methyl uridine and N1-Methylpseudourodine-5’-triphosphate (m1 ⁇ TP).
  • E28 The immunogenic composition of embodiment E27, wherein the modified nucleotide comprises N1-Methylpseudourodine-5’-triphosphate (m1 ⁇ TP).
  • E29 The immunogenic composition according to any of embodiments E21-E28, wherein the open reading frame is codon-optimized.
  • E30 The immunogenic composition of embodiment E21, wherein the 5’ UTR comprises SEQ ID NO: 1.
  • the immunogenic composition of embodiment E21, wherein the 3’ UTR comprises SEQ ID NO: 2.
  • E32 The immunogenic composition of embodiment E21, wherein the 3′ polyadenylation tail comprises SEQ ID NO: 3. E33.
  • E34 The immunogenic composition of any preceding embodiment, wherein the RNA polynucleotide has a purity of greater than 85%.
  • E35 The immunogenic composition according to any one of embodiments E21-E34, wherein at least 80% of the total RNA in the immunogenic composition is encapsulated.
  • E36 The immunogenic composition according to any one of embodiments E21-E35, wherein the percentage of intact mRNA encapsulated in the LNP is at least 80%.
  • E37 The immunogenic composition according to any one of any of the preceding embodiments, wherein the LNP comprises an N:P ratio of from about 2:1 to about 30:1.
  • E38 The immunogenic composition of embodiment E37, wherein the LNP has an N:P ratio of at least 5. E39. The immunogenic composition of embodiment E37, wherein the LNP has an N:P ratio of at least 6. E40. The immunogenic composition of embodiment E37, wherein the LNP has an N:P ratio of 10. E41. The immunogenic composition according to any one of embodiments E21-E40, wherein the immunogenic composition comprises Tris. E42. The immunogenic composition according to any one of embodiments E21-E41, wherein the immunogenic composition comprises sucrose. E43. The immunogenic composition according to any one of embodiments E21-E42, wherein the immunogenic composition does not further comprise sodium chloride. E44.
  • the immunogenic composition according to any one of embodiments E21-E46, wherein the immunogenic composition has less than or equal to 12.5 EU/mL of bacterial endotoxins.
  • E48. The immunogenic composition according to any one of embodiments E21-E47, wherein the immunogenic composition is stable up to about 5 oC. E49.
  • E50. A method for delivering a nucleic acid to a subject in need thereof comprising administering to the subject an immunogenic composition of any one of embodiments E21 to E49 comprising a nucleic acid.
  • E51. A method for delivering a therapeutic peptide or protein to a subject in need thereof, the method comprising administering to the subject an immunogenic composition of any one of embodiments E21 to E49 comprising a nucleic acid, wherein the nucleic acid encodes the therapeutic peptide or protein.
  • a method for treating or preventing a disease or disorder in a subject comprising administering to the subject an immunogenic composition of any one of embodiments E21 to E49 comprising a nucleic acid, wherein administering the nucleic acid to cells of the subject is beneficial in treating or preventing the disease or disorder.
  • E53. A method for treating or preventing a disease or disorder in a subject comprising administering to the subject an immunoenic composition of any one of embodiments E21 to E49 comprising a nucleic acid, wherein the nucleic acid encodes a therapeutic peptide or protein and wherein delivering the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
  • the RNA polynucleotides are mixed in desired ratios in a single vessel and are subsequently formulated into lipid nanoparticles.
  • the inventors surprisingly discovered that the initial input of different RNA polynucleotides at a known ratio to be formulated in a single LNP process surprisingly resulted in LNPs encapsulating the different RNA polynucleotides in about the same ratio as the input ratio.
  • the results were surprising in view of the potential for the manufacturing process to favor one RNA polynucleotide to another when encapsulating the RNA polynucleotides into an LNP. Such embodiments may be referred herein as "pre-mix”.
  • first and second RNA polynucleotides are formulated in a single lipid nanoparticle.
  • the first, second, third, and fourth RNA polynucleotides are formulated in a single LNP.
  • the first, second, third, fourth, and fifth RNA polynucleotides are formulated in a single LNP.
  • the first, second, third, fourth, fifth, and sixth RNA polynucleotides are formulated in a single LNP.
  • the first, second, third, fourth, fifth, sixth, and seventh RNA polynucleotides are formulated in a single LNP.
  • the first, second, third, fourth, fifth, sixth, seventh, and eighth RNA polynucleotides are formulated in a single LNP.
  • the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1.
  • the molar ratio of the first RNA polynucleotide to the second RNA polynucleotide is greater than 1:1.
  • the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1.
  • the molar ratio of the first RNA polynucleotide to the third RNA polynucleotide is greater than 1:1.
  • the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the sixth RNA polynucleotide is greater than 1:1.
  • the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1. In some embodiments, the molar ratio of the first RNA polynucleotide to the seventh RNA polynucleotide is greater than 1:1.
  • the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide in the mix of RNA polynucleotides prior to formulation into LNPs is about 1:50, about 1:25, about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, or about 5:1, about 10:1, about 25:1 or about 50:1.
  • the molar ratio of the first RNA polynucleotide to the eighth RNA polynucleotide is greater than 1:1.
  • the RNA molecule such as the first RNA molecule, is an saRNA.
  • RNA refers to RNA with the ability to replicate itself.
  • Self- amplifying RNA molecules may be produced by using replication elements derived from a virus or viruses, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest.
  • a self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs.
  • the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins.
  • the self-amplifying RNA may also include 5'- and 3 '-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest).
  • a subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA.
  • the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and/or may be under the control of an internal ribosome entry site (IRES).
  • the self-amplifying RNA molecule is not encapsulated in a virus- like particle.
  • Self-amplifying RNA molecules described herein may be designed so that the self- amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA.
  • a self-amplifying RNA molecule described herein encodes (i) an RNA-dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen.
  • the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus protein nsP1, nsP2, nsP3, nsP4, and any combination thereof.
  • the self-amplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5-methyluridine). In some embodiments, the self- amplifying RNA molecules does not include a modified nucleotide (e.g., pseudouridine, N6- methyladenosine, 5- methylcytidine, 5-methyluridine).
  • the saRNA construct may encode at least one non-structural protein (NSP), disposed 5’ or 3’ of the sequence encoding at least one peptide or polypeptide of interest.
  • the sequence encoding at least one NSP is disposed 5’ of the sequences encoding the peptide or polypeptide of interest.
  • the sequence encoding at least one NSP may be disposed at the 5’ end of the RNA construct.
  • at least one non-structural protein encoded by the RNA construct may be the RNA polymerase nsP4.
  • the saRNA construct encodes nsP1, nsP2, nsP3 and, nsP4.
  • nsP1 is the viral capping enzyme and membrane anchor of the replication complex (RC).
  • nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing.
  • nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP-ribosylation.
  • nsP4 is the core viral RNA-dependent RNA polymerase.
  • the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsP1, nsP2, nsP3, and nsP4.
  • the self-amplifying RNA molecules do not encode alphavirus structural proteins.
  • the self-amplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the self-amplifying RNA molecule cannot perpetuate itself in infectious form.
  • the second RNA or the saRNA molecule further includes (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence.
  • the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.
  • self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.
  • the saRNA molecule is alphavirus-based. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family.
  • alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR
  • the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., saRNA).
  • the self-amplifying RNA molecules described herein include at least about 4 kb.
  • the self-amplifying RNA may be equal to any one of, at least any one of, at most any one of, or between any two of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb.
  • the self-amplifying RNA may include at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb.
  • the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb
  • the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence.
  • the polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.
  • the saRNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides.
  • the self-amplifying RNA described herein may encode epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both.
  • the saRNA molecule is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/diafiltration.
  • compositions comprising a self- amplifying RNA molecule comprising a 5’ Cap, a 5’ untranslated region, a coding region comprising a sequence encoding an RNA-dependent RNA polymerase (also referred to as a “replicase”), a subgenomic promoter, such as one derived from an alphavirus, an open reading frame encoding a gene of interest (e.g., an antigen derived from a virus), a 3’ untranslated region, and a 3’ poly A sequence.
  • RNA-dependent RNA polymerase also referred to as a “replicase”
  • a subgenomic promoter such as one derived from an alphavirus
  • an open reading frame encoding a gene of interest (e.g., an antigen derived from a virus)
  • a 3’ untranslated region e.g., an antigen derived from a virus
  • the saRNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below.
  • the saRNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below.
  • the RNA may include a 5' cap comprising a 7'-methylguanosine, and the first 1, 2 or 35' ribonucleotides may be methylated at the 2' position of the ribose.
  • the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; the fifth RNA polynucleotide is formulated in a fifth LNP; the sixth RNA polynucleotide is formulated in a sixth LNP; the seventh RNA polynucleotide is formulated in a seventh LNP; and the eighth RNA polynucleotide is formulated in an eighth LNP.
  • the molar ratio of the first LNP to the third LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the third LNP is greater than 1:1.
  • the molar ratio of the first LNP to the fourth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the fourth LNP is greater than 1:1.
  • the molar ratio of the first LNP to the sixth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the sixth LNP is greater than 1:1.
  • the molar ratio of the first LNP to the eighth LNP in the mix of LNPs prior to formulation into LNPs is about 1:50, about 1:25, about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1, about 10: 1, about 25: 1 or about 50: 1. In some embodiments, the molar ratio of the first LNP to the eighth LNP is greater than 1:1.
  • the resulting ratio of RNA polynucleotide is comparable whether the plurality of RNA polynucleotides are mixed prior to formulation in an LNP (pre-mixed) or whether the RNA polynucleotides encoding a particular antigen are formulated in an individual LNP and the plurality of LNPs for different antigens are mixed (post-mixed).
  • pre-mixed the RNA polynucleotides encoding a particular antigen are formulated in an individual LNP and the plurality of LNPs for different antigens are mixed (post-mixed).
  • the mRNAs encoding antigens are separately encapsulated (e.g., separate encapsulation of each mRNA encoding an antigen; encapsulation of mRNA(s) encoding a first antigen (e.g. influenza type A antigen) in a first population of nanoparticles (e.g., LNPs) and encapsulation of mRNA(s) encoding a second antigen (e.g. influenza type B antigen) in a second population of nanoparticles (e.g., LNPs); or encapsulation of mRNA(s) encoding a first antigen (e.g.
  • the composition comprises coencapsulated mRNAs encoding an antigen.
  • a composition comprising (i) two or more different RNAs, each encoding an antigenic polypeptide (e.g., an HA protein) of a first virus (e.g., influenza type A virus), e.g. so that the two or more different RNAs, together, encode two or more different polypeptides (e.g.
  • influenza type A virus is encapsulated in a first population of nanoparticles and each RNA encoding an antigenic polypeptide of a second virus (e.g. influenza type B virus) is encapsulated in a second population of nanoparticles; each RNA is encapsulated in a separate nanoparticle; or each RNA encoding a polypeptide of a first antigen (e.g. influenza type A antigenic polypeptide)is encapsulated in a first population of nanoparticles and the two RNAs encoding a polypeptide of second antigen (e.g. influenza type B antigenic polypeptide) are each encapsulated in separate populations of nanoparticles.
  • the present disclosure relates to mRNA vaccines in general.
  • IVT in vitro transcribed
  • ORF protein-encoding open reading frame
  • UTRs 5′ and 3′ untranslated regions
  • iii a 7- methyl guanosine 5′ cap structure
  • iv a 3′ poly(A) tail.
  • ORF open reading frame
  • UTRs untranslated regions
  • iv a 7- methyl guanosine 5′ cap structure
  • iv a 3′ poly(A) tail.
  • the non-coding structural features play important roles in the pharmacology of mRNA and can be individually optimized to modulate the mRNA stability, translation efficiency, and immunogenicity.
  • nucleoside-modified mRNA By incorporating modified nucleosides, mRNA transcripts referred to as “nucleoside-modified mRNA” can be produced with reduced immunostimulatory activity, and therefore an improved safety profile can be obtained.
  • modified nucleosides allow the design of mRNA vaccines with strongly enhanced stability and translation capacity, as they can avoid the direct antiviral pathways that are induced by type IFNs and are programmed to degrade and inhibit invading mRNA. For instance, the replacement of uridine with pseudouridine in IVT mRNA reduces the activity of 2′- 5′-oligoadenylate synthetase, which regulates the mRNA cleavage by RNase L.
  • mRNA expression can be strongly increased by sequence optimizations in the ORF and UTRs of mRNA, for instance by enriching the GC content, or by selecting the UTRs of natural long-lived mRNA molecules.
  • sequence-engineered mRNA mRNA expression can be strongly increased by sequence optimizations in the ORF and UTRs of mRNA, for instance by enriching the GC content, or by selecting the UTRs of natural long-lived mRNA molecules.
  • Another approach is the design of “self-amplifying mRNA” constructs.
  • Anti- reverse cap (ARCA) modifications can ensure the correct cap orientation at the 5′ end, which yields almost complete fractions of mRNA that can efficiently bind the ribosomes.
  • Other cap modifications such as phosphorothioate cap analogs, can further improve the affinity towards the eukaryotic translation initiation factor 4E, and increase the resistance against the RNA decapping complex.
  • the invention relates to an immunogenic composition
  • an mRNA molecule that encodes one or more polypeptides or fragments thereof of an antigen.
  • the mRNA molecule comprises a nucleoside-modified mRNA.
  • mRNA useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 '-terminus of the first region (e.g., a 5 -UTR), a second flanking region located at the 3 '-terminus of the first region (e.g., a 3 -UTR), at least one 5 '-cap region, and a 3 '-stabilizing region.
  • the mRNA of the disclosure further includes a poly-A region or a Kozak sequence (e.g., in the 5 '-UTR).
  • mRNA of the disclosure may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide.
  • mRNA of the disclosure may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence, and/or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside).
  • the 3 '-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-0-methyl nucleoside and/or the coding region, 5 '-UTR, 3 '-UTR, or cap region may include an alternative nucleoside such as a 5-substituted uridine (e.g., 5-methoxyuridine), a 1-substituted pseudouridine (e.g., 1-methyl-pseudouridine), and/or a 5- substituted cytidine (e.g., 5-methyl-cytidine).
  • a 5-substituted uridine e.g., 5-methoxyuridine
  • a 1-substituted pseudouridine e.g., 1-methyl-pseudouridine
  • a 5- substituted cytidine e.g., 5-methyl-cytidine
  • compositions described herein comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA).
  • mRNA for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.”
  • an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a polyA tail.
  • UTR untranslated
  • a “5′ untranslated region” refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
  • a “3′ untranslated region” refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
  • An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.
  • a “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates.
  • a polyA tail may contain 10 to 300 adenosine monophosphates.
  • a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
  • a polyA tail contains 50 to 250 adenosine monophosphates.
  • the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.
  • a polynucleotide includes 200 to 3,000 nucleotides.
  • a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).
  • a LNP includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio.
  • the N:P ratio may be from about 2: 1 to about 8: 1. In other embodiments, the N:P ratio is from about 5 : 1 to about 8: 1. For example, the N:P ratio may be about 5.0: 1 , about 5.5 : 1, about 5.67: 1, about 6.0: 1, about 6.5: 1 , or about 7.0: 1. For example, the N:P ratio may be about 5.67: 1.
  • the composition comprises a second LNP, wherein the second LNP does not encapsulate a polynucleotide as described in WO2023057930, the entirety of which is incorporated herein by reference.
  • compositions and methods described herein relate to frozen or lyophilized lipid nanoparticles encapsulating or associated with RNA in the presence of a cryoprotectant, preferably a carbohydrate cryoprotectant, and/or further in the presence of lipid nanoparticles that are devoid of nucleic acid, (e.g., not encapsulating and not associated with RNA (also referred herein as “blank” LNPs), or liposomes, or a higher cryoprotectant concentration) resulting in a composition comprising LNPs encapsulating RNA or associated with RNA that is characterized by, among other things, an improved integrity of the RNA after completion of the respective freezing or lyophilization process and which is further characterized by increased storage stability, such as, for example, with respect to storage for extended periods and/or under non-cooling conditions, as compared to a composition comprising lipid nanoparticles encapsulating or associated with RNA in the absence of the blank LNPs, or liposomes
  • the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA, a second lipid nanoparticle that is devoid of nucleic acid, and a cryoprotectant that results in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine.
  • the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA and an increased cryoprotectant concentration that results in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine.
  • the compositions include a mixture of a first lipid nanoparticle encapsulating or associated with RNA, a liposome, and an increased cryoprotectant concentration that result in improved characteristics of the encapsulated RNA after freezing or lyophilization processes, preferably for use as a pharmaceutical composition, such as, for example, an immunogenic composition or vaccine.
  • a pharmaceutical composition such as, for example, an immunogenic composition or vaccine.
  • the compositions and methods thereof described herein are suitable for use at an industrial scale. The methods described herein may be used to produce, for example, a frozen or lyophilized composition comprising LNPs encapsulating or associated with RNA having the above-mentioned properties in a reproducible and cost-effective manner.
  • nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3 '-UTR, (d) the poly A tail, and any combination of (a, b, c, or d above) comprise naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).
  • mRNA of the disclosure may include one or more alternative components, as described herein, which impart useful properties including increased stability and/or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
  • a modRNA may exhibit reduced degradation in a cell into which the modRNA is introduced, relative to a corresponding unaltered mRNA.
  • These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and/or viability of contacted cells, as well as possess reduced immunogenicity.
  • mRNA of the disclosure may include one or more modified (e.g., altered or alternative) nucleobases, nucleosides, nucleotides, or combinations thereof.
  • the mRNA useful in a LNP can include any useful modification or alteration, such as to the nucleobase, the sugar, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone).
  • alterations e.g., one or more alterations are present in each of the nucleobase, the sugar, and the internucleoside linkage.
  • RNAs ribonucleic acids
  • TAAs threose nucleic acids
  • GAAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotide X in a mRNA may or may not be uniformly altered in a mRNA, or in a given predetermined sequence region thereof.
  • all nucleotides X in a mRNA are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.
  • An alteration may also be a 5'- or 3 '-terminal alteration.
  • the polynucleotide includes an alteration at the 3 '-terminus.
  • the polynucleotide may contain from about 1% to about 100% alternative 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, 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
  • Polynucleotides may contain at a minimum zero and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least 5% alternative nucleotides, at least 10% alternative nucleotides, at least 25% alternative nucleotides, at least 50% alternative nucleotides, at least 80% alternative nucleotides, or at least 90% alternative nucleotides.
  • polynucleotides may contain an alternative pyrimidine such as an alternative uracil or cytosine.
  • At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in a polynucleotide is replaced with an alternative uracil (e.g., a 5-substituted uracil).
  • the alternative uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • cytosine in the polynucleotide is replaced with an alternative cytosine (e.g., a 5-substituted cytosine).
  • the alternative cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
  • nucleic acids do not substantially induce an innate immune response of a cell into which the polynucleotide (e.g., mRNA) is introduced.
  • nucleobases and nucleosides having an alternative uracil include pseudouridine ( ⁇ ), pyridin-4- one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio- uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uracil (ho5U), 5-aminoallyl- uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m U), 5-methoxy- uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1 -
  • the nucleobase is an alternative adenine.
  • Exemplary nucleobases and nucleosides having an alternative 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-adenine, 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 -methy 1-adenine (ml A), 2-methyl-adenine (m2A), N6- methyl-adenine (m6A), 2-
  • the nucleobase is an alternative guanine.
  • Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl- inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl), archae
  • the alternative nucleobase of a nucleotide can be independently a purine, a pyrimidine, a purine or pyrimidine analog.
  • the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine.
  • each letter refers to the representative base and/or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine).
  • the mRNA may include a 5 '-cap structure.
  • the 5 '-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide 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.
  • Endogenous polynucleotide molecules may be 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 polynucleotide.
  • This 5 '-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5 ' end of the polynucleotide may optionally also be 2'-0-methylated.5 '-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. Alterations to polynucleotides may generate a non-hydrolyzable cap structure preventing decapping and thus increasing polynucleotide half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, alternative nucleotides may be used during the capping reaction.
  • a Vaccinia Capping Enzyme from New England Biolabs 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 alternative guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides.
  • Additional alterations include, but are not limited to, 2'-0-methylation of the ribose sugars of 5'-terminal and/or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxy group of the sugar.
  • Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of an mRNA molecule.
  • 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.
  • the 3'-0 atom of the other, unaltered, guanosine becomes linked to the 5 '- terminal nucleotide of the capped polynucleotide (e.g., an mRNA).
  • the N7- and 3'-0-methylated guanosine provides the terminal moiety of the capped polynucleotide (e.g., mRNA).
  • Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine, m7Gm- ppp-G).
  • Non-limiting examples of N7- (4-chlorophenoxy ethyl) substituted dinucleotide cap analogs include a N7-(4- chlorophenoxyethyl)-G(5 )ppp(5 ')G and a N7-(4-chlorophenoxyethyl)-m3 '-OG(5 )ppp(5 ')G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321 :4570-4574; the cap structures of which are herein incorporated by reference).
  • the phrase "more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function, and/or structure as compared to synthetic features or analogs of the prior art, or which outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects.
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '- triphosphate linkage between the 5 '-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide wherein the cap guanosine contains an N7-methylation and the 5 '-terminal nucleotide of the polynucleotide contains a 2'-0-methyl.
  • Capl structure Such a structure is termed the Capl structure.
  • 5 '-terminal caps may include endogenous caps or cap analogs.
  • a 5 '- terminal cap may include a guanosine analog.
  • Useful guanosine analogs include inosine, N1- methyl- guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA- guanosine, and 2-azido-guanosine.
  • a polynucleotide contains a modified 5 '-cap. A modification on the 5 '-cap may increase the stability of polynucleotide, increase the half-life of the polynucleotide, and could increase the polynucleotide translational efficiency.
  • the modified 5 '-cap may include, but is not limited to, one or more of the following modifications: modification at the 2'- and/or 3 '-position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.
  • GTP capped guanosine triphosphate
  • CH2 methylene moiety
  • a 5'-UTR may be provided as a flanking region to the mRNA.
  • a 5’ -UTR may be homologous or heterologous to the coding region found in a polynucleotide.
  • flanking region Multiple 5 '-UTRs may be included in the flanking region and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and/or after codon optimization.
  • an ORF encoding an antigen of the disclosure is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized.
  • Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias 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; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • encoded protein e.g., glycosylation sites
  • add, remove or shuffle protein domains add or delete restriction sites
  • modify ribosome binding sites and mRNA degradation sites adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization tools, algorithms and services are known in the art—non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods.
  • the open reading frame (ORF) sequence is optimized using optimization algorithms.
  • 5 '-UTRs which are heterologous to the coding region of an mRNA may be engineered.
  • the mRNA may then be administered to cells, tissue or organisms and outcomes such as protein level, localization, and/or half-life may be measured to evaluate the beneficial effects the heterologous 5 ' -UTR may have on the mRNA.
  • mRNAs may include a stem loop such as, but not limited to, a histone stem loop.
  • the stem loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length.
  • the histone stem loop may be located 3 '-relative to the coding region (e.g., at the 3 '-terminus of the coding region).
  • the addition of at least one chain terminating nucleoside may slow the degradation of a polynucleotide and thus can increase the half-life of the polynucleotide.
  • a mRNA, which includes the histone stem loop may be stabilized by an alteration to the 3 '-region of the polynucleotide that can prevent and/or inhibit the addition of oligio(U).
  • a mRNA, which includes the histone stem loop may be stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2',3 '-dideoxynucleoside 3 '-0- methylnucleosides, 3 -0- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • the mRNA of the present disclosure may include a histone stem loop, a poly-A region, and/or a 5 '-cap structure. The histone stem loop may be before and/or after the poly-A region.
  • the polynucleotide encoding for a histone stem loop and a poly-A region or a polyadenylation signal may code for an allergenic antigen or an autoimmune self-antigen.
  • An mRNA may include a polyA sequence and/or polyadenylation signal.
  • a polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof.
  • a polyA sequence may be a tail located adjacent to a 3' untranslated region of a nucleic acid.
  • a long chain of adenosine nucleotides is normally added to messenger RNA (mRNA) molecules to increase the stability of the molecule.
  • the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 70 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides.
  • the length is at least 3000 nucleotides.
  • the poly-A region may be 80 nucleotides, 120 nucleotides, 160 nucleotides in length on an alternative polynucleotide molecule described herein. In other instances, the poly-A region may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length on an alternative polynucleotide molecule described herein. In some cases, the poly-A region is designed relative to the length of the overall alternative polynucleotide.
  • This design may be based on the length of the coding region of the alternative polynucleotide, the length of a particular feature or region of the alternative polynucleotide (such as mRNA) or based on the length of the ultimate product expressed from the alternative polynucleotide.
  • the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature.
  • the poly-A region may also be designed as a fraction of the alternative polynucleotide to which it belongs.
  • multiple distinct mRNA may be linked together to the PABP (poly-A binding protein) through the 3'-end using alternative nucleotides at the 3'- terminus of the poly-A region.
  • Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and day 7 post-transfection.
  • the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site.
  • a poly-A region may be used to modulate translation initiation.
  • an mRNA may include a polyA-G quartet.
  • the G- quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly-A region. The resultant mRNA may be assayed for stability, protein production and other parameters including half-life at various time points.
  • mRNA may include a poly-A region and may be stabilized by the addition of a 3 '-stabilizing region.
  • the mRNA with a poly-A region may further include a 5 '-cap structure.
  • mRNA may include a poly- A-G quartet.
  • the mRNA with a poly-A-G quartet may further include a 5 '-cap structure.
  • the 3 '-stabilizing region which may be used to stabilize mRNA includes a poly-A region or poly-A-G quartet.
  • mRNA which includes a polyA region or a poly-A-G quartet may be stabilized by an alteration to the 3 '-region of the polynucleotide that can prevent and/or inhibit the addition of oligio(U).
  • mRNA which includes a poly-A region or a poly-A-G quartet may be stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2',3 '-dideoxynucleoside 3 -O- methylnucleosides, 3 '-O- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • the lipids can encapsulate the mRNA in the form of a lipid nanoparticle (LNP) to aid cell entry and stability of the RNA/lipid nanoparticles.
  • LNP lipid nanoparticle
  • Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and/or prophylactic.
  • a LNP may be designed for one or more specific applications or targets.
  • the elements of a LNP may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements.
  • the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements.
  • the therapeutic and/or prophylactic included in a LNP may also be selected based on the desired delivery target or targets.
  • a therapeutic and/or prophylactic may be selected for a particular indication, condition, disease, or disorder and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery).
  • a LNP may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest.
  • Such a composition may be designed to be specifically delivered to a particular organ.
  • a composition may be designed to be specifically delivered to a mammalian liver.
  • a composition may be designed to be specifically delivered to a lymph node.
  • a composition may be designed to be specifically delivered to a mammalian spleen.
  • a LNP may include one or more components described herein.
  • the LNP formulation of the disclosure includes at least one lipid nanoparticle component.
  • Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and/or prophylactic, such as a nucleic acid.
  • a LNP may be designed for one or more specific applications or targets. The elements of a LNP may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements.
  • a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L- lactic acid) (PLLA), poly(gly colic acid) (PGA), poly(lactic acid-co-gly colic acid) (PLGA), poly(L- lactic acid-co-gly colic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L- lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co- glycolide), poly(D,L- lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HP)
  • the amount of polymer-lipid in the lipid composition of a pharmaceutical composition disclosed herein ranges from about 0.1 mol % to about 5 mol %, from about 0.5 mol % to about 5 mol %, from about 1 mol % to about 5 mol %, from about 1.5 mol % to about 5 mol %, from about 2 mol % to about 5 mol %, from about 0.1 mol % to about 4 mol %, from about 0.5 mol % to about 4 mol %, from about 1 mol % to about 4 mol %, from about 1.5 mol % to about 4 mol %, from about 2 mol % to about 4 mol %, from about 0.1 mol % to about 3 mol %, from about 0.5 mol % to about 3 mol %, from about 1 mol % to about 3 mol %, from about 1.5 mol % to about 3 mol %, from about 2 mol % to about 3 mol %, from
  • the amount of polymer-lipid (or polymer-conjugated lipid) in the lipid composition is 1.8 mol %.
  • the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.
  • Surface altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl- ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin ⁇ 4, dornase alfa, neltenexine, and erdosteine), and DNases (e.
  • a surface altering agent may be disposed within a nanoparticle and/or on the surface of a LNP (e.g., by coating, adsorption, covalent linkage, or other process).
  • a LNP may also comprise one or more functionalized lipids.
  • a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction.
  • a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging.
  • the surface of a LNP may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.
  • lipid nanoparticles may include any substance useful in pharmaceutical compositions.
  • the lipid nanoparticle may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surface active agents, buffering agents, preservatives, and other species.
  • preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
  • 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.
  • 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.
  • alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONETM, KATHONTM, and/or EUXYL®.
  • An exemplary free radical scavenger includes butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine.
  • buffering agents 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, calcium lactobionate, 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
  • the formulation including a LNP may further include a salt, such as a chloride salt.
  • the formulation including a LNP may further includes a sugar such as a disaccharide.
  • the formulation further includes a sugar but not a salt, such as a chloride salt.
  • a LNP may further include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol.
  • Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). The characteristics of a LNP may depend on the components thereof.
  • the sterol component of a LNP of the disclosure is a single phytosterol.
  • the phytosterol component of a LNP of the disclosure is a mixture of different phytosterols (e.g.2, 3, 4, 5 or 6 different phytosterols).
  • the phytosterol component of an LNP of the disclosure is a blend of one or more phytosterols and one or more zoosterols, such as a blend of a phytosterol (e.g., a sitosterol, such as beta-sitosterol) and cholesterol.
  • the phytosterol is ⁇ -sitosterol, campesterol, sigmastanol, or any combination thereof.
  • the phytosterol is ⁇ -sitosterol.
  • the cholesterol analog comprises ⁇ -sitosterol, campesterol, and stigmasterol.
  • the mixture of structural lipids comprises a mixture of ⁇ -sitosterol and cholesterol.
  • the mixture of structural lipids is a mixture of ⁇ -sitosterol and cholesterol.
  • the mixture of structural lipids comprises about 35% to about 85% of ⁇ -sitosterol, about 5% to about 35% stigmasterol, and about 5% to about 35% of campesterol.
  • the mixture of structural lipids comprises about 40% to about 80% of ⁇ -sitosterol, about 10% to about 30% stigmasterol, and about 10% to about 30% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 70% of ⁇ -sitosterol, about 10% to about 25% stigmasterol, and about 10% to about 25% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 70% of ⁇ -sitosterol, about 15% to about 25% stigmasterol, and about 15% to about 25% of campesterol.
  • the mixture of structural lipids comprises about 35% to about 45% of ⁇ -sitosterol, about 20% to about 30% stigmasterol, and about 20% to about 30% of campesterol. In some embodiments, the the mixture of structural lipids comprises about 40% to about 50% of ⁇ -sitosterol, about 25% to about 35% stigmasterol, and about 25% to about 35% of campesterol. In some embodiments, the mixture of structural lipids comprises about 65% to about 75% of ⁇ -sitosterol, about 5% to about 15% stigmasterol, and about 5% to about 15% of campesterol.
  • the mixture of structural lipids comprises about 35% to about 45% of ⁇ -sitosterol, about 20% to about 30% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 40% to about 50% of ⁇ -sitosterol, about 25% to about 35% stigmasterol, and 0% of campesterol. In some embodiments, the mixture of structural lipids comprises about 65% to about 75% of ⁇ -sitosterol, about 5% to about 15% stigmasterol, and 0% of campesterol. Accordingly, in some preferred embodiments, the composition does not comprise campesterol.
  • the composition comprises a mixture of structural lipids comprising about 10% to about 30% of cholesterol, about 10% to about 30% ⁇ -sitosterol, and about 10% to about 30% stigmasterol, and 0% campesterol. See, for example, Table 2.
  • the composition further comprises about 30-50% cationic lipid and about 5-25% phospholipid.
  • the mixture of structural lipids is between about 30 mol% and about 60 mol%.
  • the mixture of structural lipids is between about 40 mol% and about 60 mol%.
  • the mixture of structural lipids is between about 30 mol% and about 50 mol%.
  • the mixture of structural lipids is between about 40 mol% and about 50 mol%. In some embodiments, the mixture of structural lipids is about 30 mol%. In some embodiments, the mixture of structural lipids is about 40 mol%. In some embodiments, the mixture of structural lipids is about 50 mol%. In some embodiments, the mixture of structural lipids is about 60 mol%. In some embodiments, the mol % of the structural lipids is between about 1% and 50% of the mol % of the compound having the structure of any of the foregoing compounds present in the lipid nanoparticle.
  • the lipid nanoparticle compositions comprise about 30 mol% to about 50 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 35 mol% to about 45 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 37 mol% to about 42 mol% of structural lipids. In some embodiments, the lipid nanoparticle compositions comprise about 35, about 36, about 37, about 38, about 39, or about 40 mol% of structural lipids. In some embodiments, the nanoparticle comprises about 39 to about 40 mol% structural lipids.
  • the composition comprising a mixture of structural lipids does not comprise a mixture of cholesterol and ⁇ -sitosterol of about 38.5 mol%.
  • the composition comprises a mixture of structural lipids comprising cholesterol and a cholesterol analog, wherein the mixture is about 15 mol% to about 25 mol% of cholesterol and about 15 mol% to about 25 mol% cholesterol analog. See, for example, Tables 2, 5 or 6.
  • the amount of cholesterol in the mixture of structural lipids in the composition disclosed herein is at least about 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6,
  • the cholesterol analog is ⁇ - sitosterol, stigmasterol or campesterol. In a preferred embodiment, the cholesterol analog is ⁇ - sitosterol.
  • the amount of cholesterol in the mixture of structural lipids in the composition disclosed herein is at least about 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7,15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 2
  • a LNP of the invention comprises an N:P ratio of about 3:1, 4:1, or 5:1.
  • the characteristics of a LNP may depend on the absolute or relative amounts of its components. For instance, a LNP including a higher molar fraction of a phospholipid may have different characteristics than a LNP including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the lipid nanoparticle. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, and 2-lysophosphatidyl choline.
  • a phospholipid moiety for the lipid nanoparticle include a lipid that is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1- carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DSPC
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidyl-ethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.
  • a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC.
  • Lipid nanoparticles may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a LNP.
  • Dynamic light scattering or potentiometry may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a LNP, such as particle size, polydispersity index, and zeta potential. The mean size of a LNP may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
  • the mean size of a LNP may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm.
  • the mean size of a LNP may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.
  • a LNP may be relatively homogenous.
  • a polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • the efficiency of encapsulation of a therapeutic and/or prophylactic describes the amount of therapeutic and/or prophylactic that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided.
  • the encapsulation efficiency is desirably high (e.g., close to 100%).
  • the encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and/or prophylactic in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and/or prophylactic (e.g., RNA) in a solution.
  • a LNP may be formulated in a capsule, film, or tablet having a coating.
  • a capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.
  • Formulations comprising amphiphilic polymers and lipid nanoparticles may be formulated in whole or in part as pharmaceutical compositions.
  • Pharmaceutical compositions may include one or more amphiphilic polymers and one or more lipid nanoparticles.
  • a pharmaceutical composition may include one or more amphiphilic polymers and one or more lipid nanoparticles including one or more different therapeutics and/or prophylactics.
  • Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein.
  • one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a LNP.
  • the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention.
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • Relative amounts of the one or more amphiphilic polymers, the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and/or any additional ingredients in a pharmaceutical composition in accordance with the present 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.
  • a pharmaceutical composition may comprise between 0.1% and 100% (wt/wt) of one or more lipid nanoparticles.
  • a pharmaceutical composition may comprise between 0.1% and 15% (wt/vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w/v).
  • the lipid nanoparticles and/or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and/or shipment (e.g., being stored at a temperature of about 5 °C or lower, such as a temperature between about -150 °C and about 5 °C or between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about 5 °C, 0 °C, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, - 80 °C, -90
  • the pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and/or shipment at, for example, about - 20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C.
  • RNA integrity is a measure of RNA quality that quantitates intact RNA.
  • the method is also capable of detecting potential degradation products. RNA integrity is preferably determined by capillary gel electrophoresis. The initial specification is set to ensure sufficient RNA integrity in drug product preparations.
  • the RNA polynucleotide has an integrity of at least about 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 95%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 98%. In some embodiments, the RNA polynucleotide has an integrity of or greater than about 99%. In preferred embodiments, the RNA polynucleotide has a clinical grade purity. In some embodiments, the purity of the RNA polynucleotide is between about 60% and about 100%.
  • the method of producing the RNA polynucleotides removes long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA residual solvent and/or residual salt.
  • the short abortive transcript contaminants comprise less than 15 bases.
  • the short abortive transcript contaminants comprise about 8-12 bases.
  • the method of the invention also removes RNAse inhibitor.
  • the purified RNA polynucleotide comprises 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less or is substantially free of protein contaminants as determined by capillary electrophoresis.
  • the purified RNA polynucleotide has integrity of 60% or greater, 70% or greater, 80% or greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater as determined by a known method, such as, e.g., capillary electrophoresis.
  • Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules include, for example, m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1-methyladenosine); m2A (2- methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2- methylthio-N6- methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopenten
  • modified nucleosides in the list may be excluded.
  • Additional exemplary modified nucleotides include any one of N-1-methylpseudouridine; pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine.
  • the modified nucleotide is N-1-methylpseudouridine.
  • the RNA molecule may include phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.
  • the modified or unnatural nucleotides are selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyl uridine.
  • the modified or unnatural nucleotides are selected from the group consisting of 5- methyluridine, N1-methylpseudouridine, 5-methoxyuridine, and 5-methylcytosine. In some embodiments, at least 10% of a total population of a particular nucleotide in the RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 75% of a total population of a particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of the particular nucleotide population in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least a portion, or all of a total population of a particular nucleotide in the RNA molecule has been replaced with two modified or unnatural nucleotides.
  • the two modified or unnatural nucleotides are provided in a ratio equal to any one of, at least any one of, at most any one of, or between any two of 1:99 to 99:1, including 1:99; 2:98; 3:97; 4:96; 5:95; 6:94; 7:93; 8:92; 9:91; 10:90; 11:89; 12:88; 13:87; 14:86; 15:85; 16:84; 17:83; 18:82, 19:81; 20:80; 21:79; 22:78; 23:77; 24:76; 25:75; 26:74; 27:73; 28:72; 29:71; 30:70; 31:69; 32:68; 33:67; 34:66; 35:65; 36:64; 37:63; 38:62; 39:61; 40:60; 41:59; 42:58; 43:57; 44:56; 45:55; 46:54; 47:53; 48:52; 49:51; 50:50; 51:
  • At least 10% of a total population of a first particular nucleotide in a RNA molecule as disclosed herein has been replaced with one or more modified or unnatural nucleotides, and at least 10% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides
  • at least 25% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 10% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides
  • at least 25% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 25% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides
  • at least 50% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 75% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and at least 75% of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • essentially all of a total population of a first particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides
  • essentially all of a total population of a second particular nucleotide in the molecule has been replaced with one or more modified or unnatural nucleotides.
  • at least 25% of a total population of uridine nucleotides in the RNA molecule has been replaced with N1-methylpseudouridine.
  • at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1- methylpseudouridine.
  • essentially all uridine nucleotides in the molecule have been replaced with 5-methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the molecule has been replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 2-thiouridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 2-thiouridine.
  • At least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1-methylpseudouridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methoxyuridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5-methyluridine and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine.
  • the 5′ UTR and the 3′ UTR are from a wild-type alphavirus. Examples of alphaviruses are described below.
  • the first RNA molecule includes a 5′ UTR and the 3′ UTR derived from a naturally abundant mRNA in a tissue.
  • the first RNA molecule includes a 5′ UTR and the 3′ UTR derived from an alphavirus.
  • the second RNA or the saRNA molecule includes a 5′ UTR and the 3′ UTR derived from an alphavirus.
  • the second RNA or the saRNA molecule includes a 5′ UTR and the 3′ UTR from a wild-type alphavirus.
  • the RNA molecule includes a 5’ cap. In some embodiments, the RNA molecule includes a 5’UTR or 3’UTR as disclosed in WO2024/154061, which is hereby incorporated herein by reference in its entirety, including all sequences set forth therein. In some embodiments, the RNA molecule comprises a 5’ UTR having the sequence GAA ⁇ AAAC ⁇ AG ⁇ A ⁇ C ⁇ C ⁇ GG ⁇ CCCCA CAGAC ⁇ CAGA GAGAACCCGC CACC (SEQ ID NO: 1) , wherein ⁇ is 1-methyl-3'-pseudouridylyl.
  • the RNA molecule comprises a 3’ UTR comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCC CGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCAC CACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAG CCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUU AACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUA GC (3’ WHO UTR2). (SEQ ID NO: 7).
  • the RNA molecule comprises a 3’ UTR comprises the sequence C ⁇ CGAGC ⁇ GG ⁇ AC ⁇ GCA ⁇ GCACGCAA ⁇ GC ⁇ AGC ⁇ GCCCC ⁇ CCCG ⁇ CC ⁇ GGG ⁇ AC CCCGAG ⁇ C ⁇ CCCCCGACC ⁇ CGGG ⁇ CCCAGG ⁇ A ⁇ GC ⁇ CCCACC ⁇ CCACC ⁇ GCCCCAC ⁇ CACCACC ⁇ C ⁇ GC ⁇ AG ⁇ CCAGACACC ⁇ CCCAAGCACGCAGCAA ⁇ GCAGC ⁇ CAAAAC GC ⁇ AGCC ⁇ AGCCACACCCCCACGGGAAACAGCAG ⁇ GA ⁇ AACC ⁇ AGCAA ⁇ AAAC GAAAG ⁇ AAC ⁇ AAGC ⁇ A ⁇ AC ⁇ AACCCCAGGG ⁇ GG ⁇ CAA ⁇ CG ⁇ GCCAGCCACA CCC ⁇ GGAGC ⁇ AGC (3’ WHO ⁇ TR2) (SEQ ID NO: 8), wherein ⁇ is 1-methyl-3'-pseudouridylyl.
  • the 5′ and 3′ UTRs may be operably linked to an ORF, which may be a sequence of codons derived from a gene of interest that is capable of being translated into a polypeptide of interest.
  • ORF Open reading frame
  • the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs).
  • the ORF encodes a non-structural viral gene.
  • the ORF further includes one or more subgenomic promoters.
  • the RNA molecule includes a subgenomic promoter operably linked to the ORF.
  • the subgenomic promoter comprises a cis-acting regulatory element.
  • the cis-acting regulatory element is immediately downstream (5’-3’) of B 2 .
  • the cis-acting regulatory element is immediately downstream (5’-3’) of a guanine that is immediately downstream of B 2 .
  • the cis-acting regulatory element is an AU-rich element.
  • the AU-rich element is au, auaaaagau, auaaaagau, auag, auauauauau, auauauauauau, augaugaugau, augau, augau, auaaagaua, or auaaaagaug.
  • the RNA molecule includes alphavirus nonstructural protein nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP4. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsP1, nsP2, and nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4.
  • the gene of interest encodes a polypeptide of interest selected from, e.g., biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
  • the sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GenBank.
  • the RNA molecule includes a coding region for an antigen preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia
  • the saRNA molecule described herein includes a 5’ cap.
  • the 5'-cap moiety is a natural 5'-cap.
  • a “natural 5'-cap” is defined as a cap that includes 7-methylguanosine connected to the 5’ end of an mRNA molecule through a 5′ to 5′ triphosphate linkage.
  • the 5'-cap moiety is a 5'- cap analog.
  • capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.
  • An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyl- transferase, and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures.
  • VCE Vaccinia Virus Capping Enzyme
  • Cap 0 structure can help maintaining the stability and translational efficacy of the RNA molecule.
  • the 5' cap of the RNA molecule may be further modified by a 2 '-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '- ⁇ ] N), which may further increase translation efficacy.
  • the RNA molecule may be enzymatically capped at the 5′ end using Vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to yield cap 0 structure.
  • An inverted 7- methylguanosine cap is added via a 5′ to 5′ triphosphate bridge.
  • a 2′O- methyltransferase with Vaccinia guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2′OH group is methylated on the penultimate nucleotide.
  • S-adenosyl- L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent.
  • Non-limiting examples of 5′ cap structures are those which, among other things, have enhanced binding of cap binding polypeptides, increased half-life, reduced susceptibility to 5′ endonucleases and/or reduced 5′ decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme may create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5′-terminal nucleotide of the mRNA includes a 2′-O- methyl.
  • Cap1 structure is termed the Cap1 structure.
  • Cap structures include, but are not limited to, 7mG(5′)ppp(5′)N,pN2p (cap 0) and 7mG(5′)ppp(5′)N1mpNp (cap 1).
  • Cap 0 is a N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, typically referred to as m7G cap or m7Gppp.
  • the cap 0 structure can help provide for efficient translation of the mRNA that carries the cap.
  • An additional methylation on the 2′O position of the initiating nucleotide generates Cap 1, or refers to as m7GpppNm-, wherein Nm denotes any nucleotide with a 2′O methylation.
  • the 5′ terminal cap includes a cap analog, for example, a 5′ terminal cap may include a guanine analog.
  • Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
  • the capping region may include a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or fewer nucleotides in length.
  • the cap is absent.
  • the first and second operational regions may be equal to any one of, at least any one of, at most any one of, or between any two of 3 to 40, e.g., 5-30, 10-20, 15, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
  • the 5’ Cap is represented by Formula I:
  • R 1 and R 2 ar ndependently guanine, adenine, or uracil.
  • B 1 and B 2 are naturally-occurring bases.
  • R 1 is methyl and R 2 is hydrogen.
  • B 1 is guanine.
  • B 1 is adenine.
  • B 2 is adenine.
  • B 2 is uracil.
  • B 2 is uracil and at least 5% of a total population of uracil nucleotides in the molecule that are downstream of B 2 have been replaced with one or more modified or unnatural nucleotides.
  • the RNA molecule comprises at least 7000 nucleotides. In some embodiments, the RNA molecule comprises at least 8000 nucleotides. In some embodiments, at least 80% of the total RNA molecules are full length.
  • the alphavirus is Venezuelan equine encephalitis virus. In some embodiments, the alphavirus is Semliki Forest virus.
  • the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with N1- methylpseudouridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine.
  • the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, at least 50% of a total population of uridine nucleotides in the molecule has been replaced with 5- methyluridine, and essentially all cytosine nucleotides in the molecule have been replaced with 5-methylcytosine.
  • the nucleotide immediately downstream (5’ to 3’) of the 5’ Cap comprises guanine, B 1 is adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen, essentially all uridine nucleotides in the molecule have been replaced with about 75% 5-methoxyuridine and about 25% N1-methylpseudouridine.
  • the poly A tail nucleotide length may be equal to any one of, at least any one of, at most any one of, or between any two of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400.
  • the RNA molecule includes a poly A tail that includes about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides.
  • the RNA molecule includes a poly A tail includes a sequence of greater than 30 adenosine nucleotides (“As”).
  • the RNA molecule includes about 70 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 80 consecutive adenosine residues.
  • Compositions In some instances, the compositions described herein include at least one RNA as described herein.
  • RNA molecules (capped and uncapped) in the composition are capped.
  • RNA molecules in the composition are full length RNA transcripts.
  • the composition comprises an amount of the first RNA molecule that is at least about 1 to 100 times greater than the amount of the second RNA molecule.
  • the composition further includes a pharmaceutically acceptable carrier.
  • the composition further includes a pharmaceutically acceptable vehicle.
  • the composition further includes a lipid-based delivery system, which delivers an RNA molecule to the interior of a cell, where it can then replicate and/or express the encoded polypeptide of interest. The delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen.
  • the composition further includes one or more each of neutral lipids, cationic lipids, structural lipids (e.g.
  • the composition further includes any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil- in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion.
  • compositions described herein include at least two RNA molecules: a first RNA molecule and a second RNA molecule as described herein.
  • a combination vaccine composition may be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first virus or organism and further includes a second RNA molecule encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second virus or organism.
  • RNA can be co-formulated, for example, in a single lipid nanoparticle (LNP) or can be formulated in separate LNPs for co-administration.
  • the second RNA molecule includes any one of a 5’ cap, a 5’ UTR, an open reading frame, a 3’ UTR, and a poly A sequence, or any combination thereof.
  • the second RNA molecule includes a 5’ cap moiety.
  • the second RNA molecule includes a 5’ UTR and a 3’UTR.
  • the second RNA molecule includes a 5’UTR, an open reading frame, a 3’UTR, and does not further include a 5’ cap.
  • the second RNA molecule includes a 5’ cap moiety, 5’ UTR, coding region, 3’ UTR, and a 3’ poly A sequence.
  • compositions comprising (i) first RNA molecule encoding a gene of interest; and (ii) a second RNA molecule comprising a modified or unnatural nucleotide.
  • the first RNA molecule is any one of the saRNA molecules described herein.
  • the first RNA molecule comprises a 5’ Cap, a 5’ untranslated region, a coding region for a nonstructural protein comprising a RNA replicase, a subgenomic promoter, an open reading frame encoding a gene of interest, a 3’ untranslated region, and a 3’ poly A sequence.
  • RNA molecule comprises natural, unmodified nucleotides and does not include a modified or unnatural nucleotide.
  • B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, the nucleotide immediately downstream (5’ to 3’ direction) of the 5’ Cap comprises guanine. In some embodiments, B 1 is adenine and B 2 is uracil. In some embodiments, B 1 is adenine, B 2 is uracil, R 1 is methyl, and R 2 is hydrogen.
  • At least 25% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 50% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, at least 75% of a total population of a particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides. In some embodiments, essentially all of a particular nucleotide population in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides.
  • the one or more modified or unnatural replacement nucleotides comprise two modified or unnatural nucleotides provided in a ratio ranging from 1:99 to 99:1, or any derivable range therein. In some embodiments, at least 10% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and at least 10% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides
  • at least 25% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides
  • at least 25% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides
  • at least 50% of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides.
  • At least 75% of a total population of a first particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides, and essentially all of a total population of a second particular nucleotide in the first or second RNA molecule has been replaced with one or more modified or unnatural nucleotides.
  • at least 25% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine.
  • at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine.
  • At least 75% of a total population of uridine nucleotides in the first RNA molecule has been replaced with N1-methylpseudouridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 5-methoxyuridine. In some embodiments, essentially all uridine nucleotides in the molecule have been replaced with 5- methoxyuridine.
  • At least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 5-methyluridine. In some embodiments, essentially all uridine nucleotides in the first RNA molecule have been replaced with 5- methyluridine. In some embodiments, at least 50% of a total population of cytosine nucleotides in the first RNA molecule has been replaced with 5-methylcytosine. In some embodiments, essentially all cytosine nucleotides in the first RNA molecule have been replaced with 5- methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the first RNA molecule has been replaced with 2-thiouridine.
  • At least 50% of a total population of cytosine nucleotides in the second RNA molecule has been replaced with 5- methylcytosine. In some embodiments, essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 2- thiouridine. In some embodiments, essentially all uridine nucleotides in the second RNA molecule have been replaced with 2-thiouridine.
  • At least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with N1-methylpseudouridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine. In some embodiments, at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine and essentially all cytosine nucleotides in the second RNA molecule have been replaced with 5-methylcytosine.
  • essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and essentially all uridine nucleotides in the second RNA molecule have been replaced with N1-methylpseudouridine.
  • essentially all uridine nucleotides in the first RNA molecule have been replaced with N1-methylpseudouridine and at least 50% of a total population of uridine nucleotides in the second RNA molecule has been replaced with 5-methoxyuridine.
  • RNA compositions may be utilized to treat and/or prevent a disease, disorder or infection comprising administering to a subject any of the RNA compositions described herein.
  • the RNA composition produces an antigen specific immune response.
  • the antigen specific immune response comprises a T cell response.
  • the antigen specific immune response comprises a B cell response.
  • the antigen specific immune response comprises both a T cell response and a B cell response.
  • the method of producing an antigen specific immune response involves a single administration of the RNA composition.
  • the RNA composition is administered to the subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.
  • the nanoparticle has a net neutral charge at a neutral pH value.
  • the RNA (e.g., mRNA) vaccine is multivalent.
  • the RNA polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof as an antigen.
  • One aspect of the disclosure is directed to a method comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein, wherein the amount of the composition is an amount effective to produce an immune response, to treat or to prevent a disease, disorder or infection i.e. “an effective amount”.
  • Some aspects of the disclosure are directed to a method of vaccinating a subject, comprising administering to the subject in need thereof an effective amount of a composition as disclosed herein.
  • the compounds of the invention may be useful for treating or preventing a disease, disorder, or condition or delivering an active agent to treat or prevent a disease, disorder, or condition.
  • such compositions may be useful in treating or preventing a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity.
  • Diseases, disorders, and/or conditions characterized by dysfunctional or aberrant protein or polypeptide activity for which a composition may be administered include, but are not limited to rare diseases, infectious diseases (as both vaccines and therapeutics), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.
  • Administration and Dosing The term "treating”, “treat” or “treatment” as used herein embraces both preventative, e.g., prophylactic, and palliative treatment, e.g., relieve, alleviate, or slow the progression of the patient’s disease (or condition) or any tissue damage associated with the disease.
  • the terms, “subject, “individual” or “patient,” used interchangeably, refer to any animal, including mammals. Mammals according to the invention include canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, humans and the like, and encompass mammals in utero. In an embodiment, humans are suitable subjects. Human subjects may be of any gender and at any stage of development.
  • the phrase “therapeutically effective amount” or “effective amount” refers to the amount of active compound or pharmaceutical agent, such as a nucleic acid, that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include one or more of the following: (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (e.g., arresting (or slowing) further development of the pathology or symptomatology or both); and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (e.
  • an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater.
  • Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • test sample e.g., a sample of cells in culture expressing the target gene
  • test mammal e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model
  • a nucleic acid that silences, reduces, or inhibits expression of the target gene.
  • Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • a compound of the invention is administered in an amount effective to treat a condition as described herein or to deliver an active agent to treat a condition as described herein.
  • the compounds of the invention may be administered as compound per se, or alternatively, as a pharmaceutically acceptable salt.
  • the compounds of the invention may be administered rectally or vaginally. In another embodiment, the compounds of the invention may also be administered directly to the eye or ear.
  • the dosage regimen for the compounds of the invention or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary widely.
  • the total daily dose of a compound of the invention is typically from about 0.01 to about 100 mg/kg (e.g., mg compound of the invention per kg body weight) for the treatment of the indicated conditions discussed herein or to deliver an active agent by using the compounds of the invention.
  • total daily dose of the compound of the invention is from about 0.00001 to about 50 mg/kg, and in another embodiment, from about 0.0001 to about 30 mg/kg. It is not uncommon that the administration of the compounds of the invention will be repeated a plurality of times in a day (typically no greater than 4 times). Multiple doses per day typically may be used to increase the total daily dose, if desired.
  • nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, mRNA (e.g.
  • polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar polypeptide.
  • polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 95% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • nucleic acid sequences regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the nucleic acids can be any length.
  • the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is higher than the LNP integrity of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more, about 10 folds or more, about 20 folds or more, about 30 folds or more, about 40 folds or more, about 50 folds or more, about 100 folds or more, about 200 folds or more, about 300 folds or more, about 400 folds or more, about 500 folds or more, about 1000 folds or more, about 2000 folds or more, about 3000 folds or more, about 4
  • the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, about 120 months or longer.
  • the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present disclosure is longer than the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more
  • “Tx” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about X of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension
  • “T8o%” refers to the amount of time lasted for the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation to degrade to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used for the preparation of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.
  • nucleic acid integrity e.g., mRNA integrity
  • the elements of a LNP may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements.
  • the particular formulation of a LNP may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements.
  • the efficacy and tolerability of a LNP formulation may be affected by the stability of the formulation.
  • the lipid component of a LNP may include, for example, a cationic lipid, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC), a PEG lipid, and a structural lipid.
  • the lipid component of the lipid nanoparticle includes about 30 mol % to about 60 mol % cationic lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipids, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%.
  • the lipid component includes about 40 mol % said cationic lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipids, and about 1.5 mol % of PEG lipid.
  • the phospholipid may be DOPE or DSPC.
  • the PEG lipid may be PEG-DMG and/or the structural lipids may be cholesterol.
  • the amount of a therapeutic and/or prophylactic in a LNP may depend on the size, composition, desired target and/or application, or other properties of the lipid nanoparticle as well as on the properties of the therapeutic and/or prophylactic.
  • the ionizable lipid is: may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • PEG lipid refers to polyethylene glycol (PEG) -modified lipids.
  • PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCl4 or PEG-CerC20), PEG- modified dialkylamines and PEG-modified l,2-diacyloxypropan-3 -amines.
  • lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG- modified lipids are a modified form of PEG DMG.
  • the PEG-modified lipid is PEG lipid with the formula (IV): interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a mixture of two structural lipids and a non-cationic lipid.
  • a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the structural lipid is a sterol.
  • the sterol is cholesterol.
  • the sterol is a cholesterol analog.
  • a cationic lipid is selected from the group consisting of ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 2,2- dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)—N,N-dimethyl-2- nonylhenicosa-12,15-dien-1-amine (L608), and N,N-dimethyl-1-[(1S,2R)-2- oc
  • the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% neutral lipid; 35-45 mol% mixture of cholesterol and a cholesterol analog; and 1-5 mol% PEG- modified lipid.
  • the lipid mixture and thus the lipid nanoparticle, comprises 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-15 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral lipid.
  • the lipid mixture comprises 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% mixture of cholesterol and a cholesterol analog.
  • the lipid mixture and thus the lipid nanoparticle, comprises 0.5-10 mol%, 0.5-5 mol%, 0.5-1 mol%, 1-15%, 1-10 mol%, 1-5 mol%, 1.5-15%, 1.5- 10 mol%, 1.5-5 mol%, 2-15%, 2-10 mol%, 2-5 mol%, 2.5-15%, 2.5-10 mol%, 2.5-5 mol%, 3- 15%, 3-10 mol%, or 3-5 mol%, PEG-modified lipid.
  • the lipid mixture comprises: 50 mol% ionizable amino lipid; 10 mol% neutral lipid; 38.5 mol% mixture of cholesterol and a cholesterol analog; and 1.5 mol% PEG-modified lipid.
  • the ionizable amino lipid is heptadecan-9-yl 8 ((2 hydroxyethyl)(6 oxo 6- (undecyloxy)hexyl)amino)octanoate.
  • the neutral lipid is 1,2 distearoyl sn glycero-3 phosphocholine (DSPC).
  • the sterol is cholesterol.
  • the PEG-modified lipid is 1-monomethoxypolyethyleneglycol-2,3- dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG).
  • a composition may further include a pharmaceutically-acceptable excipient, inert or active.
  • a pharmaceutically acceptable excipient after administered to a subject, does not cause undesirable physiological effects.
  • the excipient in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with mRNA and can be capable of stabilizing it.
  • One or more excipients e.g., solubilizing agents
  • examples of a pharmaceutically acceptable excipients include, but are not limited to, biocompatible vehicles (e.g., LNPs), carriers, adjuvants, additives, and diluents to achieve a composition usable as a dosage form.
  • an mRNA is 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); (4) alter the biodistribution (e.g., target 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 (antigen) in vivo.
  • excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
  • a composition comprising mRNA does not include an adjuvant (the composition is adjuvant-free).
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprising two or more RNA molecules, comprises each RNA molecule in the same amount (i.e., at a 1:1 ratio).
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprising two or more RNA molecules, comprises a different amount of each RNA molecule.
  • a nucleic acid containing particle comprises a first RNA molecule and a second RNA molecule, where the first RNA molecule is present in an amount that is 0.01 to 100 times that of the second RNA molecule (e.g., wherein the amount of the first RNA molecule is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times higher than the second RNA molecule).
  • the amount of the first RNA molecule is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6,
  • a nucleic acid containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 10 times that of the second RNA molecule. In some embodiments, a nucleic acid containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 5 times that of the second RNA molecule. In some embodiments, a nucleic acid containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 3 times that of the second RNA molecule.
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprising three RNA molecules, comprises a different amount of each RNA molecule.
  • the ratio of first RNA molecule: second RNA molecule: third RNA molecule is 1: 0.01-100: 0.01-100 (e.g., 1: 0.01-50: 0.01-50; 1: 0.01-40: 0.01-40; 1: 0.01-30: 0.01-25; 1: 0.01-25: 0.01-25; 1: 0.01-20: 0.01-20; 1: 0.01-15: 0.01-15; 1: 0.01-10: 0.01-9; 1: 0.01-9: 0.01-9; 1: 0.01-8: 0.01-8; 1: 0.01-7: 0.01-7; 1: 0.01-6: 0.01-6; 1: 0.01-5: 0.01-5; 1: 0.01-4: 0.01-4; 1: 0.01-3: 0.01-3; 1: 0.01-2: 0.01-2; or 1: 0.01-1.5:
  • each of a buffer, stabilizing agent, and optionally a salt may be included in the immunogenic composition including a lipid-based delivery system.
  • any one or more of a buffer, stabilizing agent, salt, surfactant, preservative, and excipient may be excluded from the immunogenic composition including a lipid-based delivery system.
  • the immunogenic composition including a lipid-based delivery system further comprises a stabilizing agent.
  • the concentration of the stabilizing agent is equal to at least, at most, exactly, or between any two of 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, or more.
  • the mass amount of the stabilizing agent and the mass amount of the RNA are in a specific ratio.
  • the buffer is a HEPES buffer, a Tris buffer, or a PBS buffer.
  • the composition further includes an adjuvant, e.g., aluminum-containing compounds, such as, for example, any of the adjuvants listed herein, including aluminum hydroxide and AlPO4.
  • the buffer is Tris buffer.
  • the buffer is a HEPES buffer.
  • the buffer is a PBS buffer.
  • the buffer may be at least, at most, exactly, or between any two of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.
  • the buffer is at pH 7.4.
  • the immunogenic composition including a lipid-based delivery system may further comprise a salt.
  • salts include but not limited to sodium salts and/or potassium salts.
  • the salt is a sodium salt.
  • the sodium salt is sodium chloride.
  • the salt is a potassium salt.
  • the potassium salt comprises potassium chloride.
  • the salt concentration includes, but is not limited to, a concentration of about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 15 mg/mL.
  • the concentration of the salt is equal to at least, at most, exactly, or between any two of 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, or more.
  • the salt may be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6.
  • the salt may be at a pH equal to at least, at most, exactly, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5.
  • the immunogenic composition including a lipid-based delivery system further comprises a surfactant, a preservative, any other excipient, or a combination thereof.
  • any other excipient includes, but is not limited to, antioxidants, glutathione, EDTA, methionine, desferal, antioxidants, metal scavengers, or free radical scavengers.
  • the surfactant, preservative, excipient or combination thereof is sterile water for injection (sWFI), bacteriostatic water for injection (BWFI), saline, dextrose solution, polysorbates, poloxamers, Triton, divalent cations, Ringer’s lactate, amino acids, sugars, polyols, polymers, or cyclodextrins.
  • sWFI sterile water for injection
  • BWFI bacteriostatic water for injection
  • saline dextrose solution
  • polysorbates poloxamers
  • Triton Triton
  • divalent cations divalent cations
  • Ringer s lactate
  • amino acids amino acids
  • sugars polyols
  • polymers polymers
  • Ringer lactate
  • excipients which refer to ingredients in the immunogenic compositions that are not active ingredients, include but are not limited to carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants.
  • Preservatives for use in the compositions disclosed herein include but are not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • Diluents include but are not limited to ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, corn rice, and potato; and celluloses such as microcrystalline cellulose.
  • the amount of diluent in the composition may range from about 10% to about 90% by weight of the total composition, about 25% to about 75%, about 30% to about 60% by weight, or about 12% to about 60%.
  • the pH and exact concentration of the various components in the immunogenic composition including a lipid-based delivery system are adjusted according to well-known parameters. The use of such media and agents for pharmaceutical active substances is well known in the art.
  • compositions and methods for their use may “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure.
  • LNP Lipid Nanoparticle
  • the benchmark LNP formulation contains a cationic lipid (ALC-0315), a PEG-lipid (ALC- 0159), a neutral lipid (DSPC (1,2distearoyl-sn-glycero-3-phosphocholine)) and a Sterol (cholesterol).
  • ALC-0315 cationic lipid
  • ALC- 0159 PEG-lipid
  • DSPC 1,2distearoyl-sn-glycero-3-phosphocholine
  • Sterol cholesterol
  • EXAMPLE 2 Enhancing Flu B modRNA Immunogenicity with LNPs Containing Cholesterol C-24 Alkyl Derivatives This study was conducted to compare LNPs containing cholesterol C-24 alkyl derivatives, wherein the LNPs encompass a modified RNA encoding an influenza HA polypeptide from an influenza B strain (Austria).
  • mRNA-LNPs containing cholesterol analogues were formulated by combining an mRNA containing aqueous phase and a lipid containing organic phase using methods known in the art. The organic phase was prepared by solubilizing a mixture of ionizable lipid, DSPC, PEG-lipid, cholesterol and cholesterol analogs at various ratios in ethanol.
  • the organic phase and aqueous phase were mixed at a flow rate ratio of 3:1 by syringe pumps.
  • the resultant solution was dialyzed against 10 mM Tris buffer (pH 7.4).
  • Post- dialysis solution was concentrated and spiked with cryo-protectant to a final mRNA-LNP solution with RNA concentration of 0.1 mg/ml.
  • Immunogenicity in Balb/c mice was evaluated using Neutralization and HAI assays.
  • the test articles are listed in Table 2. Each dose was 50 ul administered to mice via IM on days 0 and 28, according to the respective dose listed in Table 2.
  • Each formulation described throughout the Examples section herein comprises ALC- 0159 pegylated lipid at 1.8% molar percentage in the LNP formulation.
  • the objective of this Example was to assess in vitro expression (IVE) of hemagglutinin (HA) from lipid nanoparticles (LNPs) formulated with alternative cholesterols and compare this in vitro expression performance to LNPs formulated with novel cholesterols and/or N/P ratios.
  • This Example helped to inform whether or not changing certain cholesterols, and/or N/P ratios, can alter the in vitro expression of HA from an unmodified bi-cistronic saRNA encoding HA and NA of H1N1 A/Wisconsin, in HeLa cells.
  • An exemplary bicistronic saRNA encoding HA and NA include the sequence set forth in SEQ ID NO: 9.
  • Another exemplary bicistronic saRNA includes the sequence set forth in SEQ ID NO: 10. See, for example, International patent application PCT/IB2023/057034, published as WO2024/013625, entitled, “Self-amplifying rna encoding an influenza virus antigen,” (Pfizer Inc.) filed on July 7, 2023, which is incorporated by reference herein in its entirety and describes saRNA molecules and bicistronic saRNA.
  • Method An 11-point, 2-fold dilution of LNPs in HeLa cell cultures was performed. Geometric mean fluorescence intensity (GMFI) of HA positive cells, the percentage of total HA positive cells and cell viability was measured.
  • GMFI Geometric mean fluorescence intensity
  • the EC50s for LNP 2 through 5 were significantly lower than the EC50 of the benchmark LNP comprising cholesterol (LNP 1) (Table 4).
  • the in vitro activity (IVA) of the cells treated with LNPs 2 through 5 was significantly higher than the activity of the cells treated with the benchmark LNP comprising cholesterol (LNP 1).
  • GMFI from HA staining was higher for all LNPs containing alternative sterols, with the b-Sitosterol/Cholesterol (6:4) LNP performing the best.
  • Cell viability was significantly lower for all LNPs containing alternative sterols, as compared to the benchmark LNP 1.
  • beta-Sitosterol/Cholesterol (6:4) LNPs demonstrated the best relative ratio of live cells vs. % HA positive.
  • EXAMPLE 4 In vitro expression of hemagglutinin (HA) from LNPs formulated with alternative sterols.
  • HEK293T Human embryonic kidney cells are seeded on one to two 12-well culture plates per assay instance and transfected with control and drug product (DP) test samples across two assay instances. After 21-24 hours, cells are harvested from the 12-well plates and transferred to 96-well assay plates. The cells are stained with fixable aqua viability dye before being permeabilized and fixed. After the fixative is washed from the cells, a fluorophore-conjugated HA antibody cocktail is added which binds to influenza HA antigens.
  • DP control and drug product
  • the LNPs encapsulated modified mRNA encoding GFP The LNPs encapsulated modified mRNA encoding GFP.
  • mRNA-LNPs containing cholesterol analogues were formulated by combining an mRNA containing aqueous phase and a lipid containing organic phase using methods known in the art.
  • the organic phase was prepared by solubilizing a mixture of ionizable lipid, DSPC, PEG-lipid, cholesterol and cholesterol analogs at various ratios in ethanol.
  • the organic phase and aqueous phase were mixed at a flow rate ratio of 3:1 by syringe pumps.
  • the resulted solution was dialyzed against 10 mM Tris buffer (pH ⁇ 7.4-7.6).
  • Post-dialysis solution was concentrated and spiked with cryo-protectant to a final mRNA-LNP solution with RNA concentration as described herein (mg/ml).
  • the formulations described in this Example comprise ALC-0159 PEGylated lipid at 1.8% molar percentage in the LNP formulations. Results are shown in Table 11. Decreasing sitosterol percentage in the sterol mixture appeared to result in slightly higher %EE and smaller LNP size. Sito/chol 1:1 mixture showed slightly better EC50 than the other two ratios in human embryonic kidney (HEK293T) cells.
  • Table 13 Experiment input Factors Levels Benchmark Cat% 30 40 50 47.5 Phos% 10 15 20 10 Chol Mix Cholesterol Beta-Sitosterol/Cholesterol Cholesterol (1:1) N/P ratio 6 10 14 6 Table 14 Experiment output Critical quality Range Analytical method attribute Size (nm) 50-150 DLS PDI 0-0.3 DLS EC50 0-500, minimize IVE MFI 0-100,000 IVE maximize %EE 70-100, maximize Ribogreen assay A total of 15 formulation candidates (Candidates A01-A14 and benchmark) were formulated and evaluated using the analytical methods listed. The detailed composition and critical attributes are listed in the Table 15 below.
  • sitosterol-containing formulations compared to the cholesterol benchmark.
  • the four sitosterol LNP formulations exhibited comparable EC50 values for both total and preF-specific protein expression.
  • Each formulation described throughout this Example comprises ALC-315, DSPC , sterol and ALC-0159 PEGylated lipidin the LNP formulation.
  • the sterol component is a mixture of cholesterol and cholesterol analogues listed in Table 17 at a 6:4 or 1:1 molar ratio. All sterol-candidate containing formulations showed favorable critical attributes except for SN792-formulation showing relatively low % encapsulation of the RSV preF modRNA.
  • the in vitro expression of antigen-of-interest was measured with two antibodies in HEK293T cells, L4-6 antibody detecting total F protein and in- house mAb-1 antibody detecting prefusion-specific protein specifically.
  • FIG.9A-9F summarize the critical attributes of these LNPs (see Table 17 formulations) over 6-month storage at 5 °C.
  • Sterol- containing LNPs were mostly colloidally stable except for Sito-4, Sito-5 and stigmasterol formulations which showed a considerable increase in both size and PDI.
  • the decrease rate of % encapsulation and integrity of the mRNA was comparable among sterol formulations and the cholesterol benchmark formulations.
  • SN792-LNP was not further characterized due to its low encapsulation.
  • sterol-containing LNPs continued to show significantly enhanced expression of both total F protein (measured by L4-6) and preF-specific protein (measured by mAb-1) after 6-month refrigeration, outforming cholesterol benchmark formulation that exhibited the fastest increase in the EC50 values in IVE with both antibodies.
  • This result demonstrated improved 5 °C stability for various sterol incorporated LNPs over cholesterol formulation.
  • An immunogenic composition comprising at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, formulated in a lipid nanoparticle comprising one or more structural lipids and/or analogs thereof.
  • RNA ribonucleic acid
  • the structural lipids comprise a mixture of cholesterol and a cholesterol analog.
  • the RNA further comprises a 5’ cap analog.
  • the 5’ cap analog comprises m2 7,3’-O Gppp(m1 2’-O )ApG.
  • the RNA further comprises a modified nucleotide.
  • the modified nucleotide comprises N1-Methylpseudourodine-5’-triphosphate (m1 ⁇ TP).
  • the immunogenic composition of paragraph 1, wherein the antigenic polypeptide is encoded by an open reading frame (ORF) of a gene of interest. 7.
  • the immunogenic composition further comprises a cationic lipid.
  • the immunogenic composition comprises a lipid nanoparticle encompassing the mRNA molecule.
  • the immunogenic composition comprises a lipid nanoparticle encompassing at least one ribonucleic acid (RNA) polynucleotide having an open reading frame derived from a gene of interest encoding at least one antigenic polypeptide.
  • RNA ribonucleic acid
  • the immunogenic composition of paragraph 10 wherein the lipid nanoparticle size is at most 180 nm. 13. The immunogenic composition of paragraph 10, wherein at least 80% of the total RNA in the immunogenic composition is encapsulated. 14. The immunogenic composition of paragraph 1, wherein the immunogenic composition comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate). 15. The immunogenic composition of paragraph 1, wherein the immunogenic composition comprises ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). 16.
  • the LNP comprises a structural lipid selected from the group consisting of: cholesterol, fecosterol, sitosterol, ⁇ - sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.
  • the LNP comprises an N:P ratio of from about 2:1 to about 30:1. 21.
  • the immunogenic composition of paragraph 19, wherein the immunogenic composition comprises a combination of cholesterol and any one of beta-sitosterol or campesterol. 22.
  • the immunogenic composition of paragraph 20, wherein the immunogenic composition comprises Cholesterol: ⁇ -Sitosterol 0.4:0.6.
  • the LNP comprises ⁇ -sitosterol and cholesterol having an N:P ratio of at least 6.
  • 26. The immunogenic composition according to any one of paragraphs 1-22, wherein the LNP comprises ⁇ -sitosterol and cholesterol having an N:P ratio of 10.
  • the lipid nanoparticle comprises about 30 mol % to about 60 mol % one or more ionizable lipids, about 0 mol % to about 30 mol % one or more non-cationic helper lipids, about 18.5 mol % to about 48.5 mol % structural component, and about 0 mol % to about 10 mol % one or more PG- lipids. 31.
  • the immunogenic composition comprises Tris.
  • the immunogenic composition comprises sucrose.
  • the immunogenic composition does not further comprise sodium chloride.
  • the immunogenic composition of paragraph 1 wherein the immunogenic composition comprises 300 mM sucrose. 37. The immunogenic composition of paragraph 1, wherein the immunogenic composition has a pH 7.4. 38. The immunogenic composition of paragraph 1, wherein the immunogenic composition has less than or equal to 12.5 EU/mL of bacterial endotoxins. 39. The immunogenic composition of paragraph 1, wherein the RNA polynucleotide comprises a 5’ cap, 5’ UTR, 3’ UTR, histone stem-loop and poly-A tail. 40.
  • An immunogenic composition comprising: (i) a first ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a first antigen polypeptide or an immunogenic fragment thereof, and (ii) a second RNA polynucleotide having an open reading frame encoding a second antigen polypeptide or an immunogenic fragment thereof, wherein the first and second RNA polynucleotides are formulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the immunogenic composition of any preceding paragraph further comprising: (iii) a third antigen comprising at least one antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is from a different virus, bacteria or strain of the virus or bacteria to both the first and second antigens.
  • a third antigen comprising at least one antigenic polypeptide or an immunogenic fragment thereof, wherein the third antigen is from a different virus, bacteria or strain of the virus or bacteria to both the first and second antigens.
  • the immunogenic composition of any preceding paragraph further comprising: (v) a fifth RNA polynucleotide having an open reading frame encoding a fifth antigen, said antigen comprising at least one antigenic polypeptide or an immunogenic fragment thereof, wherein the fifth antigen is from a different virus, bacteria or strain of the virus or bacteria to the first, second, third, and fourth antigens.
  • a fifth RNA polynucleotide having an open reading frame encoding a fifth antigen said antigen comprising at least one antigenic polypeptide or an immunogenic fragment thereof, wherein the fifth antigen is from a different virus, bacteria or strain of the virus or bacteria to the first, second, third, and fourth antigens.
  • 49 The immunogenic composition of paragraph 48, wherein the first, second, third, fourth, and fifth RNA polynucleotides are formulated in a lipid nanoparticle.
  • the first, second, third, fourth, and fifth RNA polynucleotides are formulated in a single
  • RNA polynucleotide having an open reading frame encoding an eighth antigen, said antigen comprising at least one antigenic polypeptide or an immunogenic fragment thereof, wherein the eighth antigen is from a different virus, bacteria or strain of the virus or bacteria to the first, second, third, fourth, fifth, sixth and seventh antigens.
  • the immunogenic composition of any one of the preceding paragraphs, wherein the ratio of the first RNA polynucleotide to the second RNA polynucleotide is 1: greater than 1. 59. The immunogenic composition of paragraph 58, wherein the RNA polynucleotides are not present in equal ratios. 60. The immunogenic composition of any one of any of the preceding paragraphs, wherein the ratio of the first RNA polynucleotide to the second RNA polynucleotide is 1:2. 61. The immunogenic composition of any one of the preceding paragraphs, wherein the ratio of the first RNA polynucleotide to the second RNA polynucleotide is 1:4. 62.
  • each RNA polynucleotide comprises a 5′ terminal cap, a 5’ UTR, a 3’UTR, and a 3′ polyadenylation tail.
  • the immunogenic composition of paragraph 67, wherein the 3’ UTR comprises SEQ ID NO: 2.
  • the immunogenic composition of paragraph 67, wherein the 3′ polyadenylation tail comprises SEQ ID NO: 3.
  • the immunogenic composition of any preceding paragraph, wherein the RNA polynucleotide has an integrity greater than 85%. 73.
  • RNA polynucleotide has a purity of greater than 85%.
  • the lipid nanoparticle comprises 20-60 mol % ionizable cationic lipid, 5-25 mol % neutral lipid, 25-55 mol % cholesterol, and 0.5-5 mol % PG-modified lipid.
  • the cationic lipid comprises: .
  • the PEG- modified lipid comprises: .
  • the at least the first and second RNA polynucleotides are formulated in a single lipid nanoparticle. 78.
  • the immunogenic composition of paragraph 76 wherein the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; and the fourth RNA polynucleotide is formulated in a fourth LNP. 85.
  • the immunogenic composition of paragraph 76 wherein the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; and the fifth RNA polynucleotide is formulated in a fifth LNP.
  • the first RNA polynucleotide is formulated in a first LNP
  • the second RNA polynucleotide is formulated in a second LNP
  • the third RNA polynucleotide is formulated in a third LNP
  • the fourth RNA polynucleotide is formulated in a fourth LNP
  • the fifth RNA polynucleotide is formulated in a fifth LNP.
  • the immunogenic composition of paragraph 76 wherein the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; the fifth RNA polynucleotide is formulated in a fifth LNP; and the sixth RNA polynucleotide is formulated in a sixth LNP. 87.
  • the immunogenic composition of paragraph 76 wherein the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; the fifth RNA polynucleotide is formulated in a fifth LNP; the sixth RNA polynucleotide is formulated in a sixth LNP; and the seventh RNA polynucleotide is formulated in a seventh LNP. 88.
  • the immunogenic composition of paragraph 76 wherein the first RNA polynucleotide is formulated in a first LNP; the second RNA polynucleotide is formulated in a second LNP; the third RNA polynucleotide is formulated in a third LNP; the fourth RNA polynucleotide is formulated in a fourth LNP; the fifth RNA polynucleotide is formulated in a fifth LNP; the sixth RNA polynucleotide is formulated in a sixth LNP; the seventh RNA polynucleotide is formulated in a seventh LNP; and the eighth RNA polynucleotide is formulated in an eighth LNP. 89.
  • the immunogenic composition of any preceding paragraphs for use in the eliciting an immune response against an antigen.

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Abstract

L'invention concerne des nanoparticules lipidiques, des compositions immunogènes et leurs méthodes d'utilisation.
PCT/IB2024/059673 2023-10-06 2024-10-03 Compositions immunogènes Pending WO2025074292A2 (fr)

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