WO2023212696A1 - Vaccins lyophilisés contre le cytomégalovirus humain - Google Patents

Vaccins lyophilisés contre le cytomégalovirus humain Download PDF

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Publication number
WO2023212696A1
WO2023212696A1 PCT/US2023/066366 US2023066366W WO2023212696A1 WO 2023212696 A1 WO2023212696 A1 WO 2023212696A1 US 2023066366 W US2023066366 W US 2023066366W WO 2023212696 A1 WO2023212696 A1 WO 2023212696A1
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Prior art keywords
mrna
composition
less
lipid
hcmv
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PCT/US2023/066366
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English (en)
Inventor
Johnathan GOLDMAN
Kimberly HASSETT
Sarah Sullivan
Xiaohan PENG
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Modernatx, Inc.
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Publication of WO2023212696A1 publication Critical patent/WO2023212696A1/fr

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Classifications

    • 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
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • LNPs lipid nanoparticles
  • desired proteins such as viral or bacterial antigens
  • LNPs lipid nanoparticles
  • environmental conditions such as the presence of water or environmental nucleases. This instability raises challenges for extended storage of LNP-mRNA compositions.
  • LNPs lipid nanoparticles
  • hCMV human cytomegalovirus
  • Lyophilization or freeze-drying, is a method of preserving a composition by freezing the composition, incubating the frozen composition at a low temperature and pressure to remove water by sublimation (primary drying phase), and finally incubating the composition at a higher temperature to remove residual water that is bound to the composition (secondary drying phase).
  • Remaining water in a lyophilized composition can facilitate the degradation of nucleic acids via hydrolysis, and thus minimizing the moisture content of a lyophilized composition is important for producing lyophilized compositions in which nucleic acids are stable during long-term storage.
  • the final moisture content of a lyophilized composition can be reduced by heating the composition to a higher temperature during the secondary drying phase, but doing so reduces the integrity of the composition.
  • the introduction of an annealing step during the freezing phase in which lipid nanoparticles were held at a temperature near the freezing point of water prior to deep freezing, allowed for the use of a higher temperature during the secondary drying phase without compromising nucleic acid integrity.
  • sucrose as a lyoprotectant unexpectedly resulted in a reduction in LNP size, increasing the efficiency of the LNP-mRNA production process.
  • LNPs e.g., LNPs containing mRNAs encoding human cytomegalovirus (hCMV) antigens
  • hCMV human cytomegalovirus
  • the mRNA in the resulting lyophilized compositions thus decays more slowly, allowing these compositions to remain stable during extended storage, while still retaining the ability to be translated into an encoded protein following reconstitution of the composition and introduction of the mRNA into cells.
  • some aspects of the disclosure relate to a method of preparing a lyophilized composition, the method comprising lyophilizing a lipid nanoparticle composition that comprises a lipid nanoparticle and an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
  • hCMV human cytomegalovirus
  • the mRNA comprises a nucleotide sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the mRNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the one or more hCMV antigens comprises greater than or equal to 1 and less than or equal to 6 hCMV antigens.
  • the composition comprises (a) a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding a hCMV gH polypeptide; (b) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gL polypeptide; (c) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL128 polypeptide; (d) a mRNA polynucleotide comprising an open reading frame encoding a hCMV ULI 30 polypeptide; (e) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL131A polypeptide; and (f) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gB polypeptide.
  • mRNA messenger ribonucleic acid
  • the molar ratio of (a):(f) within the composition is about 1:1; the molar ratio of (b):(c):(d):(e) within the composition is about 1:1: 1:1; and the molar ratio of each of (a) and (f) to any one of (b), (c), (d), or (e) within the composition is about 1.5:1 to about 2:1.
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
  • the mRNA of (a) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 5 or 11;
  • the mRNA of (b) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 6 or 12;
  • the mRNA of (c) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 2 or 8;
  • the mRNA of (d) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 3 or 9;
  • the mRNA of (e) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 4 or 10;
  • the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
  • the lipid nanoparticle composition comprises a lyoprotectant.
  • the lyoprotectant comprises a sugar
  • the lyoprotectant comprises sucrose.
  • the lipid nanoparticle comprises a lipid
  • the lipid nanoparticle composition comprises a lyoprotectant mass:lipid mass ratio of at least 5:1.
  • the lyoprotectant mass:lipid mass ratio is about 6:1 to about 40:1, optionally wherein the lyoprotectant mass:lipid mass ratio is about 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 20:1, 30:1, or 40:1.
  • the composition comprises a buffer.
  • the buffer is selected from the group consisting of a Tris buffer, citrate buffer, and phosphate buffer.
  • the buffer is a Tris buffer.
  • the concentration of the buffer in the composition is about 1 mM to about 100 mM.
  • the concentration of the buffer is about 10 mM to about 20 mM.
  • the composition has a pH of about 6.5 to about 8.5.
  • the composition has a pH of about 7 to about 8.
  • the composition has a pH of about 7.4 to about 8.
  • the lyophilizing comprises an annealing step.
  • the annealing step comprises exposing the lipid nanoparticle composition to a first temperature above the freezing temperature of the composition and a second temperature below the freezing temperature of the composition.
  • the first temperature is from about -30 °C to about 0 °C.
  • the first temperature is about -30 °C, about -25 °C, about -20 °C, about -15 °C, about -10 °C, about -5 °C, or about 0 °C.
  • the second temperature is from about -100 °C to about -30 °C.
  • the second temperature is about -100 °C, about -90 °C, about -80 °C, about -70 °C, about -60 °C, about -50 °C, about -40 °C, or about -30 °C.
  • the method comprises exposing the lipid nanoparticle composition to a third temperature before exposing the lipid nanoparticle composition to the first temperature, wherein the third temperature is from about -100 °C to about -30 °C.
  • the third temperature is about -100 °C, about -90 °C, about -80 °C, about -70 °C, about -60 °C, about -50 °C, about -40 °C, or about -30 °C.
  • the annealing step is conducted for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, or at least 15 hours.
  • the lipid nanoparticle composition is exposed to the first temperature for a first period of from about 2 to about 6 hours, the second temperature for a second period of from about 2 to about 6 hours, and/or the third temperature for a third period of from about 2 to about 6 hours.
  • the lyophilizing comprises a sublimation step.
  • the sublimation is performed at a vacuum pressure of from about 50 mTorr and about 300 mTorr.
  • the lyophilizing comprises a desorption step, wherein the desorption step comprises exposing the composition to a desorption temperature.
  • the desorption temperature is about 10 °C or higher.
  • the desorption temperature is about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, or about 60 °C.
  • the desorption step comprises exposing a sublimated composition to a desorption temperature of about 40 °C.
  • the desorption is conducted for at least 2 hours.
  • the desorption is conducted for about 5 hours, about 10 hours, about 15 hours, or about 20 hours.
  • the desorption is conducted until a Pirani gauge measuring a relative vacuum at the desorption temperature produces a reading that is about the same as the reading produced by a capacitance manometer measuring absolute pressure at the desorption temperature.
  • the desorption is performed at a vacuum pressure of from about 50 mTorr and about 300 mTorr.
  • the lyophilized composition has a moisture content of 6.0% w/w or less 5.0% w/w or less, 4.0% w/w or less, 3.5% w/w or less, 3.0% w/w or less, 2.5% w/w or less, 2.0% w/w or less, 1% or less, 0.5% or less, 0.3% or less, or 0.25% or less.
  • a coefficient of degradation at 5 °C of the nucleic acid in the lyophilized composition is 0.05 month 1 or less, 0.04 month 1 or less, 0.03 month 1 or less, or 0.02 month 1 or less.
  • the coefficient of degradation is 0.01 month 1 or less, 0.008 month 1 or less, 0.006 month 1 or less, or 0.004 month 1 or less.
  • the mRNA in the lyophilized composition is at least 50% pure after least 12 months, at least 18 months, at least 21 months, at least 24 months, at least 27 months, at least 30 months, at least 33 months, or at least 36 months of storage.
  • the storage is conducted at a temperature between about 2 °C and about 8 °C.
  • the storage is conducted at about 5 °C.
  • the mRNA in the lyophilized composition is at least 50% pure after at least 48 months or at least 60 months of storage at about 5 °C.
  • the lipid nanoparticle comprises: an ionizable amino lipid.
  • the lipid nanoparticle further comprises: a non-cationic lipid; a sterol; and a polyethylene glycol (PEG)-modified lipid.
  • the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% non-cationic lipid; 35-45 mol% sterol; and 1-5 mol% PEG-modified lipid.
  • Some aspects of the disclosure relate to a lyophilized composition produced by a method of any one of the methods described herein.
  • a lyophilized pharmaceutical composition comprising a human cytomegalovirus (hCMV) immunogenic composition comprising a lipid nanoparticle and: (a) a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding a hCMV gH polypeptide; (b) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gL polypeptide; (c) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL128 polypeptide; (d) a mRNA polynucleotide comprising an open reading frame encoding a hCMV ULI 30 polypeptide; (e) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL131A polypeptide; and (f) a mRNA polynucleo
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
  • the mRNA of (a) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 5 or 11;
  • the mRNA of (b) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 6 or 12;
  • the mRNA of (c) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 2 or 8;
  • the mRNA of (d) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 3 or 9;
  • the mRNA of (e) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 4 or 10;
  • the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
  • Some aspects of the disclosure relate to a lyophilized pharmaceutical composition
  • a lyophilized pharmaceutical composition comprising a lipid nanoparticle encapsulating an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
  • hCMV human cytomegalovirus
  • the mRNA comprises a nucleotide sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the mRNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the one or more hCMV antigens comprises greater than or equal to 1 and less than or equal to 6 hCMV antigens.
  • the pharmaceutical composition comprises (a) a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding a hCMV gH polypeptide; (b) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gL polypeptide; (c) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL128 polypeptide; (d) a mRNA polynucleotide comprising an open reading frame encoding a hCMV ULI 30 polypeptide; (e) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL131A polypeptide; and (f) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gB polypeptide.
  • mRNA messenger ribonucleic acid
  • the molar ratio of (a):(f) within the composition is about 1:1; the molar ratio of (b):(c):(d):(e) within the composition is about 1:1: 1:1; and the molar ratio of each of (a) and (f) to any one of (b), (c), (d), or (e) within the composition is about 1.5:1 to about 2:1.
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
  • the mRNA of (a) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 5 or 11;
  • the mRNA of (b) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 6 or 12;
  • the mRNA of (c) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 2 or 8;
  • the mRNA of (d) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 3 or 9;
  • the mRNA of (e) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 4 or 10;
  • the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
  • Some aspects of the disclosure relate to a lyophilized pharmaceutical composition
  • a lyophilized pharmaceutical composition comprising a lipid nanoparticle and an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
  • hCMV human cytomegalovirus
  • the lyophilized pharmaceutical composition comprises a lyoprotectant.
  • the lyoprotectant comprises a sugar
  • the lyoprotectant comprises sucrose.
  • the lipid nanoparticle comprises a lipid
  • the lyophilized pharmaceutical composition comprises a lyoprotectant mass:lipid mass ratio of at least 5:1.
  • the lyoprotectant mass:lipid mass ratio is about 6:1 to about 40:1, optionally wherein the lyoprotectant mass:lipid mass ratio is about 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 20:1, 30:1, or 40:1.
  • the lyophilized composition has a moisture content of 6.0% w/w or less, 5.0% w/w or less, 4.0% w/w or less, 3.5% w/w or less, 3.0% w/w or less, 2.5% w/w or less, or 2.0% w/w or less, 1% or less, 0.5% or less, 0.3% or less, or 0.25% or less.
  • a coefficient of degradation at 5 °C of the nucleic acid in the lyophilized composition is 0.05 month 1 or less, 0.04 month 1 or less, 0.03 month 1 or less, or 0.02 month 1 or less.
  • the coefficient of degradation is 0.01 month 1 or less, 0.008 month 1 or less, 0.006 month 1 or less, or 0.004 month 1 or less.
  • the mRNA in the lyophilized composition is at least 50% pure after least 12 months, at least 18 months, at least 21 months, at least 24 months, at least 27 months, at least 30 months, at least 33 months, or at least 36 months of storage.
  • the storage is conducted at a temperature between about 2 °C and about 8 °C.
  • the storage is conducted at about 5 °C.
  • the mRNA in the lyophilized composition is at least 50% pure after at least 48 months or at least 60 months of storage at about 5 °C.
  • the lipid nanoparticle comprises: an ionizable amino lipid.
  • the lipid nanoparticle further comprises: a non-cationic lipid; a sterol; and a polyethylene glycol (PEG)-modified lipid.
  • the lipid nanoparticle comprises: 40-55 mol% ionizable amino lipid; 5-15 mol% non-cationic lipid; 35-45 mol% sterol; and 1-5 mol% PEG-modified lipid.
  • Some aspects of the disclosure relate to a method comprising reconstituting any one of the lyophilized pharmaceutical compositions described herein.
  • FIG. 1 shows the delta diameter of lipid nanoparticles comprising mRNA, which varies based on the concentration of mRNA to be encapsulated, the sucrosedipid ratio, and the liquid in which the nanoparticles are reconstituted.
  • FIGs. 2A-2C show the parameters of two example lyophilization processes, one with an annealing step and one without, and the effects of each process on the characteristics of lipid nanoparticles.
  • FIG. 2A shows the temperature and pressures used in a lyophilization process that includes an annealing step, in which the composition to be lyophilized is held at -10 °C for 5 hours during freezing.
  • FIG. 2B shows the temperatures and pressures used in a lyophilization process that does not include an annealing step, in which the composition to be lyophilized is directly cooled to -50 °C.
  • FIG. 1A shows the temperature and pressures used in a lyophilization process that includes an annealing step, in which the composition to be lyophilized is held at -10 °C for 5 hours during freezing.
  • FIG. 2B shows the temperatures and pressures used in a lyophilization process that does not include an annealing step, in which the composition to be lyophilized is directly
  • 2C shows the delta diameter (left y-axis, data in columns) and percentage encapsulation efficiency (right y-axis, data in lines) of lipid nanoparticles in compositions lyophilized by the processes shown in FIGs. 2A-2B, across a range of sucrose concentrations.
  • FIG 3 shows the effect of moisture content on stability of lyophilized compositions comprising nucleic acids.
  • the 1 st order rate constant of mRNA degradation (month 1 ) for mRNA in lyophilized compositions is shown for a range of moisture contents (% w/w).
  • FIGs. 4A-4B show the effects of secondary drying temperatures on lyophilized compositions comprising nucleic acids.
  • FIG. 4A shows an overview of a lyophilization process comprising a freezing phase with an annealing step, a primary drying phase, and a secondary drying phase.
  • FIG. 4B shows data relating to the moisture content of lyophilized compositions subjected to secondary drying phases with temperatures of 25 °C (squares) or 40 °C (circles) for up to 24 hours.
  • FIGs. 4C-4F show characteristics of lyophilized compositions prepared by secondary drying phases with temperatures of 25 °C (left bar of each group) or 40 °C (right bar of each group).
  • FIG. 4C shows lipid nanoparticle size, as measured by unfiltered dynamic light scattering.
  • FIG. 4D shows percent encapsulation efficiency, as measured by RiboGreen.
  • FIG. 4E shows reduction in mRNA purity.
  • FIG. 4F shows relative in vitro potency.
  • FIGs. 5A-5C show the effect of moisture content on the stability of mRNA in lyophilized compositions.
  • FIG. 5A shows the stability over time of mRNA (encoding hCMV glycoprotein B (gB)) in lyophilized compositions with moisture contents of 0.3% - 5.7% (% w/w).
  • FIG. 5B shows the stability of mRNA encoding hCMV gB in lyophilized compositions from multiple lots, containing moisture contents between 0.20-0.65 (% w/w), during 12 to 24 months of storage at 5 °C.
  • FIG. C shows the stability of mRNA encoding hCMV glycoprotein H (gH) in lyophilized compositions from the same lots as FIG. 5B.
  • FIG. 6 shows the efficiency of expression of hCMV pentamer encoded by mRNAs in lyophilized and reconstituted compositions (1 st , left group of bars), compositions stored at 4 °C (2 nd group), compositions frozen at -20 °C and thawed (3 rd group), and compositions frozen at - 80 °C and thawed (4 th group).
  • compositions were diluted to deliver varying amounts of mRNA, shown on the x-axis.
  • the height of each bar shows the geometric mean fluorescence intensity of cells stained with hCMV pentamer- specific antibodies and analyzed by flow cytometry.
  • NT no treatment (no RNA delivered to cells).
  • FIG. 7 shows hCMV pentamer- specific IgG titers in sera of mice that were administered one or two doses of lyophilized or frozen/thawed compositions containing antigen-encoding mRNA. Doses were administered on day 0 (prime) and day 22 (boost), with sera collected on day 21 (3 weeks post-prime dose, before boost) and 36 (2 weeks post-boost dose), and antigenspecific IgG titers were measured by ELISA.
  • nucleic acids such as mRNA
  • the nucleic acid is combined with a lipid, e.g., an ionizable lipid (e.g., in a lipid nanoparticle (LNP)).
  • a lipid e.g., an ionizable lipid (e.g., in a lipid nanoparticle (LNP)).
  • LNP lipid nanoparticle
  • One issue that arises in lyophilized compositions is the presence of water, which can facilitate the degradation of nucleic acids via hydrolysis.
  • Some embodiments comprise minimizing the moisture content of a lyophilized composition, thus producing lyophilized compositions in which nucleic acids are stable during long-term storage.
  • the pharmaceutical compositions may be characterized as being stable relative to an equivalent composition prepared by a lyophilization step that does not involve an annealing step, a high level of lyoprotectant, and/or high drying temperatures.
  • the stability of the lyophilized product may be determined, for instance, with reference to the particle size of the lipid nanoparticles comprising such composition.
  • lyophilization of the lipid nanoparticles produces a smaller particle size of the lipid nanoparticles following lyophilization and/or reconstitution.
  • Some embodiments comprise lyophilizing a composition in a system that comprises a refrigeration system, a vacuum system, and a condenser system.
  • the lyophilization is in a freeze dryer.
  • the lyophilization comprises one or more thermal treatment steps.
  • the thermal treatment comprises one or more freezing steps (e.g., a first freezing step, a second freezing step, etc.).
  • the thermal treatment step(s) are performed at atmospheric pressure.
  • the thermal treatment comprises an annealing step, in which the temperature of the composition is cycled between two or more temperatures (e.g., a first temperature and a second temperature).
  • the annealing step comprises cooling the composition to a temperature that is higher than the freezing temperature of the composition prior to being solidified by freezing at a lower temperature. That is, in some embodiments the annealing comprises exposing the composition to a first temperature above the freezing temperature and then exposing the composition to a second temperature below the freezing temperature of the composition.
  • the annealing step comprises exposing the composition to a first temperature below the freezing temperature of the composition, a second temperature (e.g., an annealing temperature) above the freezing temperature of the composition, and a third temperature below the freezing temperature of the composition. That is, in some embodiments, the first and third temperatures (e.g., freezing temperatures) are below the freezing temperature of the composition and the second temperature (e.g., annealing temperature) is above the freezing temperature of the composition.
  • a first temperature below the freezing temperature of the composition
  • a second temperature e.g., annealing temperature
  • the annealing temperature that is above the freezing temperature of the composition is a temperature of from about -30 °C to about 0 °C, such as about -30 °C, about -25 °C, about -20 °C, about -15 °C, about -10 °C, about -5 °C, or about 0 °C.
  • the temperature that is below the freezing temperature of the composition is a temperature of from about -100 °C to about -30 °C, such as about -100 °C, about -90 °C, about -80 °C, about -70 °C, about -60 °C, about -50 °C, about -40 °C, or about -30 Of
  • the annealing step is conducted for at least about 30 minutes, such as about 1 hour, about 2 hours, such as about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, or about 20 hours or more.
  • the annealing comprises exposing the composition to two or more temperatures (e.g., a first freezing temperature, a second annealing temperature, a third freezing temperature, etc.), wherein the composition is exposed to each of the various temperatures for a period of about 30 minutes, about 1 hour, about 2 hours, about 3 hours about 4 hours, about 5 hours, about 6 hours, about 8 hours, or about 10 hours or more.
  • the composition is exposed to each of the temperatures for the same time periods (/'. ⁇ ?., the first, second, etc. time periods are the same).
  • the composition is exposed to each of the temperatures for different time periods (e.g., one or more of the first, second, third, etc. temperatures are for a period of time that is different from the others).
  • an annealing step Compared to cooling the composition directly to the final freezing temperature, an annealing step, without being bound by theory, is believed to allow water molecules to form a more ordered arrangement prior to freezing, resulting in the formation of larger ice crystals in the frozen composition. These larger ice crystals may be less likely to damage the nucleic acids of the composition during sublimation or secondary drying.
  • an annealing step enables the use of harsher conditions during primary and/or secondary drying that may otherwise compromise nucleic acid integrity.
  • an annealing step allows for crystallization of bulking agents and/or lyoprotectants (e.g., sucrose), which provide a framework to support the integrity of lyophilized compositions and prevent collapse of a lyophilized composition during primary or secondary drying.
  • the lyophilization comprises a sublimation step (e.g., conducted at temperatures below the compositions critical collapse temperature and performed under a vacuum).
  • the sublimation occurs in a first drying step.
  • the sublimation comprises using one or more of conduction, convection, and radiation to provide heat energy sufficient to drive a phase change from solid to gas. Without being bound by theory, it is believed that the sublimation step removes free ice crystals and/or organic solvents in the composition.
  • the sublimation comprises exposing a thermally-treated composition to a temperature that is below the melting point of water, but higher than the temperature used to freeze the composition, under vacuum.
  • the primary drying phase is conducted at a temperature of about -40 °C to about 0 °C, such as about -40 °C, about -35 °C, about -32 °C, about -30 °C, about -25 °C, about -20 °C, about -15 °C, about -10 °C, about -5 °C, or about 0 °C.
  • the primary drying phase is conducted at a temperature of from about -35 °C to about -25 °C.
  • the primary drying phase is conducted at a temperature of from about -32 to about -25 °C. At low pressure and temperature, ice can sublimate from the composition, such that majority of water is removed. In some embodiments, the primary drying phase is carried for at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 hours.
  • the sublimation is conducted at a pressure that is 20% to 30% of the vapor pressure of ice at the sublimation temperature. In some embodiments, the sublimation is performed at a vacuum pressure of between about 50 mTorr and about 300 mTorr, such as about 75 mTorr, about 100 mTorr, about 125 mTorr, about 150 mTorr, about 175 mTorr, or about 200 mTorr.
  • Some embodiments comprise determining that primary drying is complete when a Pirani gauge, which measures the relative vacuum of an environment, outputs a reading at the sublimation temperature that is similar to the reading produced by a capacitance manometer, which measures the absolute pressure of an environment, at the sublimation temperature.
  • a Pirani gauge measures the relative vacuum of an environment using a sensor wire, which is heated by electric current, and measuring the current required to maintain a constant temperature. When more gas is present, a higher a current is required to maintain the temperature.
  • a capacitance manometer measures absolute pressure using a metal diaphragm under tension, with one side of the diaphragm exposed to the gas whose pressure is to be measured, and the other side exposed to a sealed vacuum, which serves as a reference.
  • Pirani gauges are particularly sensitive to the presence of water vapor, and thus produce higher readings than a capacitance manometer when water vapor is still present in the environment, indicating primary drying is not complete.
  • a Pirani gauge yields a similar measurement to that of a capacitance manometer, which is not sensitive to the presence of water vapor, this convergence indicates a lack of water vapor. This absence of water vapor at the current temperature and pressure indicates that no more ice is being sublimated and primary drying is complete.
  • the lyophilization comprises a desorption step.
  • the desorption occurs in a secondary drying step.
  • a composition can be warmed to a higher temperature (e.g., as compared to a primary drying step) to facilitate removal of remaining water molecules that are bound to the composition.
  • the desorption is conducted at a desorption temperature of at least about 20 °C, such as about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, or more.
  • the secondary drying phase is conducted at a desorption temperature of about 40 °C. In some embodiments, the secondary drying phase is conducted for at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, or at least 20 hours.
  • the desorption is conducted at a pressure that is 20% to 30% of the vapor pressure of water at the desorption temperature. In some embodiments, the desorption is performed at a vacuum pressure of between about 50 mTorr and about 300 mTorr, such as about 75 mTorr, about 100 mTorr, about 125 mTorr, about 150 mTorr, about 175 mTorr, or about 200 mTorr.
  • the secondary drying phase is conducted until a Pirani gauge measuring a relative vacuum at the desorption temperature produces a reading that is about the same as the reading produced by a capacitance manometer measuring absolute pressure at the desorption temperature.
  • Warming the composition to a higher temperature than the temperature used in primary drying promotes the evaporation of residual water from the composition, causing the reading of a Pirani gauge to again become elevated relative to the reading of a capacitance manometer.
  • Convergence of the Pirani gauge reading to that of a capacitance manometer indicates an absence of water vapor, as no more water is being evaporated at the current desorption temperature.
  • the secondary drying phase is conducted until the moisture content of the composition is less than about 6% w/w, less than about 5.5% w/w, less than about 5% w/w, less than about 4.5% w/w, less than about 4% w/w, less than about 3.5% w/w, less than about 3% w/w, less than about 2.5% w/w, less than about 2% w/w, less than about 1% w/w, or less than about 0.5% w/w.
  • compositions e.g., lipid nanoparticle compositions
  • methods of preparing lyophilized compositions wherein the composition comprises a lyoprotectant.
  • the compositions comprise a lipid nanoparticle comprising a nucleic acid, such as mRNA.
  • a lyoprotectant is added to the nucleic acid (e.g., mRNA) before the nucleic acid is combined with the lipid or lipid nanoparticles and prior to lyophilization, such that the final composition includes the lyoprotectant.
  • the lyoprotectant is added to the lipid or lipid nanoparticle before the lipid nanoparticle is combined with the nucleic acid.
  • the lyoprotectant, lipid, and nucleic acid are combined prior to formation of the lipid nanoparticle.
  • the lyoprotectant is added to a composition comprising a lipid nanoparticle and a nucleic acid.
  • a lyoprotectant refers to a composition that mitigates the effects of lyophilization on product integrity.
  • the processes of thermal treatment, sublimation, and desorption expose a composition to harsh conditions, which may reduce the integrity of one or more components of the lyophilized composition.
  • Inclusion of a lyoprotectant in a composition to be lyophilized may prevent or reduce some of this reduction in integrity through multiple mechanisms of action.
  • a lyoprotectant may alter the structure of ice crystals in a frozen composition and/or act as a buffer between a composition to be preserved and ice crystals formed during freezing, such that sublimating ice crystals are less likely to damage the composition to be preserved. Additionally, a lyoprotectant may slow the rate of temperature increase in a composition during desorption, reducing the likelihood of undesired chemical reactions that could compromise product integrity.
  • the lyoprotectant is a sugar.
  • a sugar is a carbohydrate molecule comprising one or more monosaccharide monomers.
  • sugars include sucrose, trehalose, maltose, lactose, glucose, fructose, and galactose.
  • the sugar is sucrose.
  • Sucrose refers to a compound of the formula C12H22O11 that is a dimer of a glucose monomer and a fructose monomer.
  • Trehalose refers to a compound of the formula C12H22O11 that is a dimer of two glucose monomers joined by a 1,1- glycosidic linkage.
  • Maltose refers to a compound of the formula C12H22O11 that is a dimer of two glucose monomers joined by a 1,4-glycosidic linkage.
  • Lactose refers to a compound of the formula C12H22O11 that is a dimer of a glucose monomer and a galactose monomer.
  • the lyoprotectant is a polyol. In some embodiments, the lyoprotectant is mannitol. In some embodiments, the lyoprotectant is malitol. In some embodiments, the lyoprotectant is glycine. In some embodiments, the lyoprotectant is cyclodextrin. In some embodiments, the lyoprotectant is maltodextrin.
  • the ratio of sugar mass (e.g., sucrose) to lipid mass (e.g., total mass of ionizable lipids, non-cationic lipids, sterols, and PEG-modified lipids) in the composition is about 5:1 to about 40:1 (5 g sugar: 1 g lipids to 40 g sugar: 1 g lipids). In some embodiments, the ratio of sugar mass to lipid mass is about 8:1 to about 20:1. In some embodiments, the ratio of sugar mass to lipid mass is about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, or about 12:1. In some embodiments, the ratio of sugar mass to lipid mass is at least 8:1. In some embodiments, the ratio of sugar mass to lipid mass is at least 10:1.
  • sucrose sucrose
  • lipid mass e.g., total mass of ionizable lipids, non-cationic lipids, sterols, and PEG-
  • the ratio of lyoprotectant mass to lipid mass in the composition is about 5:1 to about 40:1 (5 g lyoprotectant: 1 g lipids to 40 g lyoprotectant: 1 g lipids). In some embodiments, the ratio of lyoprotectant mass to lipid mass is about 8:1 to about 20:1. In some embodiments, the ratio of lyoprotectant mass to lipid mass is about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, or about 12:1. In some embodiments, the ratio of lyoprotectant mass to lipid mass is at least 8:1. In some embodiments, the ratio of lyoprotectant mass to lipid mass is at least 10:1.
  • the ratio of the sugar mass to a lipid nanoparticle mass is from about 5:1 to about 40:1. In some embodiments, the ratio of sugar mass to lipid nanoparticle mass is about 8:1 to about 20:1. In some embodiments, the ratio of sugar mass to lipid nanoparticle mass is about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, or about 12:1. In some embodiments, the ratio of sugar mass to lipid nanoparticle mass is at least 8:1. In some embodiments, the ratio of sugar mass to lipid nanoparticle mass is at least 10: 1.
  • the composition contains at least about 5% w/w sucrose, such as about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10% w/w, about 11% w/w, about 12% w/w, about 15% w/w, or more sucrose.
  • the sucrose is added to a nucleic acid (e.g., mRNA) before the nucleic acid is combined with a lipid or lipid nanoparticles and prior to lyophilization, such that the final composition includes the sucrose.
  • the sucrose is added to the lipid or lipid nanoparticle before the lipid nanoparticle is combined with the nucleic acid.
  • the sucrose, lipid, and nucleic acid are combined prior to formation of the lipid nanoparticle.
  • the sucrose is added to a composition comprising a lipid nanoparticle and a nucleic acid.
  • compositions e.g., lipid nanoparticle compositions
  • methods of preparing lyophilized compositions wherein the composition comprises a buffer and/or other components.
  • the buffer and/or other components are present in pre-lyophilized compositions.
  • the buffer and/or other components are present in reconstituted lyophilized compositions (e.g., they can be used to reconstitute a lyophilized composition).
  • the compositions comprise a lipid nanoparticle comprising a nucleic acid, such as mRNA, and a buffer.
  • a buffer is added to the nucleic acid (e.g., mRNA) before the nucleic acid is combined with the lipid or lipid nanoparticles and prior to lyophilization, such that the final composition includes the buffer.
  • the buffer is added to the lipid or lipid nanoparticle before the lipid nanoparticle is combined with the nucleic acid.
  • the buffer, lipid, and nucleic acid are combined prior to formation of the lipid nanoparticle.
  • the buffer is added to a composition comprising a lipid nanoparticle and a nucleic acid.
  • a buffer refers to a composition that mitigates the effects of acid or bases on the pH of a composition containing the buffer.
  • the processes of thermal treatment, sublimation, and desorption alter the temperature and amount of water in a composition, both of which may change the pH of the composition and facilitate undesired chemical reactions that compromise the integrity of the composition.
  • Inclusion of a buffer in a composition to be lyophilized may prevent or reduce some of this reduction in integrity through multiple mechanisms of action.
  • a buffer may absorb free hydrogen or hydroxide ions that are released from other components of the composition before, during, or after lyophilization.
  • a buffer prevents the ions from reacting with other components of the composition, such as nucleic acids or lipids, which may otherwise facilitate nucleic acid cleavage, modification of nucleic acid structure, or disruption of lipid nanoparticle integrity. Additionally, a buffer may slow the rate of pH change in a composition after lyophilization, reducing the likelihood of undesired pH change that could compromise product integrity.
  • the LNP, nucleic acid, or LNP-encapsulated nucleic acid is exposed to a lyophilization buffer via TFF diafiltration. In some embodiments, the LNP, nucleic acid, or LNP-encapsulated nucleic acid is exposed to a lyophilization buffer via dialysis, the LNP, nucleic acid, or LNP-encapsulated nucleic acid is exposed to a lyophilization buffer via a combination of TFF diafiltration and dialysis.
  • the buffer is selected from the group consisting of a Tris buffer, citrate buffer, phosphate buffer, triethylammonium bicarbonate (TEAB), and histidine buffer.
  • the buffer is a Tris buffer.
  • the buffer is a citrate buffer.
  • the buffer is a phosphate buffer.
  • the buffer is a TEAB buffer.
  • the buffer is a histidine buffer.
  • the concentration of the buffer in the composition prior to lyophilization is about 1 mM to about 100 mM. In some embodiments, the concentration of the buffer in the composition prior to lyophilization is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.
  • the concentration of the buffer in the composition prior to lyophilization is about 10 mM. In some embodiments, the concentration of the buffer in the composition prior to lyophilization is about 20 mM. In some embodiments, the concentration of the buffer in the composition prior to lyophilization is about 25 mM. In some embodiments, the concentration of the buffer in the composition prior to lyophilization is about 30 mM. In some embodiments, the buffer concentration is about 100 mM or less, such as about 75 mM or less, about 50 mM or less, about 25 mM or less, or about 10 mM or less. In some embodiments, the buffer concentration is from about 5 mM to about 100 mM, such as about 10 mM to about 50 mM, about 5 mM to about 20 mM. or about 20 mM to about 30 mM.
  • the concentration of the buffer in the composition after lyophilization and reconstitution is about 1 mM to about 100 mM. In some embodiments, the concentration of the buffer in the composition after lyophilization and reconstitution is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.
  • the concentration of the buffer in the composition after lyophilization and reconstitution is about 10 mM. In some embodiments, the concentration of the buffer in the composition after lyophilization and reconstitution is about 20 mM. In some embodiments, the concentration of the buffer in the composition prior to lyophilization is about 25 mM. In some embodiments, the concentration of the buffer in the composition after lyophilization and reconstitution is about 30 mM. In some embodiments, the buffer concentration is about 100 mM or less, such as about 75 mM or less, about 50 mM or less, about 25 mM or less, or about 10 mM or less.
  • the buffer concentration is from about 5 mM to about 100 mM, such as about 10 mM to about 50 mM, about 5 mM to about 20 mM. or about 20 mM to about 30 mM.
  • the pH of the composition prior to lyophilization is about 6 to about 9. In some embodiments, the pH is about 6 to about 7. In some embodiments, the pH is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 6.0. In some embodiments, the pH is about 7 to about 8.
  • the pH is about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0.
  • the pH is about 8 to about 9.
  • the pH is about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.
  • the pH is about 7.0 to about 7.2, about 7.2 to about 7.4, about 7.4 to about 7.6, about 7.6 to about 7.8, or about 7.8 to about 8.0.
  • the pH of the composition after lyophilization and reconstitution is about 6 to about 9.
  • the pH is about 6 to about 7.
  • the pH is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 6.0.
  • the pH is about 7 to about 8.
  • the pH is about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0.
  • the pH is about 8 to about 9.
  • the pH is about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In some embodiments, the pH is about 7.0 to about 7.2, about 7.2 to about 7.4, about 7.4 to about 7.6, about 7.6 to about 7.8, or about 7.8 to about 8.0.
  • the pH is below the pKa of an amino lipid in an ionizable lipid in a LNP, such as about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, or more pH units below the pKa of an amino lipid in the ionizable lipid.
  • the composition (e.g., prior to lyophilization) comprises a salt, such as sodium chloride.
  • the salt concentration in the composition is from about 0.1 mM to about 300 mM.
  • the salt concentration in a pre-lyophilized composition is about 50 mM or less, such as from about 0 mM to about 50 mM, or about 0.1 mM to about 50 mM.
  • the salt concentration is preferably from about 25 mM to about 200 mM, such as about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM.
  • the composition (e.g., prior to lyophilization) comprises a surfactant.
  • exemplary surfactants include Tween20, Tween80, BrijS200, and PEG-DMG.
  • the composition comprises from about 0.0001wt% to about 0.5wt% surfactant, such as about 0.001wt% to about 0.4wt%, about 0.002wt% to about 0.3wt%, about 0.003 wt% to about 0.2wt%, or about 0.005 wt% to about 0.1 wt% surfactant.
  • the composition comprises about 0.005wt%, about 0.05wt%, about 0.01wt%, about 0.1 wt%, or about 0.5wt% surfactant.
  • the composition (e.g., prior to lyophilization) comprises a metal chelator.
  • metal chelators include EDTA (ethylenediaminetetraacetic acid) and DTPA (diethylenetriaminepentaacetic acid).
  • the composition comprises from 0.1 mM to about 5 mM or more of the metal chelator, such as about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM of the metal chelator.
  • the nucleic acids are present in a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and/or a lipoplex.
  • nucleic acids are present in lipid nanoparticle (LNP) compositions.
  • LNP lipid nanoparticle
  • Lipid nanoparticles typically comprise amino lipid, non-cationic lipid, structural lipid, and PEG lipid components along with the nucleic acid cargo of interest.
  • the lipid nanoparticles can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551;
  • PCT/US2015/027400 PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575; PCT/US2016/069491; PCT/US2016/069493; and PCT/US2014/66242, all of which are incorporated by reference herein in their entirety.
  • the lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)- modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% structural lipid, and 0.5-15% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-30% non-cationic lipid, 10-55% structural lipid, and 0.5-15% PEG-modified lipid.
  • the lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.
  • the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid.
  • the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid.
  • the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid.
  • the lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipid.
  • the lipid nanoparticle comprises 45 - 55 mole percent (mol%) ionizable amino lipid.
  • lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.
  • the ionizable amino lipid is a compound of Formula (Al): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched - wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
  • R 4 is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consisting wherein denotes a point of attachment;
  • R 10 is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2- 3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C1-3 alkyl,
  • each R 6 is independently selected from the group consisting of C1-3 alkyl,
  • M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;
  • R’ is a C1-12 alkyl or C2-12 alkenyl
  • 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.
  • R’ a is R’ branched ;
  • R’branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , R ay , and R a ⁇ are each H;
  • R 2 and R 3 are each Ci-14 alkyl;
  • R 4 is -(CFH2)H;
  • n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each - C(O)O-;
  • R’ is a Ci-12 alkyl; 1 is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , R ay , and R a ⁇ are each H;
  • R 2 and R 3 are each Ci-14 alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -
  • R’ is a Ci-12 alkyl; 1 is 3; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ is C2-12 alkyl;
  • R a ⁇ , R ay , and R a ⁇ are each H;
  • R 2 and R 3 are each C 1-14 alkyl alkyl);
  • n2 is 2;
  • R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R' is a Ci-12 alkyl; 1 is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , and R a ⁇ are each H;
  • R a ⁇ is C2-12 alkyl;
  • R 2 and R 3 are each Ci-14 alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M' are each -C(O)O-;
  • R’ is a Ci-12 alkyl; 1 is 5; and m is 7.
  • the compound of Formula (I) is selected from:
  • the ionizable amino lipid is a compound of Formula (Ala): its N-oxide, or a salt or isomer thereof, wherein R’ a is R ,branched ; wherein
  • R’ branched denotes a point of attachment; wherein R a ⁇ , R ay , and R a ⁇ are each independently selected from the group consisting of H,
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
  • R 4 is selected from the group consisting of -(CFhkOH wherein n is selected from the group consisting wherein ? denotes a point of attachment; wherein
  • R 10 is N(R)2; each R is independently selected from the group consisting of C 1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C1-3 alkyl,
  • M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;
  • R’ is a C1-12 alkyl or C2-12 alkenyl
  • 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.
  • the ionizable amino lipid is a compound of Formula (Alb): its N-oxide, or a salt or isomer thereof, wherein R’ a is R ,branched ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R ay , and R a ⁇ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
  • R 4 is -(CH 2 ) n OH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of C1-3 alkyl,
  • each R 6 is independently selected from the group consisting of C1-3 alkyl,
  • M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;
  • R’ is a C1-12 alkyl or C2-12 alkenyl
  • 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.
  • R’ a is R’ branched ;
  • R’branched is denotes a point of attachment;
  • R a ⁇ , R ay , and R a ⁇ are each H;
  • R 2 and R 3 are each Ci-14 alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each - C(O)O-;
  • R’ is a Ci-12 alkyl; 1 is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ , R ay , and R a ⁇ are each H;
  • R 2 and R 3 are each Ci-14 alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each - C(O)O-;
  • R’ is a Ci-12 alkyl; 1 is 3; and m is 7.
  • R’ a is R’ branched ;
  • R’branched is denotes a point of attachment;
  • R a ⁇ and R a ⁇ are each H;
  • R ay is C2-12 alkyl;
  • R 2 and R 3 are each Ci-14 alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R is a Ci-12 alkyl; 1 is 5; and m is 7.
  • the ionizable amino lipid is a compound of Formula (Ale): its N-oxide, or a salt or isomer thereof, wherein R’ a is R ,branched ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R ay , and R a ⁇ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl; wherein ? denotes a point of attachment; whereinR 10 is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C1-3 alkyl,
  • M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;
  • R’ is a C1-12 alkyl or C2-12 alkenyl
  • 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.
  • point of attachment; R a ⁇ , R ay , and R a ⁇ are each H; R a ⁇ is C2-12 alkyl; R 2 and R 3 are each Ci-14 alkyl; denotes a point of attachment; R 10 is NH(CI-6 alkyl); n2 is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a Ci-12 alkyl; 1 is 5; and m is
  • the compound of Formula (Ale) is:
  • the ionizable amino lipid is a compound of Formula (All): wherein R’ a is R’ branched O r R’ cyclic ; wherein wherein ? denotes a point of attachment;
  • R ay and R a ⁇ are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of R ay and R a ⁇ is selected from the group consisting of Ci-
  • R by and R b5 are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of R by and R b5 is selected from the group consisting of Ci-
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
  • R 4 is selected from the group consisting of -(CFFjnOH wherein n is selected from the group consisting wherein denotes a point of attachment; wherein R 10 is N(R)2; each R is independently selected from the group consisting of Ci-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a Ci-12 alkyl or C2-12 alkenyl;
  • Y a is a C3-6 carbocycle
  • R*” a is selected from the group consisting of Ci-15 alkyl and C2-15 alkenyl; and s is 2 or 3; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable amino lipid is a compound of Formula (All- a): wherein R’ a is R’ branched O r R’ cyclic ; wherein wherein ? denotes a point of attachment;
  • R ay and R a ⁇ are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of R ay and R a ⁇ is selected from the group consisting of Ci-
  • R by and R b5 are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of R by and R b5 is selected from the group consisting of Ci- 12 alkyl and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
  • R 4 is selected from the group consisting of -(CFFjnOH wherein n is selected from the group consisting wherein denotes a point of attachment; wherein R 10 is N(R)2; each R is independently selected from the group consisting of Ci-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a Ci-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable amino lipid is a compound of Formula (All-b): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched O r R’ cyclic ; wherein wherein denotes a point of attachment;
  • R ay and R by are each independently selected from the group consisting of Ci-12 alkyl and
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and
  • R 4 is selected from the group consisting of -(CFFjnOH wherein n is selected from the group consisting wherein denotes a point of attachment; wherein R 10 is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a Ci-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • the ionizable amino lipid is a compound of Formula (AII-c): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched O r R’ cyclic ; wherein wherein denotes a point of attachment; wherein R ay is selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
  • R 4 is selected from the group consisting of - (CH 2 ) n OH wherein n is selected from the group consisting wherein denotes a point of attachment; wherein R 10 is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
  • R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable amino lipid is a compound of Formula (All-d): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched O r R' cyclic ; wherein wherein denotes a point of attachment; wherein R ay and R by are each independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl;
  • R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting wherein denotes a point of attachment; wherein R 10 is N(R) 2 ; each R is independently selected from the group consisting of C1-6 alkyl, C 2 -3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a Ci-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable amino lipid is a compound of Formula (All-e): its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched O r R’ cyclic ; wherein wherein ? denotes a point of attachment; wherein R ay is selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of Ci-14 alkyl and
  • R 4 is -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;
  • R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • m and 1 are each independently selected from 4, 5, and 6. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), m and 1 are each 5. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), each R’ independently is a Ci-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), each R’ independently is a C2-5 alkyl.
  • R’ b is: R 3 ⁇ R 2 an d R 2 and R 3 are each independently a Ci-14 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e),
  • R’ b is: and R 2 and R 3 are each independently a C 6-10 alkyl.
  • R’ b is: R 3 ⁇ X R 2 and R 2 and R 3 are each a Cs alkyl.
  • (All), (All-a), (All-b), (AII-c), (AII- and R 3 are each independently a C 6-10 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- and R 3 are each independently a C 6-10 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- and R 3 are each independently a C 6-10 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- and R 3 are each independently a C 6-10 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- and R 3 are each independently a C 6-10 alkyl.
  • R 3 ⁇ R 2 , R ay is a C2-6 alkyl and R 2 and R 3 are each independently a C 6-10 alkyl.
  • R 2 and R 3 are each a
  • R’ branched is; a Ci-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All- d), or (All-e), R’ branched is; a Ci-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c),
  • R’ branched is; each a C2-6 alkyl.
  • m and 1 are each independently selected from 4, 5, and 6 and each R’ independently is a Ci-12 alkyl. In some embodiments of the compound of Formula (All), (All-a),
  • m and 1 are each 5 and each R’ independently is a C2-5 alkyl.
  • m and 1 are each 5 and each R’ independently is a C2-5 alkyl.
  • R’ branched is; are each independently selected from 4, 5, and 6, each R’ independently is a Ci- 12 alkyl, and R ay and R by are each a Ci-12 alkyl.
  • R ay and R by are each a Ci-12 alkyl.
  • R’ branched is; are each 5, each R’ independently is a C2-5 alkyl, and R ay and R by are each a C2-6 alkyl.
  • R’ branched is; are each independently selected from 4, 5, and 6, R’ is a Ci-12 alkyl, R ay is a Ci-12 alkyl and R 2 and R 3 are each independently a C 6-10 alkyl.
  • R’ branched is; are each 5, R’ is a C2- alkyl, R ay is a C2-6 alkyl, and R 2 and R 3 are each a C 8 alkyl.
  • R 10 is NH(CI-6 alkyl) and n2 is 2.
  • R 4 wherein R 10 is NH(CH3) and n2 is 2.
  • R’ branched is; independently selected from 4, 5, and 6, each R’ independently is a Ci- 12 alkyl, R ay and R by are each a Ci-12 alkyl, wherein R 10 is NH(CI-6 alkyl), and n2 is 2.
  • each R’ independently is a C2-5 alkyl
  • R ay and R by are each a C2-6 alkyl wherein R 10 is NH(CH3) and n2 is 2.
  • R’ branched is; are each independently selected from 4, 5, and 6, R’ is a Ci-12 alkyl, R 2 and R 3 are each independently a C 6-10 alkyl, R ay is a Ci-12 alkyl wherein R 10 is NH(CI-6 alkyl) and n2 is 2.
  • R’ branched is; are each 5, R’ is a C2-
  • R y is a C2-6 alkyl
  • R 2 and R 3 are each a Cs alkyl
  • R 10 is NH(CH 3 ) and n2 is 2.
  • R 4 is -(CH 2 ) n OH and n is 2, 3, or 4. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), R 4 is -(CH 2 ) n OH and n is 2.
  • R’ branched is independently selected from 4, 5, and 6, each R’ independently is a Ci- 12 alkyl, R ay and R by are each a Ci-12 alkyl, R 4 is -(CH 2 ) n OH, and n is 2, 3, or 4.
  • R’ branched i s: . m and 1 are each 5, each R’ independently is a C2-5 alkyl, R ay and R b ⁇ are each a C2-6 alkyl, R 4 is -(CH 2 ) n OH, and n is 2.
  • the ionizable amino lipid is a compound of Formula (All-f): wherein R’ a is R’ branched O r R’ cyclic ; wherein wherein denotes a point of attachment;
  • R ay is a C1-12 alkyl
  • R 2 and R 3 are each independently a Ci-14 alkyl
  • R 4 is -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;
  • R’ is a C1-12 alkyl; m is selected from 4, 5, and 6; and
  • 1 is selected from 4, 5, and 6.
  • n and 1 are each 5, and n is 2, 3, or 4.
  • R’ is a C2-5 alkyl
  • R ay is a C2-6 alkyl
  • R 2 and R 3 are each a C 6-10 alkyl.
  • m and 1 are each 5, n is 2, 3, or 4, R’ is a C2-5 alkyl, R ay is a C2-6 alkyl, and R 2 and R 3 are each a C 6-10 alkyl.
  • the ionizable amino lipid is a compound of Formula (All-g):
  • R’ is a C2-5 alkyl; and R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting wherein denotes a point of attachment, R 10 is NH(CI-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.
  • the ionizable amino lipid is a compound of Formula (All-h): wherein
  • R ay and R hy are each independently a C2-6 alkyl; each R’ independently is a C2-5 alkyl; and
  • R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting wherein denotes a point of attachment, R 10 is NH(CI-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.
  • R 4 is , wherein
  • R 10 is NH(CH 3 ) and n2 is 2.
  • R 4 is -(CH2)OH.
  • the ionizable amino lipids may be one or more of compounds of Formula (VI): (VI), or their N-oxides, or salts or isomers thereof, wherein: Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R4 is selected from the group consisting of hydrogen, a C3-6 carbocycle, -(CH2) n Q, - (CH 2 )nCHQR,
  • M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, - C(O)N(R’)-,
  • R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
  • Rs is selected from the group consisting of C3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O) 2 N(R) 2 , C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, - R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-15 alkyl and
  • each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl
  • each Y is independently a C3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I
  • m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R4 is -(CH 2 ) n Q, - (CH 2 ) n CHQR, -CHQR, or -CQ(R) 2 , then (i) Q is not -N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
  • another subset of compounds of Formula (VI) includes those in which:
  • Ri is selected from the group consisting of C5-30 alkyl, Cs- 2 o alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R 2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R 2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R4 is selected from the group consisting of a C3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R)
  • M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(0)N(R’)-, -N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, an aryl group, and a heteroaryl group;
  • R7 is selected from the group consisting of C1-3 alkyl, C 2 -3 alkenyl, and H;
  • Rs is selected from the group consisting of C3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C1-6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2 -6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and
  • each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, - R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
  • another subset of compounds of Formula (VI) includes those in which:
  • Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R4 is selected from the group consisting of a C3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R) 2 C(
  • R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
  • Rs is selected from the group consisting of C3-6 carbocycle and heterocycle
  • R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, - R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
  • another subset of compounds of Formula (VI) includes those in which:
  • Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R4 is selected from the group consisting of a C3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH 2 ) n N(R) 2 , -C(O)OR, -OC(O)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -C(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(R)C(S)N(R) 2 , -CRN(R)
  • M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, an aryl group, and a heteroaryl group;
  • R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
  • Rs is selected from the group consisting of C3-6 carbocycle and heterocycle
  • R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O) 2 N(R) 2 , C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, - R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
  • another subset of compounds of Formula (VI) includes those in which
  • Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R2 and R3 are independently selected from the group consisting of H, C2-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R4 is -(CFDiiQ or -(CH 2 ) n CHQR, where Q is -N(R)2, and n is selected from 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, an aryl group, and a heteroaryl group;
  • R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-18 alkyl, C2-I8 alkenyl, - R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
  • another subset of compounds of Formula (VI) includes those in which
  • Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R2 and R3 are independently selected from the group consisting of Ci-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R4 is selected from the group consisting of -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, and - CQ(R) 2 , where Q is -N(R) 2 , and n is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
  • M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O) 2 -, -S-S-, an aryl group, and a heteroaryl group;
  • R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of Ci-is alkyl, C2-I8 alkenyl, - R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of Ci-12 alkyl and Ci-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
  • m is 5, 7, or 9.
  • Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2.
  • Q is -N(R)C(O)R, or -N(R)S(O) 2 R.
  • a subset of compounds of Formula (VI) includes those of Formula (VI-B): or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein.
  • m is selected from 5, 6, 7, 8, and 9;
  • R4 is hydrogen, unsubstituted C1-3 alkyl, or - (CH 2 ) n Q, in which Q is H, -NHC(S)N(R) 2 , -NHC(O)N(R) 2 , -N(R)C(O)R, -N(R)S(O) 2 R, - N(R)R 8 ,
  • M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”- C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R 2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C2-14 alkenyl.
  • m is 5, 7, or 9.
  • Q is OH, -NHC(S)N(R) 2 , or -NHC(O)N(R) 2 .
  • Q is -N(R)C(O)R, or -N(R)S(O) 2 R.
  • the compounds of Formula (VI) are of Formula (Vila), or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
  • the compounds of Formula (VI) are of Formula (Vllb), or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
  • the compounds of Formula (VI) are of Formula (Vile) or (Vile): or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
  • the compounds of Formula (VI) are of Formula (Vllf): (Vllf) or their N-oxides, or salts or isomers thereof, wherein M is -C(O)O- or -OC(O)-, M” is C1-6 alkyl or C2-6 alkenyl, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, and n is selected from 2, 3, and 4.
  • the compounds of Formula (VI) are of Formula (Vlld), (Vlld), or their N-oxides, or salts or isomers thereof, wherein n is 2, 3, or 4; and m, R’, R”, and
  • R2 through Re are as described herein.
  • each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
  • an ionizable amino lipid comprises a compound having structure:
  • an ionizable amino lipid comprises a compound having structure:
  • the compounds of Formula (VI) are of Formula (Vllg), (Vllg), or their N-oxides, or salts or isomers thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, - C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, Ci-14 alkyl, and C2-14 alkenyl.
  • M is C1-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl).
  • R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
  • the ionizable amino lipids are one or more of the compounds described in U.S. Application Nos. 62/220,091, 62/252,316, 62/253,433, 62/266,460, 62/333,557, 62/382,740, 62/393,940, 62/471,937, 62/471,949, 62/475,140, and 62/475,166, and PCT Application No. PCT/US2016/052352.
  • the central amine moiety of a lipid according to Formula (VI), (VI-A), (VI-B), (VII), (Vila), (Vllb), (Vile), (Vlld), (Vile), (Vllf), or (Vllg) may be protonated at a physiological pH.
  • a lipid may have a positive or partial positive charge at physiological pH.
  • Such amino lipids may be referred to as cationic lipids, ionizable lipids, cationic amino lipids, or ionizable amino lipids.
  • Amino lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.
  • the ionizable amino lipids may be one or more of compounds of formula (VIII), or salts or isomers thereof, wherein
  • t 1 or 2;
  • Ai and A2 are each independently selected from CH or N;
  • Z is CH2 or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • Ri, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20 alkyl, C5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”;
  • Rxi and Rx2 are each independently H or C1-3 alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, - OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group;
  • M* is Ci-Ce alkyl
  • W 1 and W 2 are each independently selected from the group consisting of -O- and -N(Re)-; each Re is independently selected from the group consisting of H and C1-5 alkyl;
  • the compound is of any of formulae (Villa l)-(VIIIa8):
  • the ionizable amino lipid is salt thereof.
  • the central amine moiety of a lipid according to Formula (VIII), (Vlllal), (VIIIa2), (VIIIa3), (VIIIa4), (VIIIa5), (VIIIa6), (VIIIa7), or (VIIIa8) may be protonated at a physiological pH.
  • a lipid may have a positive or partial positive charge at physiological pH.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:
  • R 1 is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl
  • R 2 and R 3 are each independently optionally substituted C1-C36 alkyl
  • R 4 and R 5 are each independently optionally substituted Ci-Ce alkyl, or R 4 and R 5 join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;
  • L 1 , L 2 , and L 3 are each independently optionally substituted Ci-Cis alkylene;
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: G 1 is -N(R 3 )R 4 or -OR 5 ;
  • R 1 is optionally substituted branched, saturated or unsaturated C12-C36 alkyl
  • R 3 and R 4 are each independently H, optionally substituted branched or unbranched, saturated or unsaturated Ci-Ce alkyl; or R 3 and R 4 are each independently optionally substituted branched or unbranched, saturated or unsaturated Ci-Ce alkyl when L is C6-C12 alkylene, Cf>- C12 alkenylene, or C2-C6 alkynylene; or R 3 and R 4 , together with the nitrogen to which they are attached, join to form a heterocyclyl;
  • R 5 is H or optionally substituted Ci-Ce alkyl
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt thereof, wherein: each R la is independently hydrogen, R lc , or R ld ; each R lb is independently R lc or R ld ; each R lc is independently -[CH2]2C(O)X 1 R 3 ; each R ld Is independently -C(O)R 4 ; each R 2 is independently -[C(R 2a )2]cR 2b ; each R 2a is independently hydrogen or Ci-Ce alkyl;
  • R 2b is -N(LI-B) 2 ; -(OCH 2 CH 2 )6OH; or -(OCH 2 CH 2 )bOCH 3 ; each R 3 and R 4 is independently C6-C30 aliphatic; each I.3 is independently C1-C10 alkylene; each B is independently hydrogen or an ionizable nitrogen-containing group; each X 1 is independently a covalent bond or O; each a is independently an integer of 1-10; each b is independently an integer of 1-10; and each c is independently an integer of 1-10.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
  • G 1 and G 2 are each independently C2-C12 alkylene or C2-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C2-C24 alkenylene, C1-C24 heteroalkylene or C2-
  • G 3 is C1-C24 heteroalkylene or C2- C24 heteroalkenylene when X is N, and Y is absent;
  • R a , R b , R d and R e are each independently H or C1-C12 alkyl or C1-C12 alkenyl;
  • R c and R f are each independently C1-C12 alkyl or C2-C12 alkenyl; each R is independently H or C1-C12 alkyl;
  • R 1 , R 2 and R 3 are each independently C1-C24 alkyl or C2-C24 alkenyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
  • G 3 is Ci-Ce alkylene
  • R a is H or C1-C12 alkyl
  • R la and R lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R la is H or C1-C12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R 3a is H or C1-C12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently H or methyl
  • R 7 is H or C1-C20 alkyl
  • R 9 and R 10 are each independently H or C1-C12 alkyl
  • R" is aralkyl; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2, wherein each alkyl, alkylene and aralkyl is optionally substituted.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
  • X and X' are each independently N or CR;
  • G 1 , G 2 and G 2 are each independently C2-Ci2 alkylene or C2-C12 alkenylene;
  • G is C2-C24 heteroalkylene or C2-C24 heteroalkenylene
  • R a , R b , R d and R e are, at each occurrence, independently H, C1-C12 alkyl or C2-C12 alkenyl;
  • R c and R f are, at each occurrence, independently C1-C12 alkyl or C2-C12 alkenyl;
  • R is, at each occurrence, independently H or C1-C12 alkyl
  • R 1 and R 2 are, at each occurrence, independently branched C6-C24 alkyl or branched Cf>- C24 alkenyl; z is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, hetero alkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
  • G 1 and G 2 are each independently C2-C12 alkylene or C2-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
  • R 1 and R 2 are each independently branched C6-C24 alkyl or branched Ce- C24 alkenyl
  • R 3 is -N(R 4 )R 5 ;
  • R 4 is C1-C12 alkyl
  • R 5 is substituted C1-C12 alkyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted unless otherwise specified.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
  • G la and G 2b are each independently C2-C12 alkylene or C2-C12 alkenylene;
  • G lb and G 2b are each independently C1-C12 alkylene or C2-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
  • R a , R b , R d and R e are each independently H or C1-C12 alkyl or C2-C12 alkenyl;
  • R c and R f are each independently C1-C12 alkyl or C2-C12 alkenyl
  • R 1 and R 2 are each independently branched C6-C24 alkyl or branched Ce- C24 alkenyl
  • R 4a is C1-C12 alkyl
  • R 4b is H, C1-C12 alkyl or C2-C12 alkenyl
  • R 5a is H, Ci-C 8 alkyl or C 2 -C 8 alkenyl
  • R 5b is C2-C12 alkyl or C2-C12 alkenyl when R 4b is H; or R 5b is C1-C12 alkyl or C2- C12 alkenyl when R 4b is C1-C12 alkyl or C2-C12 alkenyl;
  • R 6 is H, aryl or aralkyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
  • R is, at each occurrence, independently H or OH
  • R 1 and R 2 are each independently optionally substituted branched, saturated or unsaturated C12-C36 alkyl
  • R 3 and R 4 are each independently H or optionally substituted straight or branched, saturated or unsaturated Ci-Ce alkyl
  • R 5 is optionally substituted straight or branched, saturated or unsaturated Ci-Ce alkyl; and n is an integer from 2 to 6.
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1 ; R a is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, Ci-
  • R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 1 and R 2 have, at each occurrence, the following structure, respectively: a 1 and a 2 are, at each occurrence, independently an integer from 3 to 12; b 1 and b 2 are, at each occurrence, independently 0 or 1 ; c 1 and c 2 are, at each occurrence, independently an integer from 5 to 10; d 1 and d 2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
  • G 1 and G 2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C1-C12 alkyl
  • R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
  • R 4 is C1-C12 alkyl
  • R 5 is H or Ci-C 6 alkyl; and x is 0, 1 or 2.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
  • G 3 is Ci-Ce alkylene
  • R a is H or C1-C12 alkyl
  • R la and R lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R la is H or Ci -C 12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R 3a is H or C1-C12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently H or methyl
  • R 7 is C4-C20 alkyl
  • R 8 and R 9 are each independently C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
  • R la and R lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R la is H or Ci -C 12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 3a is H or C1-C12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently methyl or cyclo alkyl;
  • R 7 is, at each occurrence, independently H or C1-C12 alkyl;
  • R 8 and R 9 are each independently unsubstituted C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7- membered heterocyclic ring comprising one nitrogen atom;
  • a and d are each independently an integer from 0 to 24;
  • b and c are each independently an integer from 1 to 24; and
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt thereof, wherein
  • Ri and R2 are the same or different, each a linear or branched alkyl with 1-9 carbons, or as alkenyl or alkynyl with 2 to 11 carbon atoms,
  • Li and L2 are the same or different, each a linear alkyl having 5 to 18 carbon atoms, or form a heterocycle with N,
  • Xi is a bond, or is -CG-G- whereby L2-CO-O-R2 is formed,
  • X2 is S or O
  • L3 is a bond or a lower alkyl, or form a heterocycle with N
  • R3 is a lower alkyl
  • R4 and R5 are the same or different, each a lower alkyl.
  • the lipid nanoparticle comprises an ionizable lipid having the structure: or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure:
  • the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure: (XXII-L), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle comprises a lipid having the structure:
  • the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof.
  • the lipid nanoparticles described herein comprise one or more non-cationic lipids.
  • Non-cationic lipids may be phospholipids.
  • the lipid nanoparticle comprises 5-25 mol% non-cationic lipid.
  • the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid.
  • the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.
  • a non-cationic lipid comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecen
  • the lipid nanoparticle comprises 5 - 15 mol%, 5 - 10 mol%, or 10 - 15 mol% DSPC.
  • the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.
  • the lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • 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, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a membrane e.g., a cellular or intracellular membrane.
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • 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).
  • 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, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (DSPC), 1,
  • a phospholipid is an analog or variant of DSPC.
  • a phospholipid useful or potentially is a compound of Formula (IX): or a salt thereof, wherein: each R 1 is independently optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R 1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each instance of L 2 is independently a bond or optionally substituted Ci-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ); each instance of R 2 is independently optionally substituted Ci-30 alkyl, optionally substituted Ci-30 alkenyl, or optionally substituted Ci-30 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C
  • Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2; provided that the compound is not of the formula: wherein each instance of R 2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.
  • the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT/US2018/037922.
  • the lipid nanoparticle comprises a molar ratio of 5-25% noncationic lipid relative to the other lipid components.
  • the lipid nanoparticle may comprise a molar ratio of 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% non-cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% non-cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of 5-25% phospholipid relative to the other lipid components.
  • the lipid nanoparticle may comprise a molar ratio of 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% phospholipid.
  • the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% phospholipid lipid.
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid includes sterols and also to lipids containing sterol moieties.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • “sterols” are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol.
  • the structural lipids may be one or more of the structural lipids described in U.S. Application No. 16/493,814.
  • the lipid nanoparticle comprises a molar ratio of 25-55% structural lipid relative to the other lipid components.
  • the lipid nanoparticle may comprise a molar ratio of 10- 55%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30- 50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% structural lipid.
  • the lipid nanoparticle comprises a molar ratio of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% structural lipid.
  • the lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 34-35 mol%, 35- 36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%. In some embodiments, the lipid nanoparticle comprises 25-55 mol% sterol.
  • the lipid nanoparticle may comprise 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% sterol.
  • the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.
  • the lipid nanoparticle comprises 35 - 40 mol% cholesterol.
  • the lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids.
  • PEG polyethylene glycol
  • PEG-lipid refers to polyethylene glycol (PEG) -modified lipids.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified l,2-diacyloxypropan-3- amines.
  • PEGylated 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-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxlpropyl-3 -amine (PEG-c-DM A) .
  • PEG-DMG 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol
  • PEG-DSPE l,2-distea
  • the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG-modified lipid is PEG- DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and/or PEG-DPG.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about Ci6.
  • a PEG moiety for example an mPEG-NEh, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG-lipid is PEG2k-DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • PEG lipid which is a non-diffusible PEG.
  • non-diffusible PEGs include PEG- DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • lipid components e.g., PEG lipids
  • PEG lipids lipid components of various formulae described herein may be synthesized as described International Patent Application No. PCT/US2016/000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.
  • the lipid component of a lipid nanoparticle composition 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-modified lipids are a modified form of PEG DMG.
  • PEG- DMG has the following structure:
  • PEG lipids can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment.
  • a PEG lipid is a compound of Formula (X): or salts thereof, wherein:
  • R 3 is -OR°
  • R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive;
  • L 1 is optionally substituted Ci-10 alkylene, wherein at least one methylene of the optionally substituted Ci-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N );
  • D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each instance of L 2 is independently a bond or optionally substituted Ci-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with O, N(R N ), S, C(O), C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or NR N C(O)N(R N ); each instance of R 2 is independently optionally substituted Ci-30 alkyl, optionally substituted Ci-30 alkenyl, or optionally substituted Ci-30 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C
  • Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.
  • the compound of Fomula (X) is a PEG-OH lipid (i.e., R 3 is - OR°, and R° is hydrogen). In certain embodiments, the compound of Formula (X) is of Formula (X-OH): or a salt thereof.
  • a PEG lipid is a PEGylated fatty acid. In certain embodiments, a PEG lipid is a compound of Formula (XI). Described herein are compounds of Formula (XI): or a salts thereof, wherein:
  • R 3 is-OR°
  • R° is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive;
  • the compound of Formula (XI) is of Formula (XI-OH): or a salt thereof.
  • r is 40-50.
  • the compound of Formula (XI) is: or a salt thereof.
  • the compound of Formula (XI) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.
  • the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. US 15/674,872.
  • the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG lipid relative to the other lipid components.
  • the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15% PEG lipid.
  • the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG- lipid.
  • the lipid nanoparticle comprises 1-5% PEG-modified lipid, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%.
  • the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid.
  • the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%.
  • the lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.
  • Some embodiments comprise adding PEG to a composition comprising an LNP encapsulating a nucleic acid (e.g., which already includes PEG in the amounts listed above). Without being bound by theory, it is believed that spiking a LNP composition with additional PEG can provide benefits during lyophilization. Thus, some embodiments, comprise adding additional PEG as compared to an amount used for a non-lyophilized LNP composition.
  • embodiments comprise adding about 0.5mo% or more PEG to an LNP composition, such as about lmol%, about 1.5mol%, about 2mol%, about 2.5mol%, about 3mol%, about 3.5mol%, about 4mol%, about 5mol%, or more after formation of an LNP composition (e.g., which already contains PEG in amount listed elsewhere herein).
  • the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.
  • a LNP comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.
  • a LNP comprises an ionizable amino lipid of any of Formula VI, VII or VIIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.
  • a LNP comprises an ionizable amino lipid of any of Formula VI, VII or VIII, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula XI.
  • a LNP comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula VIII, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI.
  • a LNP comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid comprising a compound having Formula IX, a structural lipid, and the PEG lipid comprising a compound having Formula X or XI.
  • a LNP comprises an ionizable amino lipid of Formula VI, VII or VIII, a phospholipid having Formula IX, a structural lipid, and a PEG lipid comprising a compound having Formula XI.
  • the lipid nanoparticle comprises 49 mol% ionizable amino lipid
  • the lipid nanoparticle comprises 49 mol% ionizable amino lipid
  • the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
  • a LNP comprises an N:P ratio of from about 2:1 to about 30:1.
  • a LNP comprises an N:P ratio of about 6:1.
  • a LNP comprises an N:P ratio of about 3:1, 4:1, or 5:1.
  • a LNP comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1. In some embodiments, a LNP comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1.
  • a LNP comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1.
  • Some embodiments comprise a composition having one or more LNPs having a diameter of about 200 nm or less, such as about 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less.
  • Some embodiments comprise a composition having a mean LNP diameter of about 180 nm or less, such as about 170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less.
  • the composition has a mean LNP diameter from about 30 nm to about 200 nm, or a mean diameter from about 60 nm to about 180.
  • a LNP may comprise or one or more types of lipids, including but not limited to amino lipids (e.g., ionizable amino lipids), neutral lipids, non-cationic lipids, charged lipids, PEG- modified lipids, phospholipids, structural lipids and sterols.
  • a LNP may further comprise one or more cargo molecules, including but not limited to nucleic acids (e.g., mRNA, plasmid DNA, DNA or RNA oligonucleotides, siRNA, shRNA, snRNA, snoRNA, IncRNA, etc.), small molecules, proteins and peptides.
  • the composition comprises a liposome.
  • a liposome is a lipid particle comprising lipids arranged into one or more concentric lipid bilayers around a central region.
  • the central region of a liposome may comprises an aqueous solution, suspension, or other aqueous composition.
  • a lipid nanoparticle may comprise two or more components (e.g., amino lipid and nucleic acid, PEG-lipid, phospholipid, structural lipid).
  • a lipid nanoparticle may comprise an amino lipid and a nucleic acid.
  • Compositions comprising the lipid nanoparticles, such as those described herein, may be used for a wide variety of applications, including the stealth delivery of therapeutic payloads with minimal adverse innate immune response.
  • nucleic acids i.e., originating from outside of a cell or organism
  • a particulate carrier e.g., lipid nanoparticles
  • the particulate carrier should have minimal particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids intracellularly, and illicit no or minimal immune response.
  • many conventional particulate carriers have relied on the presence and/or concentration of certain components (e.g., PEG-lipid).
  • nucleic acid e.g., mRNA molecules
  • the lipid nanoparticles comprise one or more of ionizable molecules, polynucleotides, and optional components, such as structural lipids, sterols, neutral lipids, phospholipids and a molecule capable of reducing particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipid), such as those described above.
  • ionizable molecules such as structural lipids, sterols, neutral lipids, phospholipids and a molecule capable of reducing particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipid), such as those described above.
  • PEG polyethylene glycol
  • a LNP described herein may include one or more ionizable molecules (e.g., amino lipids or ionizable lipids).
  • the ionizable molecule may comprise a charged group and may have a certain pKa.
  • the pKa of the ionizable molecule may be greater than or equal to about 6, greater than or equal to about 6.2, greater than or equal to about 6.5, greater than or equal to about 6.8, greater than or equal to about 7, greater than or equal to about 7.2, greater than or equal to about 7.5, greater than or equal to about 7.8, greater than or equal to about 8.
  • the pKa of the ionizable molecule may be less than or equal to about 10, less than or equal to about 9.8, less than or equal to about 9.5, less than or equal to about 9.2, less than or equal to about 9.0, less than or equal to about 8.8, or less than or equal to about 8.5. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 6 and less than or equal to about 8.5). Other ranges are also possible. In embodiments in which more than one type of ionizable molecule are present in a particle, each type of ionizable molecule may independently have a pKa in one or more of the ranges described above.
  • an ionizable molecule comprises one or more charged groups.
  • an ionizable molecule may be positively charged or negatively charged.
  • an ionizable molecule may be positively charged.
  • an ionizable molecule may comprise an amine group.
  • the term “ionizable molecule” has its ordinary meaning in the art and may refer to a molecule or matrix comprising one or more charged moiety.
  • a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc.
  • the charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).
  • positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups.
  • the charged moieties comprise amine groups.
  • negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged.
  • the charge density of the molecule and/or matrix may be selected as desired.
  • an ionizable molecule may include one or more precursor moieties that can be converted to charged moieties.
  • the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above.
  • the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively.
  • the ionizable molecule may have any suitable molecular weight.
  • the molecular weight of an ionizable molecule is less than or equal to about 2,500 g/mol, less than or equal to about 2,000 g/mol, less than or equal to about 1,500 g/mol, less than or equal to about 1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equal to about 900 g/mol, less than or equal to about 800 g/mol, less than or equal to about 700 g/mol, less than or equal to about 600 g/mol, less than or equal to about 500 g/mol, less than or equal to about 400 g/mol, less than or equal to about 300 g/mol, less than or equal to about 200 g/mol, or less than or equal to about 100 g/mol.
  • the molecular weight of an ionizable molecule is greater than or equal to about 100 g/mol, greater than or equal to about 200 g/mol, greater than or equal to about 300 g/mol, greater than or equal to about 400 g/mol, greater than or equal to about 500 g/mol, greater than or equal to about 600 g/mol, greater than or equal to about 700 g/mol, greater than or equal to about 1000 g/mol, greater than or equal to about 1,250 g/mol, greater than or equal to about 1,500 g/mol, greater than or equal to about 1,750 g/mol, greater than or equal to about 2,000 g/mol, or greater than or equal to about 2,250 g/mol.
  • each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above.
  • the percentage (e.g., by weight, or by mole) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and/or of all the ionizable molecules within a particle may be greater than or equal to about 15%, greater than or equal to about 16%, greater than or equal to about 17%, greater than or equal to about 18%, greater than or equal to about 19%, greater than or equal to about 20%, greater than or equal to about 21%, greater than or equal to about 22%, greater than or equal to about 23%, greater than or equal to about 24%, greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 35%, greater than or equal to about 40%, greater than or equal to about 42%, greater than or equal to about 45%, greater than or equal to about 48%, greater than or equal to about 50%, greater than or equal to about 52%, greater than or equal to about 55%, greater than or equal to about 58%, greater than
  • the percentage (e.g., by weight, or by mole) may be less than or equal to about 70%, less than or equal to about 68%, less than or equal to about 65%, less than or equal to about 62%, less than or equal to about 60%, less than or equal to about 58%, less than or equal to about 55%, less than or equal to about 52%, less than or equal to about 50%, or less than or equal to about 48%. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 20% and less than or equal to about 60%, greater than or equal to 40% and less than or equal to about 55%, etc.).
  • each type of ionizable molecule may independently have a percentage (e.g., by weight, or by mole) in one or more of the ranges described above.
  • the percentage e.g., by weight, or by mole
  • the percentage may be determined by extracting the ionizable molecule(s) from the dried particles using, e.g., organic solvents, and measuring the quantity of the agent using high pressure liquid chromatography (i.e., HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS).
  • HPLC may be used to quantify the amount of a component, by, e.g., comparing the area under the curve of a HPLC chromatogram to a standard curve.
  • a lipid composition may comprise one or more lipids as described herein. Such lipids may include those useful in the preparation of lipid nanoparticlesas described above or as known in the art.
  • a subject to which a composition comprising a nucleic acid and a lipid, is administered is a subject that suffers from or is at risk of suffering from a disease, disorder or condition, including a communicable or non-communicable disease, disorder or condition.
  • “treating” a subject can include either therapeutic use or prophylactic use relating to a disease, disorder or condition, and may be used to describe uses for the alleviation of symptoms of a disease, disorder or condition, uses for vaccination against a disease, disorder or condition, and uses for decreasing the contagiousness of a disease, disorder or condition, among other uses.
  • the nucleic acid is an mRNA vaccine designed to achieve particular biologic effects.
  • exemplary vaccines feature mRNAs encoding a particular antigen of interest (or an mRNA or mRNAs encoding antigens of interest).
  • the vaccines feature an mRNA or mRNAs encoding antigen(s) derived from infectious diseases.
  • the disclosure encompasses infectious disease vaccines.
  • the antigen of the infectious disease vaccine is a viral antigen.
  • the infectious agent is a human cytomegalovirus (hCMV).
  • a disease, disorder, or condition is caused by or associated with a member of the herpesvirus family.
  • the virus is a human cytomegalovirus (hCMV).
  • the antigen is a human cytomegalovirus (hCMV) antigen.
  • the article and/or pharmaceutical composition comprises one or more (e.g., greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, or greater than or equal to 5; less than or equal to 10, less than or equal to 8, less than or equal to 6, less than or equal to 4, or less than or equal to 2; combinations of these ranges are also possible, such as greater than or equal to 1 and less than or equal to 6) hCMV antigens.
  • the disease, disorder or condition is a disease or condition caused by or associated with human cytomegalovirus (hCMV).
  • a composition disclosed herein does not comprise a pharmaceutical preservative. In other embodiments, a composition disclosed herein does comprise a pharmaceutical preservative.
  • pharmaceutical preservatives include methyl paragen, ethyl paraben, propyl paraben, butyl paraben, benzyl acohol, chlorobutanol, phenol, meta cresol (m-cresol), chloro cresol, benzoic acid, sorbic acid, thiomersal, phenylmercuric nitrate, bronopol, propylene glycol, benzylkonium chloride, and benzethionium chloride.
  • compositions disclosed herein does not comprise phenol, m-cresol, or benzyl alcohol.
  • Compositions in which microbial growth is inhibited may be useful in the preparation of injectable formulations, including those intended for dispensing from multi-dose vials.
  • Multi-dose vials refer to containers of pharmaceutical compositions from which multiple doses can be taken repeatedly from the same container.
  • Compositions intended for dispensing from multi-dose vials typically must meet USP requirements for antimicrobial effectiveness.
  • administering means providing a material to a subject in a manner that is pharmacologically useful.
  • a composition disclosed herein is administered to a subject enterally.
  • an enteral administration of the composition is oral.
  • a composition disclosed herein is administered to the subject parenterally.
  • a composition disclosed herein is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracistemally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs.
  • compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result.
  • the desirable result will depend upon the active agent being administered.
  • an effective amount of a composition comprising a nucleic acid and a lipid may be an amount of the composition that is capable of increasing expression of a protein in the subject.
  • a therapeutically acceptable amount may be an amount that is capable of treating a disease or condition, e.g., a disease or condition that that can be relieved by increasing expression of a protein in a subject.
  • dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, the intended outcome of the administration, time and route of administration, general health, and other drugs being administered concurrently.
  • a subject is administered a composition comprising a nucleic acid and a lipid I in an amount sufficient to increase expression of a protein in the subject.
  • LNP preparations are analyzed for polydispersity in size (e.g., particle diameter) and/or composition (e.g., amino lipid amount or concentration, phospholipid amount or concentration, structural lipid amount or concentration, PEG-lipid amount or concentration, mRNA amount (e.g., mass) or concentration) and, optionally, further assayed for in vitro and/or in vivo activity.
  • Fractions or pools thereof can also be analyzed for accessible mRNA and/or purity (e.g., purity as determined by reverse-phase (RP) chromatography).
  • Particle size (e.g., particle diameter) can be determined by Dynamic Light Scattering (DLS). DLS measures a hydrodynamic diameter. Smaller particles diffuse more quickly, leading to faster fluctuations in the scattering intensity and shorter decay times for the autocorrelation function. Larger particles diffuse more slowly, leading to slower fluctuations in the scattering intensity and longer decay times in the autocorrelation function.
  • mRNA purity can be determined by reverse phase high-performance liquid chromatography (RP-HPLC) size based separation. This method can be used to assess mRNA integrity by a length-based gradient RP separation and UV detection of RNA at 260 nm.
  • RP-HPLC reverse phase high-performance liquid chromatography
  • main peak or “main peak purity” refers to the RP-HPLC signal detected from mRNA that corresponds to the full size mRNA molecule loaded within a given LNP. mRNA purity can also be assessed by fragmentation analysis.
  • Fragmentation analysis is a method by which nucleic acid (e.g., mRNA) fragments can be analyzed by capillary electrophoresis. Fragmentation analysis involves sizing and quantifying nucleic acids (e.g., mRNA), for example by using an intercalating dye coupled with an LED light source. Such analysis may be completed, for example, with a Fragment Analyzer from Advanced Analytical Technologies, Inc.
  • compositions formed via the methods described herein may be particularly useful for administering an agent to a subject in need thereof.
  • the compositions are used to deliver a pharmaceutically active agent.
  • the compositions are used to deliver a prophylactic agent.
  • the compositions may be administered in any way known in the art of drug delivery, for example, orally, parenterally, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intrathecally, submucosally, etc.
  • compositions may be combined with pharmaceutically acceptable excipients to form a pharmaceutical composition.
  • excipients may be chosen based on the route of administration as described below, the agent being delivered, and the time course of delivery of the agent.
  • compositions described herein and for use in accordance with the embodiments described herein may include a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient means a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Some examples of materials which can serve as pharmaceutically acceptable excipients are sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, methylcellulose, hydroxypropylmethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; citric acid, acetate salts, Ringer’s solution
  • compositions can be administered to humans and/or to animals, orally, parenterally, intracistemally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, or as an oral or nasal spray.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants
  • sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer’s solution, ethanol, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostea,
  • compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • Dosage forms for topical or transdermal administration of a pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.
  • the particles are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also possible.
  • the ointments, pastes, creams, and gels may contain, in addition to the compositions, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to the compositions, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons .
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms can be made by dissolving or dispensing the compositions in a proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the compositions in a polymer matrix or gel.
  • compositions are loaded and stored in prefilled syringes and cartridges for patient-friendly autoinjector and infusion pump devices.
  • kits for use in preparing or administering the compositions described herein may include any solvents, solutions, buffer agents, acids, bases, salts, targeting agent, etc. needed in the composition formation process. Different kits may be available for different targeting agents.
  • the kit includes materials or reagents for purifying, sizing, and/or characterizing the resulting compositions. The kit may also include instructions on how to use the materials in the kit.
  • the one or more agents (e.g., pharmaceutically active agent) to be contained within the composition are typically provided by the user of the kit.
  • kits for using or administering the compositions are also described herein.
  • the compositions may be provided in convenient dosage units for administration to a subject.
  • the kit may include multiple dosage units.
  • the kit may include 1-100 dosage units.
  • the kit includes a week supply of dosage units, or a month supply of dosage units.
  • the kit includes an even longer supply of dosage units.
  • the kits may also include devices for administering the compositions. Exemplary devices include syringes, spoons, measuring devices, etc.
  • the kit may optionally include instructions for administering the compositions (e.g., prescribing information).
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N + (CI-4 alkyl)4- salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
  • composition As disclosed herein, the terms “composition” and “formulation” are used interchangeably.
  • nucleic acid is an mRNA.
  • the nucleic acids for example mRNAs, are preferably in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles), such that the nucleic acids, e.g., mRNAs, are suitable for use in vivo.
  • nucleic acids e.g., mRNAs
  • nucleic acids are capable of being delivered to cells and/or tissues within a subject, e.g., a human subject, to effectuate translation of protein encoded by these nucleic acids.
  • nucleic acid refers to multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G))).
  • nucleic acid refers to polyribonucleotides as well as polydeoxyribonucleotides.
  • nucleic acid shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base containing polymer.
  • Non-limiting examples of nucleic acids include chromosomes, genomic loci, genes or gene segments that encode polynucleotides or polypeptides, coding sequences, non-coding sequences (e.g., intron, 5'-UTR, or 3'-UTR) of a gene, pri-mRNA, pre- mRNA, cDNA, mRNA, etc.
  • a nucleic acid e.g., mRNA
  • the substitution and/or modification is in one or more bases and/or sugars.
  • a nucleic acid e.g., mRNA
  • a nucleic acid includes nucleic acids having backbone sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 2' position and other than a phosphate group or hydroxy group at the 5' position.
  • a substituted or modified nucleic acid e.g., mRNA
  • a modified nucleic acid e.g., mRNA
  • a nucleic acid includes sugars such as hexose, 2'-F hexose, 2'-amino ribose, constrained ethyl (cEt), locked nucleic acid (LNA), arabinose or 2'-fluoroarabinose instead of ribose.
  • a nucleic acid e.g., mRNA
  • a nucleic acid is heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have an amino acid backbone with nucleic acid bases).
  • the nucleic acid sequences include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • an “engineered nucleic acid” is a nucleic acid that does not occur in nature. It should be understood, however, that while an engineered nucleic acid as a whole is not naturally-occurring, it may include nucleotide sequences that occur in nature.
  • an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species).
  • an engineered nucleic acid includes a bacterial nucleotide sequence, a human nucleotide sequence, and/or a viral nucleotide sequence.
  • Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids.
  • a “recombinant nucleic acid” is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) and, in some embodiments, can replicate in a living cell.
  • a “synthetic nucleic acid” is a molecule that is amplified or chemically, or by other means, synthesized.
  • a synthetic nucleic acid includes those that are chemically modified, or otherwise modified, but can base pair with naturally-occurring nucleic acid molecules.
  • Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
  • a nucleic may comprise naturally occurring nucleotides and/or non-naturally occurring nucleotides such as modified nucleotides.
  • nucleic acid vector When applied to a nucleic acid sequence, the term “isolated” in this context denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.
  • a nucleic acid vector may include an insert which may be an expression cassette or open reading frame (ORF).
  • 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 protein or peptide (e.g., a therapeutic protein or therapeutic peptide).
  • a start codon e.g., methionine (ATG)
  • a stop codon e.g., TAA, TAG or TGA
  • an expression cassette encodes a RNA including at least the following elements: a 5' untranslated region, an open reading frame region encoding the mRNA, a 3' untranslated region and a polyA tail.
  • the open reading frame may encode any mRNA sequence, or portion thereof.
  • a nucleic acid vector comprises a 5' untranslated region (UTR).
  • a “5' untranslated region (UTR)” 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 protein or peptide.
  • a nucleic acid vector comprises a 3' untranslated region (UTR).
  • a “3' untranslated region (UTR)” 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 protein or peptide.
  • 5' and 3' are used herein to describe features of a nucleic acid sequence related to either the position of genetic elements and/or the direction of events (5' to 3'), such as e.g. transcription by RNA polymerase or translation by the ribosome which proceeds in 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Conventionally, DNA sequences, gene maps, vector cards and RNA sequences are drawn with 5' to 3' from left to right or the 5' to 3' direction is indicated with arrows, wherein the arrowhead points in the 3' direction. Accordingly, 5' (upstream) indicates genetic elements positioned towards the left-hand side, and 3' (downstream) indicates genetic elements positioned towards the right-hand side, when following this convention.
  • a nucleic acid typically comprises a plurality of nucleotides.
  • a nucleotide includes a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group.
  • Nucleotides include nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates.
  • a nucleoside monophosphate includes a nucleobase linked to a ribose and a single phosphate; a nucleoside diphosphate (NDP) includes a nucleobase linked to a ribose and two phosphates; and a nucleoside triphosphate (NTP) includes a nucleobase linked to a ribose and three phosphates.
  • Nucleotide analogs are compounds that have the general structure of a nucleotide or are structurally similar to a nucleotide.
  • Nucleotide analogs include an analog of the nucleobase, an analog of the sugar and/or an analog of the phosphate group(s) of a nucleotide.
  • a nucleoside includes a nitrogenous base and a 5-carbon sugar. Thus, a nucleoside plus a phosphate group yields a nucleotide.
  • Nucleoside analogs are compounds that have the general structure of a nucleoside or are structurally similar to a nucleoside.
  • Nucleoside analogs for example, include an analog of the nucleobase and/or an analog of the sugar of a nucleoside.
  • nucleotide includes naturally-occurring nucleotides, synthetic nucleotides and modified nucleotides, unless indicated otherwise.
  • Non- limiting examples of naturally-occurring nucleotides used for the production of RNA include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and 5 -methyluridine triphosphate (m5UTP).
  • adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and/or uridine diphosphate (UDP) are used.
  • nucleotide analogs include, but are not limited to, antiviral nucleotide analogs, phosphate analogs (soluble or immobilized, hydrolyzable or non-hydrolyzable), dinucleotide, trinucleotide, tetranucleotide, e.g., a cap analog, or a precursor/substrate for enzymatic capping (vaccinia or ligase), a nucleotide labeled with a functional group to facilitate ligation/conjugation of cap or 5' moiety (IRES), a nucleotide labeled with a 5' PO4 to facilitate ligation of cap or 5' moiety, or a nucleotide labeled with a functional group/protecting group that can be chemically or enzymatically cleaved.
  • antiviral nucleotide/nucleoside analogs include, but are not limited, to Ganciclovir, Entecavir, Tel
  • compositions with at least about 0.25 mg/mL nucleic acid e.g., mRNA
  • nucleic acid e.g., mRNA
  • Some embodiments comprise compositions with at least about 0.25 mg/mL nucleic acid (e.g., mRNA), such as 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL, 1.5 mg/mL, or 2 mg/mL nucleic acid.
  • the nucleic acid is not self-amplifying.
  • Self-amplifying nucleic acids e.g., self-amplifying RNAs
  • proteins such as viral replicases that are capable of using the nucleic acid encoding the replicase as a template for replication, allowing copying of the self-amplifying nucleic acid within a cell.
  • a given dose of the self-amplifying nucleic acid may lead to production of more of an encoded non-replicase protein (e.g., antigen) than an equivalent dose of a nucleic acid that encodes the same protein but is not self-amplifying (e.g., an mRNA).
  • an encoded non-replicase protein e.g., antigen
  • an equivalent dose of a nucleic acid that encodes the same protein but is not self-amplifying e.g., an mRNA
  • the nucleic acids (e.g., mRNAs) of the methods and compositions encode an immunogenic protein, which may be translated in vivo from the nucleic acid following administration of the composition to a subject, e.g., a human. Translation of an immunogenic protein in vivo elicits an immune response targeting the immunogenic protein.
  • Immune responses targeting a protein may include the generation of antibodies that specifically bind to and/or neutralize the protein, the generation of B cells encoding antibodies specific to the protein, and the generation of T cells with receptors that bind peptides with sequences present in the sequence of the protein.
  • RNA e.g., mRNA
  • a lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • nucleotides and nucleosides comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U).
  • nucleotides and nucleosides comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).
  • the lyophilized hCMV immunogenic compositions comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one hCMV antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art.
  • nucleotides and nucleosides comprise modified nucleotides or nucleosides.
  • modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides.
  • modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
  • a naturally-occurring modified nucleotide or nucleoside is one as is generally known or recognized in the art.
  • Non-limiting examples of such naturally occurring modified nucleotides and nucleosides can be found, inter alia, in the widely recognized MODOMICS database.
  • a non-naturally occurring modified nucleotide or nucleoside is one that is generally known or recognized in the art.
  • Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/036773; PCT/US2015/036759; PCT/US2015/036771; or PCT/IB 2017/051367 all of which are incorporated by reference herein.
  • nucleic acids e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids
  • nucleic acids can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
  • Nucleic acids e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids
  • a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
  • a modified RNA nucleic acid e.g., a modified mRNA nucleic acid
  • introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
  • Nucleic acids e.g., RNA nucleic acids, such as mRNA nucleic acids
  • Nucleic acids in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties.
  • the modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars.
  • the modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.
  • one or more nucleic acids comprises modified nucleosides and/or nucleotides.
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a “nucleotide” refers to a nucleoside, including a phosphate group.
  • Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
  • Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification.
  • non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into nucleic acids.
  • modified nucleobases in nucleic acids comprise 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (ely), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (y).
  • modified nucleobases in nucleic acids e.g., RNA nucleic acids, such as mRNA nucleic acids
  • the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.
  • a mRNA comprises 1-methyl-pseudouridine (mly) substitutions at one or more or all uridine positions of the nucleic acid.
  • a mRNA comprises 1-methyl-pseudouridine (mly) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • a mRNA comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.
  • a mRNA comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
  • a mRNA comprises uridine at one or more or all uridine positions of the nucleic acid.
  • mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine.
  • a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • the nucleic acids may be partially or fully modified along the entire length of the molecule.
  • one or more or all or a given type of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • all nucleotides X in a nucleic acid are modified nucleotides, wherein X may be 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.
  • the nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to
  • the mRNAs may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
  • the nucleic acids may contain a modified pyrimidine such as a modified 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 the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil).
  • the modified 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 nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine).
  • the modified 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).
  • HCMV includes several surface glycoproteins that are involved in viral attachment and entry into different cell types.
  • the pentameric complex composed of gH/gL/UL128/UL130/UL131A, mediates entry into endothelial cells, epithelial cells, and myeloid cells.
  • HCMV proteins UL128, UL130, and UL131A assemble with gH and gL proteins to form a heterologous pentameric complex, designated gH/gL/UL128-131A, found on the surface of the HCMV.
  • Natural variants and deletion and mutational analyses have implicated proteins of the gH/gL/UL128-131A complex with the ability to infect certain cell types, including for example, endothelial cells, epithelial cells, and leukocytes.
  • HCMV enters cells by fusing its envelope with either the plasma membrane (fibroblasts) or the endosomal membrane (epithelial and endothelial cells).
  • HCMV initiates cell entry by attaching to the cell surface heparan sulfate proteoglycans using envelope glycoprotein M (gM) or gB. This step is followed by interaction with cell surface receptors that trigger entry or initiate intracellular signaling.
  • the entry receptor function is provided by gH/gL glycoprotein complexes. Different gH/gL complexes are known to facilitate entry into different cell types including epithelial cells, endothelial cells, or fibroblasts.
  • gB and gH/gL are necessary and sufficient for cell fusion and thus constitute the “core fusion machinery” of HCMV, which is conserved among other herpesviruses.
  • core fusion machinery of HCMV
  • hCMV glycoproteins gB, gH, gL, gM, and gN are immunogenic and involved in the immunostimulatory response in a variety of cell types.
  • UL128, UL130, and UL131A genes are relatively conserved among hCMV isolates and therefore represent an attractive target for vaccination.
  • recent studies have shown that antibodies to epitopes within the pentameric gH/gL/UL128-131 complex neutralize entry into endothelial, epithelial, and other cell types, thus blocking the ability of hCMV to infect several cell types.
  • the majority of neutralizing antibodies may be directed against envelope glycoproteins (Britt et al., 1990; Fouts et al., 2012; Macagno et al., 2010; Marshall et al., 1992, incorporated herein by reference), whereas robust T cell responses may be directed against the tegument protein pp65 and nonstructural proteins such as IE1 and IE2 (Blanco-Lobo et al., 2016; Borysiewicz et al., 1988; Kern et al., 2002, incorporated herein by reference).
  • HCMV envelope glycoprotein complexes represent major antigenic targets of antiviral immune responses.
  • RNA e.g., mRNA
  • RNA vaccines that include polynucleotides encoding an HCMV antigen, in particular an HCMV antigen from one of the HCMV glycoprotein complexes.
  • RNA e.g., mRNA
  • vaccines that include at least one polynucleotide encoding at least one hCMV antigenic polypeptide.
  • the hCMV RNA vaccines described herein may be used to induce a balanced immune response, comprising both cellular and humoral immunity, without many of the risks associated with DNA vaccines and live attenuated vaccines.
  • Some aspects of the disclosure relate to hCMV mRNA vaccines containing mRNAs encoding the hCMV pentamer (gH, gL, UL128, UL130, and UL131A) and gB in lipid nanoparticles.
  • the hCMV immunogenic compositions may comprise (a) a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding a hCMV gH polypeptide; (b) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gL polypeptide; (c) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL128 polypeptide; (d) a mRNA polynucleotide comprising an open reading frame encoding a hCMV ULI 30 polypeptide; (e) a mRNA polynucleotide comprising an open reading frame encoding a hCMV UL131A polypeptide; and (f) a mRNA polynucleotide comprising an open reading frame encoding a hCMV gB polypeptide.
  • mRNA messenger ribon
  • Each of the individual mRNAs encoding the hCMV pentamer (gH, gL, UL128, UL130, and UL131A) and gB may be included in the composition at equal mass ratios (e.g., an mRNA mass ratio for gH:gL:UL128:UL130:UL131A:gB of approximately 1:1: 1:1: 1:1).
  • an approximately equal molar ratio of gL, UL128, UL130, and UL131A, and increased molar ratios of gB and/or gH relative to the other mRNA components within an hCMV immunogenic composition is provided.
  • the molar ratio of (a) :(f) within the immunogenic composition is about 1:1; the molar ratio of (b):(c):(d):(e) within the immunogenic composition is about 1 : 1 : 1 : 1; and the molar ratio of each of (a) and (f) to any one of (b), (c), (d) or (e) within the immunogenic composition is about 1.5:1 to 2:1. In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
  • the molar ratio of each of (a) and (f) to any one of (b), (c), (d) or (e) within the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1). In some embodiments, the molar ratio of (a) to any one of (b), (c), (d) or (e) within the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1).
  • the molar ratio of (f) to any one of (b), (c), (d) or (e) within the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1).
  • the molar ratio of (a) to any one of (b), (c), (d) or (e) within the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1), and the molar ratio of (f) to any one of (b), (c), (d) or (e) within the immunogenic composition is about 1.5:1 to 2:1 (e.g., 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1).
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 1.5: 1 : 1 : 1 : 1 : 1.5.
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2
  • the hCMV vaccine components comprise the sequences provided in Table 3.
  • the mRNA encoding hCMV gH protein comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the nucleotide sequence of sequence of SEQ ID NO: 5.
  • the mRNA encoding hCMV gL protein comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the nucleotide sequence of sequence of SEQ ID NO: 6.
  • the mRNA encoding hCMV UL128 protein comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the nucleotide sequence of sequence of SEQ ID NO: 2.
  • the mRNA encoding hCMV ULI 30 protein comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the nucleotide sequence of sequence of SEQ ID NO: 3.
  • the mRNA encoding hCMV UL131A protein comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the nucleotide sequence of sequence of SEQ ID NO: 4.
  • the mRNA encoding hCMV gB protein comprises a nucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the nucleotide sequence of sequence of SEQ ID NO: 1.
  • the mRNA encoding the hCMV gH polypeptide comprises the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the mRNA encoding the hCMV gL polypeptide comprises the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the mRNA encoding the hCMV UL128 polypeptide comprises the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the mRNA encoding the hCMV UL130 polypeptide comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the mRNA encoding the hCMV UL131A polypeptide comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the mRNA encoding the hCMV gB polypeptide comprises the nucleotide sequence of SEQ ID NO: 1.
  • the open reading frame encoding the hCMV gH polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the sequence of SEQ ID NO: 11.
  • the open reading frame encoding the hCMV gL polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the sequence of SEQ ID NO: 12.
  • the open reading frame encoding the hCMV UL128 polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the sequence of SEQ ID NO: 8.
  • the open reading frame encoding the hCMV ULI 30 polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the sequence of SEQ ID NO: 9.
  • the open reading frame encoding the hCMV UL131A polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the sequence of SEQ ID NO: 10.
  • the open reading frame encoding the gB polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the sequence of SEQ ID NO: 7.
  • the mRNA encoding the hCMV gH polypeptide comprises an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the mRNA encoding the hCMV gL polypeptide comprises an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the mRNA encoding the hCMV UL128 polypeptide comprises an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the mRNA encoding the hCMV UL130 polypeptide comprises an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 9.
  • the mRNA encoding the hCMV UL131A polypeptide comprises an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the mRNA encoding the hCMV gB polypeptide comprises an open reading frame (ORF) of the nucleotide sequence of SEQ ID NO: 7.
  • the hCMV gH polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the amino acid sequence of SEQ ID NO: 19
  • the hCMV gL polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the amino acid sequence of SEQ ID NO: 20.
  • the hCMV UL128 polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the amino acid sequence of SEQ ID NO: 16.
  • the hCMV ULI 30 polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the amino acid sequence of SEQ ID NO: 17.
  • the hCMV UL131A polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the amino acid sequence of SEQ ID NO: 18.
  • the hCMV gB polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or more than 99% identity, to the amino acid sequence of SEQ ID NO: 15.
  • the hCMV gH polypeptide comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the hCMV gL polypeptide comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the hCMV UL128 polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the hCMV UL130 polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the hCMV UL131A polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the hCMV gB polypeptide comprises the amino acid sequence of SEQ ID NO: 15.
  • the mRNA components of a hCMV immunogenic composition are present in equal masses. In other embodiments, the mRNA components of a hCMV immunogenic composition (e.g., mRNA vaccine) are not present in equal masses.
  • the mRNA comprises a nucleotide sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the mRNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the lipid nanoparticle comprises (a) an mRNA polynucleotide comprising an open reading frame encoding an hCMV gH polypeptide (e.g., an mRNA comprising SEQ ID. NOs: 5 and/or 11); (b) an mRNA polynucleotide comprising an open reading frame encoding a hCMV gL polypeptide (e.g., an mRNA comprising SEQ ID. NOs: 6 and/or 12); (c) an mRNA polynucleotide comprising an open reading frame encoding an hCMV UL128 polypeptide (e.g., an mRNA comprising SEQ ID.
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV gH polypeptide e.g., an mRNA comprising SEQ ID. NOs: 5 and/or 11
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV UL130 polypeptide e.g., an mRNA comprising SEQ ID. NOs: 3 and/or 9
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV UL131A polypeptide e.g., an mRNA comprising SEQ ID. NOs: 4 and/or 10
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV gB polypeptide e.g., an mRNA comprising SEQ ID. NOs: 1 and/or 7).
  • the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f)
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about l:l:l:l:l.
  • the lipid nanoparticle comprises (a), (b), (c),
  • the molar ratio of (b):(c):(d):(e) is about 1:1: 1:1. In some embodiments where the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f), the molar ratio of (a):(f) is about 1:1. In certain embodiments where the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f), the molar ratio of each of (a) and (f) to one or more (e.g., all) of (b), (c), (d), and/or (e) is about 1.5:1 to 2:1. In some embodiments where the lipid nanoparticle comprises (a), (b), (c), (d),
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
  • the mRNA comprises a nucleotide sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the mRNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the mRNA comprises a nucleotide sequence having at least 90% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the lipid nanoparticle comprises (a) an mRNA polynucleotide comprising an open reading frame encoding an hCMV gH polypeptide (e.g., an mRNA comprising SEQ ID. NOs: 5 and/or 11); (b) an mRNA polynucleotide comprising an open reading frame encoding a hCMV gL polypeptide (e.g., an mRNA comprising SEQ ID.
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV UL128 polypeptide e.g., an mRNA comprising SEQ ID. NOs: 2 and/or 8
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV ULI 30 polypeptide e.g., an mRNA comprising SEQ ID. NOs: 3 and/or 9
  • an mRNA polynucleotide comprising an open reading frame encoding an hCMV UL131A polypeptide e.g., an mRNA comprising SEQ ID.
  • the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f), the molar ratio of (a):(b):(c):(d):(e):(f) is about l:l:l:l:l.
  • the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f)
  • the molar ratio of (b):(c):(d):(e) is about 1:1: 1:1.
  • the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f)
  • the molar ratio of (a):(f) is about 1:1.
  • the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f)
  • the molar ratio of each of (a) and (f) to one or more (e.g., all) of (b), (c), (d), and/or (e) is about 1.5:1 to 2:1.
  • the lipid nanoparticle comprises (a), (b), (c), (d), (e), and/or (f)
  • the molar ratio of (a):(b):(c):(d):(e):(f) is about 2:1:1:1:1:2.
  • mRNAs associated with hCMV immunogenic compositions described herein may further comprise a 5’ cap (e.g., 7mG(5’)ppp(5’)NlmpNp), a polyA tail (e.g., -100 nucleotides), or a 5’ cap and a polyA tail.
  • a 5’ cap e.g., 7mG(5’)ppp(5’)NlmpNp
  • a polyA tail e.g., -100 nucleotides
  • the hCMV immunogenic compositions may comprise a signal sequence.
  • the hCMV mRNA vaccines may include any 5’ untranslated region (UTR) and/or any 3’ UTR. Exemplary UTR sequences are provided in Table 3; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein. UTRs may also be omitted from the vaccine constructs provided herein.
  • the hCMV immunogenic compositions e.g., mRNA vaccines
  • molar ratios are used to determine the amounts of each mRNA component
  • the hCMV immunogenic compositions may have increased stability relative to hCMV immunogenic compositions (e.g., mRNA vaccines) in which the mRNA components are present in equal masses. This increased stability can help to ensure that the hCMV immunogenic compositions are stable throughout the shelf-life of a drug product containing these compositions and are still sufficiently efficacious for administration to a subject until the specified expiry date of the drug product.
  • Stability of mRNA constructs can be measured by any means known to one of ordinary skill in the art.
  • stability of mRNA constructs is calculated based on measuring degradation and/or purity of the mRNA construct. Longer mRNA constructs within hCMV immunogenic compositions described herein, such as gH and gB are expected to degrade faster than the shorter mRNA constructs within the same hCMV immunogenic compositions. Accordingly, in some embodiments, stability of hCMV immunogenic compositions described herein is measured by measuring the degradation and/or purity of gH and/or gB.
  • RNA refers to the amount of full-length intact mRNA (e.g., mRNA encoding gB) relative to the total input of the mRNA (e.g., mRNA encoding gB) on mass basis.
  • the RNA e.g., mRNA
  • the RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to any nucleotide sequence disclosed herein.
  • the RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to any one of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • the RNA comprises an ORF that comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12.
  • identity refers to a relationship between the sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g.. “algorithms”). Identity of related antigens or nucleic acids can be readily calculated by known methods.
  • Percent (%) identity as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSLBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402).
  • Another popular local alignment technique is based on the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197).
  • a general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453).
  • FGSAA Fast Optimal Global Sequence Alignment Algorithm
  • Sequence tags can be used for peptide detection, purification or localization.
  • Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminal or N-terminal residues may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support.
  • sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function.
  • cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids.
  • buried hydrogen bond networks may be replaced with hydrophobic residues to improve stability.
  • glycosylation sites may be removed and replaced with appropriate residues.
  • sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an RNA (e.g., mRNA) vaccine.
  • sequence tags or terminal peptide sequences e.g., at the N-terminal or C-terminal ends
  • RNA e.g., mRNA
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of antigens of interest.
  • any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical
  • the fragment is immunogenic and confers a protective immune response to the human cytomegalovirus (hCMV).
  • hCMV human cytomegalovirus
  • an antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein.
  • Antigens/antigenic polypeptides can range in length from about 4, 6, or 8 amino acids to full length proteins.
  • the methods comprise obtaining lyophilized compositions with a moisture content below a certain value.
  • moisture content refers to the percentage (by weight) of a composition that is comprised of water.
  • Methods of measuring moisture content of a lyophilized composition include loss-on-drying (LOD) analysis, thermogravimetric (TGA) analysis, and Karl Fischer titration (see, e.g., FDA, Docket No. 89D- 0140, Guideline for the Determination of Residual Moisture in Dried Biological Products (Center for Biologies Evaluation and Research, January 1990)).
  • the lyophilized composition has a moisture content of 6.0% w/w or less, 5.0% w/w or less, 4.0% w/w or less, 3.5% w/w or less, 3.0% w/w or less, 2.5% w/w or less, 2.0% w/w or less, 1.5% w/w or less, 1% w/w or less, 0.75% w/w or less, 0.5% w/w or less, or 0.25% w/w or less. In some embodiments, the lyophilized composition has a moisture content of 3.0% w/w or less.
  • the methods comprise obtaining lyophilized compositions in which the coefficient of degradation of a nucleic acid in the composition is below a predetermined value.
  • a “coefficient of degradation” refers to a parameter of an equation describing the loss of nucleic acid purity over time.
  • nucleic acid purity refers to the percentage of nucleic acid in a composition having a desired sequence and structure. Compositions are prepared using nucleic acids having a specific sequence encoding a protein to be expressed in cells. During the course of lyophilization and/or storage after lyophilization, the nucleic acid may be degraded by environmental factors such as water or nucleases.
  • Nucleases are enzymes that can facilitate this process, but nucleic acids are susceptible to degradation by water molecules even in the absence of environmental nucleases. Nucleic acid purity may be measured by any one of multiple methods known in the art, such as mass spectrometry or high- performance liquid chromatography (HPLC) (see, e.g., Papadoyannis et al. J Liq Chrom Relat Tech. 2007.
  • a sample to be analyzed such as nucleic acid
  • a solvent mobile phase
  • a column containing a solid material stationary phase
  • the rate at which molecules of the sample move through the stationary phase depends on multiple factors, including size, such that different components of the sample will be observed at different times.
  • a sample containing 100% pure nucleic acid will produce a single peak (main peak) on a chromatogram when analyzed by HPLC, while a sample containing multiple different nucleic acid molecules will produce multiple peaks, including a main peak and one or more impurity peaks, for a total of N peaks.
  • the area under the curve (A.U.C.) of each of N peaks is calculated by integration, and the percent purity is calculated using the
  • a positive value of indicates exponential decay, while a negative indicates exponential growth, with larger absolute values of indicating faster decay or growth, respectively.
  • the coefficient of degradation is expressed in units of month 1 .
  • the nucleic acid of the lyophilized composition has a coefficient of degradation at 5 °C of 0.05 month 1 or less, 0.04 month 1 or less, 0.03 month 1 or less, or 0.02 month 1 or less.
  • the coefficient of degradation is 0.02 month 1 or less.
  • the coefficient of degradation is 0.01 month 1 or less.
  • the coefficient of degradation is 0.01 month 1 or less, 0.008 month 1 or less, 0.006 month 1 or less, or 0.004 month 1 or less.
  • the coefficient of degradation is 0.004 month 1 or less.
  • the nucleic acid in a lyophilized LNP degrades (e.g., as measured by capillary electrophoresis) about 2% or less per month during storage, such as about 1% or less, about 0.75% or less, aboutO .5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less per month during storage (e.g., at 5°C).
  • the methods comprise obtaining lyophilized compositions in which the nucleic acid in the lyophilized composition is at least 50% pure (such as about 50% pure, about 55% pure, about 60% pure, about 65% pure, about 70% pure, or about 75% pure or more) after storage at 0°C or more (such as 0 °C, 2 °C, 5 °C, 8 °C, 10 °C, 15 °C, 20 °C, 25 °C, or 2-8 °C) for a given length of time.
  • the nucleic acid in the lyophilized composition is at least 50% pure (such as about 50% pure, about 55% pure, about 60% pure, about 65% pure, about 70% pure, or about 75% pure or more) after at least 12 months, at least 18 months, at least 21 months, at least 24 months, at least 27 months, at least 30 months, at least 33 months, or at least 36 months of storage.
  • the nucleic acid in the lyophilized composition is at least 50% pure (such as about 50% pure, about 55% pure, about 60% pure, about 65% pure, about 70% pure, or about 75% pure or more) after least 24 months of storage.
  • the storage is conducted at a temperature between about 2 °C and about 8 °C. In some embodiments, the storage is conducted at about 5 °C. Degradation of nucleic acids is a chemical reaction that occurs more readily at higher temperatures, and as such the coefficient of degradation depends on the temperature at which nucleic acids are stored.
  • substantially no cholesterol crystals can be detected in the formulation following 6 months or more of storage (such as after 6 months, 9 months, 12 months, 18 months, or 24 months or more of storage).
  • compositions relate to a lyophilized composition produced by any of the methods provided herein.
  • the composition is in a solid form.
  • compositions comprising an mRNA, a lyoprotectant and a lipid nanoparticle, wherein the lipid nanoparticle comprises ionizable amino lipid; non-cationic lipid; sterol; and PEG-modified lipid, wherein the composition has a moisture content of less than or equal to 6.0% w/w.
  • Some aspects of the disclosure relate to lyophilized pharmaceutical compositions comprising an mRNA, a lyoprotectant and a lipid nanoparticle, wherein the lipid nanoparticle comprises ionizable amino lipid; non-cationic lipid; sterol; and PEG-modified lipid, wherein the ratio of lyoprotectant to lipid molecules is about 8:1 to about 40:1.
  • compositions comprising an mRNA, a lyoprotectant and a lipid nanoparticle, wherein the lipid nanoparticle comprises ionizable amino lipid; non-cationic lipid; sterol; and PEG-modified lipid, wherein the PEG-modified lipid is a C18 stearic-OH -PEG, and wherein the composition is stable for at least about 9 months upon storage at about 4°C.
  • the composition has a moisture content of less than or equal to 6.0% w/w.
  • the PEG-modified lipid is a C18 stearic-OH -PEG.
  • the composition is stable for at least about 9 months upon storage at about 4°C.
  • the ratio of lyoprotectant to lipid molecules is about 8:1 to about 40:1. In some embodiments, the ratio of lyoprotectant to lipid molecules is about 8:1 to about 20:1. In some embodiments, the ratio of lyoprotectant to lipid molecules is about 8:1, about 9:1, about 10:1, about 11:1, or about 12:1. In some embodiments, the ratio of sugar molecules to lipid molecules is at least 8:1.
  • the PEG-modified lipid is:
  • the ionizable amino lipid is:
  • the lyophilized composition does not comprise protamine.
  • Previous formulations for RNA vaccine delivery included protamine complexed with RNA, and taught that such complexation of RNA with protamine was necessary to achieve sustained protective immune responses observed in experiments. See, e.g., Petsch et al., Nat Biotechnol. 2012. 30(12): 1210-1218.
  • some embodiments of lyophilized compositions comprising a lipid nanoparticle and mRNA are capable of eliciting an immune response to encoded proteins without the use of protamine for mRNA delivery, as shown in the Examples.
  • lyophilized pharmaceutical compositions and/or compositions made by lyophilization methods comprise low moisture contents.
  • reduced availability of water molecules in a lyophilized composition reduce the frequency of hydrolysis of nucleic acids (e.g., mRNAs) in a lyophilized composition, thereby improving stability of the nucleic acids in the composition, such as during extended storage over several months.
  • Moisture contents may be measured by any method known in the art, such as Karl Fischer (KF) titration, thermogravimetry (TG), and/or gas chromatography. See, e.g., Roggo et al., J Pharm Biomed Anal. 2007.
  • the composition has a moisture content of less than or equal to 6.0% w/w. In some embodiments, the composition has a moisture content of less than or equal to 5.0% w/w. In some embodiments, the composition has a moisture content of less than or equal to 4.0% w/w. In some embodiments, the composition has a moisture content of less than or equal to 3.5% w/w. In some embodiments, the composition has a moisture content of less than or equal to 3.0% w/w.
  • the composition has a moisture content of less than or equal to 2.5% w/w. In some embodiments, the composition has a moisture content of less than or equal to 2.0% w/w. In some embodiments, the composition has a moisture content of less than or equal to 1.0% w/w. In some embodiments, the composition has a moisture content of less than or equal to 0.5% w/w. In some embodiments, the composition has a moisture content between 0.01% and 6.0% w/w, between 0.01% and 5.0% w/w, between 0.01% and 4.0% w/w, between 0.01% and 3.0% w/w, between 0.01% and 2.0% w/w, or between 0.01% and 1.0% w/w.
  • the composition has a moisture content between 0.01% w/w and 2.0% w/w. In some embodiments, the composition has a moisture content between 0.01% w/w and 1.0% w/w. In some embodiments, the composition has a moisture content between 0.01% w/w and 0.5% w/w. In some embodiments, moisture content is measured within 1 month after the end of the desorption step. In some embodiments, moisture content refers to the amount of moisture in a lyophilized composition after 1 or more (e.g., 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, or more) months of storage.
  • 1 or more e.g., 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, or more
  • the lipid nanoparticle upon reconstitution has a diameter (or a composition has a mean lipid particle diameter) of 200 nm or less, such as 180 nm or less, 175 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, or 20 nm or less.
  • the lipid nanoparticle upon reconstitution the lipid nanoparticle (or a composition has a mean lipid particle diameter) has a diameter of at most 30 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 50 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 75 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 100 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 120 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 140 nm.
  • the reconstituted lipid nanoparticle has a diameter of at most 160 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 180 nm. In some embodiments, the reconstituted lipid nanoparticle has a diameter of at most 200 nm. In some embodiments, the lipid nanoparticle has a diameter from 5-200 nm, 5-180 nm, 5-160 nm, 5-150 nm, 5-140 nm, 5-120 nm, 5-100 nm, 5-80 nm, 5-60 nm, 5-40 nm, 5-30 nm, or 5-20 nm.
  • the reconstitituted lipid nanoparticle has a diameter from 40-200 nm, 40-180 nm, 40-160 nm, 40-150 nm, 40-140 nm, 40-120 nm, 40-100 nm, 40-80 nm, or 40-60 nm. In some embodiments, the reconstitituted lipid nanoparticle has a diameter from 80-200 nm, 80-180 nm, 80-160 nm, 80-150 nm, 80-140 nm, 80-120 nm, or 80-100 nm.
  • the reconstitituted lipid nanoparticle has a diameter from 120-200 nm, 120-180 nm, 120-160 nm, 120-150 nm, or 120-140 nm.
  • the reconstituted lipid nanoparticle has a dimater from 5-200 nm, 10-200 nm, 20-200 nm, 30- 200 nm, 40-200 nm, 50-200 nm, 60-200 nm, 70-200 nm, 80-200 nm, 90-200 nm, 100-200 nm, 110-200 nm, 120-200 nm, 130-200 nm, 140-200 nm, 150-200 nm, 160-200 nm, 170-200 nm, 180-200 nm, or 190-200 nm.
  • the lyophilization increases the lipid nanoparticle size (or a mean lipid particle diameter) (e.g., as determined by DLS) by about 30 nm or less, such as about 25 nm or less, about 20 nm or less, about 15 nm or less, about 10 nm or less, or about 5 nm or less. In some embodiments, the lyophilization does not measurably increase the lipid nanoparticle size (or mean lipid particle diameter).
  • a coefficient of degradation at 5 °C of the mRNA in the lyophilized composition is at most 0.05 month 1 , at most 0.04 month 1 , at most 0.03 month 1 , at most 0.02 month 1 , or at most 0.01 month 1 .
  • the coefficient of degradation is at most 0.02 month 1 .
  • the coefficient of degradation is at most 0.01 month 1 .
  • the coefficient of degradation is between 0.0001 and 0.05 month’ x , 0.0001 and 0.04 month’ 1 , 0.0001 and 0.03 month’ 1 , 0.001 and 0.02 month’ 1 , or 0.001 and 0.01 month’ 1 .
  • the coefficient of degradation is between 0.0001 and 0.02 month’ x .
  • the coefficient of degradation is between 0.001 and 0.01 month’ 1 .
  • the mRNA in the lyophilized composition is at least 50% pure after at least 12 months, at least 18 months, at least 21 months, at least 24 months, at least 27 months, at least 30 months, at least 33 months, or at least 36 months of storage. In some embodiments, the mRNA in the lyophilized composition is at least 50% pure after 12 to 120 months, 12 to 108 months, 12 to 96 months, 12 to 84 months, 12 to 72 months, 12 to 60 months, 12 to 54 months, 12 to 48 months, 12 to 42 months, 12 to 36 months, 12 to 30 months, 12 to 24 months, or 12 to 18 months of storage at a temperature between about 2 °C and about 8 °C. In some embodiments, the storage is conducted at a temperature of about 4 °C. In some embodiments, the storage is conducted at about 5 °C.
  • the mRNA in the lyophilized composition is at least 50% pure after at least 24 months of storage. In some embodiments, the mRNA in the lyophilized composition is at least 50% pure after 24 to 120 months, 24 to 108 months, 24 to 96 months, 24 to 84 months, 24 to 72 months, 24 to 60 months, 24 to 54 months, 24 to 48 months, 24 to 42 months, 24 to 36 months, or 24 to 30 months of storage at a temperature between about 2 °C and about 8 °C. In some embodiments, the storage is conducted at a temperature of about 4 °C. In some embodiments, the storage is conducted at a temperature between about 2 °C and about 8 °C. In some embodiments, the storage is conducted at a temperature of about 4 °C. In some embodiments, the storage is conducted at about 5 °C.
  • the moisture content of a lyophilized composition remains below 6.0% (w/w), 5.0%, 4.0%, 3.0%, 2.0%, 1.5%, 1.0%, or 0.5% after 3-12 months of storage.
  • the moisture content of a composition after 12 months of storage at 2-8 °C is less than or equal to 6.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 5.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 4.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 3.5% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 3.0% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 2.5% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 2.0% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 2.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 1.5% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 1.0%. In some embodiments, the moisture content of a composition after 3-12 months of storage at 2-8 °C is less than or equal to 0.5%. In some embodiments, the moisture content of a lyophilized composition is less than or equal to 2.0% w/w after 3 or more, 4 or more, 5 or more, 6 or more, 9 or more, or 12 or more months of storage at 2-8 °C.
  • the moisture content of a lyophilized composition remains below 6.0% w/w after 3-12 months of storage at a given temperature and/or relative humidity.
  • the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 6.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 5.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 4.0% w/w.
  • the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 3.5% w/w.
  • the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 3.0% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 2.5% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 2.0% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 2.0% w/w. In some embodiments, the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 1.5% w/w.
  • the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 1.0%. In some embodiments, the moisture content of a composition after 3-12 months of storage at 20-30 °C is less than or equal to 0.5%. In some embodiments, the moisture content of a lyophilized composition is less than or equal to 2.0% w/w after 3 or more, 4 or more, 5 or more, 6 or more, 9 or more, or 12 or more months of storage at 20-30 °C. In some embodiments, the storage is conducted at 50- 100%, 60-100%, 70-100%, 75%-100%, 80-100%, 90-100% or 95-100% relative humidity.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 6 months of storage.
  • the storage is conducted at a temperature between 2-8 °C.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 9 months of storage.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 12 months of storage.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 24 months of storage.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 36 months of storage.
  • the storage is conducted at a temperature between 10-20 °C. In some embodiments, the storage is conducted at a temperature between 20-30 °C. In some embodiments, the storage is conducted at a temperature between 40-50 °C. In some embodiments, the storage is conducted at a temperature between 50-60 °C.
  • the storage is conducted for 3-120 months, 3-96 months, 3-72 months, 3-60 months. 3-48 months, 3-36 months, 3-24 months, 3-12 months, 12-120 months, 12-96 months, 12-72 months, 12-60 months, 12-48 months, 12-36 months, 12-24 months, 24-120 months, 24-96 months, 24-72 months, or 24-48 months. In some embodiments, the storage is conducted at 50-100%, 60-100%, 70-100%, 75%-100%, 80- 100%, 90-100% or 95-100% relative humidity.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 3, 6, 9, 12, 18, 24, 36, 48, 60, or more months of storage above 30 °C from about 40 °C to about 50 °C). In some embodiments, the lyophilized compositions comprise mRNA with at least 96% relative purity after 1 month of storage between 30-60 °C.
  • the lyophilized compositions comprise mRNA with at least 93% relative purity after 2 months of storage between 30-60 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 90% relative purity after 3 months of storage between 30-60 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 85% relative purity after 4 months of storage between 30-60 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 85% relative purity after 5 months of storage between 30-60 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 6 months of storage between 30-60 °C.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 6 months of storage at a relative humidity above 50% (e.g., 50-100%, 50-75%, 50-80%, 80-100%, or 60-75%). In some embodiments, the lyophilized compositions comprise mRNA with at least 93% relative purity after 3 months of storage at relative humidity between 70-100%.
  • the lyophilized compositions comprise mRNA with at least 87% relative purity after 6 months of storage at relative humidity between 70-100%. In some embodiments, the lyophilized compositions comprise mRNA with at least 85% relative purity after 9 months of storage at relative humidity between 70-100%. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 12 months of storage at relative humidity between 70-100%. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 24 months of storage at relative humidity between 70-100%. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 36 months of storage at relative humidity between 70-100%.
  • the lyophilized compositions comprise mRNA with at least 80% relative purity after 48 months of storage at relative humidity between 70-100%. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 60 months of storage at relative humidity between 70-100%.
  • the lyophilized compositions comprise mRNA with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 3, 6, 9, 12, 18, 24, 36, 48, 60, or more months of storage between 2-8 °C.
  • the lyophilized compositions comprise mRNA with at least 90% relative purity after 12 months of storage between 2-8 °C.
  • the lyophilized compositions comprise mRNA with at least 90% relative purity after 24 months of storage between 2-8°C.
  • the lyophilized compositions comprise mRNA with at least 90% relative purity after 36 months of storage between 2-8 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 90% relative purity after 48 months of storage between 2-8 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 90% relative purity after 60 months of storage between 2-8 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 90% relative purity after 72, 96, 108, 120, or more, months of storage between 2-8 °C.
  • the lyophilized compositions comprise mRNA with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99% relative purity, compared to the purity of mRNA immediately after the conclusion of lyophilization, after 3, 6, 9, 12, 18, 24, 36, 48, 60, or more months of storage between 20-30 °C.
  • the lyophilized compositions comprise mRNA with at least 80% relative purity after 12 months of storage between 20-30 °C.
  • the lyophilized compositions comprise mRNA with at least 80% relative purity after 24 months of storage between 20-30 °C.
  • the lyophilized compositions comprise mRNA with at least 80% relative purity after 36 months of storage between 20-30 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 48 months of storage between 20-30 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 60 months of storage between 20-30 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 72, 96, 108, 120, or more, months of storage between 20-30 °C.
  • lipid nanoparticles of the lyophilized compositions comprise lipid nanoparticles comprising mRNA with an encapsulation efficiency of 70%.
  • encapsulation efficiency refers to the percentage of nucleic acid (e.g., mRNA) in a composition that is comprised within lipid nanoparticles. mRNA encapsulated within a lipid nanoparticle is not exposed to the environment outside the lipid nanoparticle, and is thus protected from the action of environmental factors such as nucleases, which can cleave free mRNA. Encapsulation efficiency can be measured by any one of multiple methods known in the art, such as a RiboGreen assay.
  • RiboGreen is a fluorescent dye that is fluorescent only when bound to a nucleic acid.
  • a RiboGreen assay one sample of a composition containing mRNA in lipid nanoparticles is subjected to a treatment that disrupts lipid nanoparticle integrity to release encapsulated mRNA, such as exposure to a detergent, while another sample is not disrupted.
  • RiboGreen is then added to both samples, and the fluorescence emitted from both samples is measured.
  • Encapsulation efficiency E.E.
  • the encapsulation efficiency is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100%.
  • RNA encapsulation decreases by about 10% or less, such as about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less immediately after lyophilization and/or after storage following lyophilization. In some embodiments, the amount of encapsulated RNA does not substantially decrease during lyophilization and/or storage of a lyophilized LNP.
  • the lyophilized compositions comprise mRNA with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100%, or greater than 100% in vitro potency, relative to the in vitro potency of the mRNA that was present in the composition prior to lyophilization.
  • zn vitro potency of an mRNA refers to the capacity of an mRNA to be translated into a protein encoded by the mRNA during an in vitro translation reaction.
  • in vitro translation an mRNA encoding a protein is incubated in the presence of ribosomes and aminoacyl-tRNAs, which allow the encoded protein to be produced in the absence of cells.
  • the in vitro potency of a first mRNA relative to a second mRNA encoding the same protein may be calculated by comparing the amount of protein produced by each mRNA in separate in vitro translation reactions, using the equation
  • Some embodiments comprise reconstituting a lyophilized lipid nanoparticle composition, e.g., in a reconstitution buffer suitable for pharmaceutical administration.
  • the lyophilized lipid nanoparticle composition is reconstituted in water.
  • the lyophilized lipid nanoparticle composition is reconstituted in a salt solution (e.g., a sodium chloride solution), such as an about 0.5%, about 0.9%, about 1%, about 1.5%, about 2%, about
  • the reconstitution buffer is at a physiological pH (e.g, pH 7-7.5, such as 7.4).
  • a physiological pH e.g, pH 7-7.5, such as 7.4
  • Some embodiments comprise a reconstituted lipid nanoparticle composition.
  • the lyophilized composition is reconstituted (e.g., via agitation, swirling, shaking, and/or repeated pipette aspirating and dispensing) at a temperature of from about 1°C to about 75 °C, such as about 4°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C 4 about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or about 75°C.
  • the disclosure relates to lyophilized compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention and/or treatment of hCMV infection in humans and other mammals.
  • the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • the lyophilized hCMV immunogenic compositions e.g., mRNA vaccines
  • the lyophilized hCMV immunogenic compositions e.g., mRNA vaccines
  • the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.
  • PBMCs peripheral blood mononuclear cells
  • a subject may be any mammal, including non-human primate and human subjects.
  • a subject is a human subject.
  • the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen- specific immune response.
  • a subject e.g., a mammalian subject, such as a human subject
  • the RNA encoding the hCMV antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.
  • the subject may be hCMV seropositive (e.g., has previously had a natural hCMV infection) or hCMV seronegative (e.g., has not previously had a natural hCMV infection) prior of being administered the hCMV mRNA vaccine.
  • Prophylactic protection from hCMV can be achieved following administration of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine).
  • Vaccines can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by one or more boosters). It is possible, although less desirable, to administer the vaccine to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.
  • a method of eliciting an immune response in a subject against hCMV involves administering to the subject a lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) described herein, thereby inducing in the subject an immune response specific to a hCMV antigen (e.g., the hCMV gH, gL, UL128, UL130, UL131A and/or gB).
  • a hCMV antigen e.g., the hCMV gH, gL, UL128, UL130, UL131A and/or gB.
  • the immune response is the induction of neutralizing antibodies against a hCMV antigen (e.g., the hCMV gH, gL, UL128, UL130, UL131A and/or gB).
  • the anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the hCMV.
  • An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen.
  • a prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level.
  • the effective dose is a dose listed in a package insert for the vaccine.
  • an effective dose is sufficient to produce detectable levels of hCMV antigen (e.g., gH, gL, UL128, UL130, UL131A and/or gB polypeptide) as measured in serum of the subject administered the hCMV immunogenic composition (e.g., mRNA vaccine) at 1-72 hours (e.g., 1-72 hours, 1-60 hours, 1- 45 hours, 1-30 hours, 1-15 hours, 15-72 hours, 15-60 hours, 15-45 hours, 15-30 hours, 30-72 hours, 30-60 hours, 30-45 hours, 45-72 hours, 45-60 hours, or 60-72 hours) post administration.
  • 1-72 hours e.g., 1-72 hours, 1-60 hours, 1- 45 hours, 1-30 hours, 1-15 hours, 15-72 hours, 15-60 hours, 15-45 hours, 15-30 hours
  • the effective dose is sufficient to produce neutralization titer produced by neutralizing antibody against the hCMV antigen (e.g., gH, gL, UL128, UL130, UL131A and/or gB polypeptide) as measured in serum of the subject administered the hCMV immunogenic composition (e.g., mRNA vaccine) at 1-72 hours (e.g., 1-72 hours, 1-60 hours, 1-45 hours, 1-30 hours, 1-15 hours, 15-72 hours, 15-60 hours, 15-45 hours, 15-30 hours, 30-72 hours, 30-60 hours, 30-45 hours, 45-72 hours, 45-60 hours, or 60-72 hours) post administration.
  • the hCMV antigen e.g., g., gH, gL, UL128, UL130, UL131A and/or gB polypeptide
  • 1-72 hours e.g., 1-72 hours, 1-60 hours, 1-45 hours, 1-30 hours, 1-15 hours, 15-72 hours, 15-60 hours
  • a traditional vaccine refers to a vaccine other than the mRNA vaccines described herein.
  • a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc.
  • a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).
  • FDA Food and Drug Administration
  • EMA European Medicines Agency
  • the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the hCMV or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the hCMV or an unvaccinated subject.
  • a method of eliciting an immune response in a subject against hCMV involves administering to the subject the hCMV immunogenic composition (e.g., mRNA vaccine) described herein, thereby inducing in the subject an immune response specific to hCMV antigen, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the hCMV at 2 times to 100 times the dosage level relative to the RNA vaccine.
  • the hCMV immunogenic composition e.g., mRNA vaccine
  • the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine).
  • the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine). In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine).
  • the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine).
  • a traditional vaccine at 100 times to 1000 times the dosage level relative to the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine).
  • the immune response is assessed by determining [protein] antibody titer in the subject.
  • the ability of serum or antibody from an immunized subject is tested for its ability to neutralize viral uptake or reduce hCMV transformation of human B lymphocytes.
  • the ability to promote a robust T cell response(s) is measured using art recognized techniques.
  • the disclosure provide methods of eliciting an immune response in a subject against hCMV by administering to the subject the hCMV mRNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one hCMV antigen, thereby inducing in the subject an immune response specific to hCMV antigen, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against hCMV.
  • the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to the RNA vaccine.
  • the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
  • the lyophilized hCMV immunogenic composition may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. Some aspects of the disclosure relate to methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • the lyophilized hCMV immunogenic composition is typically in dosage unit form for ease of administration and uniformity of dosage.
  • the total daily usage of the lyophilized hCMV immunogenic composition may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is administered at a dose of about 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 21 pg, 22 pg, 23
  • the dose of the lyophilized hCMV immunogenic composition refers to total pg mRNA in lipid nanoparticles.
  • Total pg mRNA refers to total dose, or the nominal dose, for a single administration with the understanding that RNA impurities, degraded mRNA, and otherwise inactive mRNA are still counted in the total. The weight of the lipid components is not included when referring to dose.
  • the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is administered at a dose of about 50-150 pg. In some embodiments, only one dose is administered, while in other embodiments, multiple doses (e.g., one, two, or three doses) are administered. In embodiments wherein multiple doses are administered, the dose between the first dose and a subsequent dose can be the same or different.
  • the effective amount of the lyophilized hCMV immunogenic composition may be as low as 150 pg, administered for example as a single dose.
  • the effective amount of lyophilized hCMV immunogenic composition is a single dose of 50-150 pg.
  • the effective amount of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) may be a single dose of 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81
  • the effective amount of the lyophilized hCMV immunogenic composition is a single dose of 50 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 100 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 150 pg.
  • the effective amount of the lyophilized hCMV immunogenic composition is either 50 pg, 100 pg or 150 pg.
  • the effective dose is administered as a primary immunization followed by a single boost of the same effective dose.
  • the effective dose is administered as a primary immunization followed by two sequential booster immunizations of the same effective dose.
  • the effective dose is 50 pg of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) and is administered as a primary immunization of 50 pg followed by two sequential booster immunizations of 50 pg.
  • the effective dose is 100 pg lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) and is administered as a primary immunization of 100 pg followed by two sequential booster immunizations of 100 pg.
  • the effective dose is 150 pg of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) and is administered as a primary immunization of 150 pg followed by two sequential booster immunizations of 150 pg.
  • the booster immunizations should be at least two weeks apart.
  • the effective amount of lyophilized hCMV immunogenic composition is two doses of 50-150 pg.
  • the effective amount of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) may be two doses of 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg
  • the effective amount of the lyophilized hCMV immunogenic composition is two doses of 50 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 100 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 150 pg.
  • the effective amount of lyophilized hCMV immunogenic composition is three doses of 50-150 pg.
  • the effective amount of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) may be three doses of 50 pg, 51 pg, 52 pg, 53 pg, 54 pg, 55 pg, 56 pg, 57 pg, 58 pg, 59 pg, 60 pg, 61 pg, 62 pg, 63 pg, 64 pg, 65 pg, 66 pg, 67 pg, 68 pg, 69 pg, 70 pg, 71 pg, 72 pg, 73 pg, 74 pg, 75 pg, 76 pg, 77 pg, 78 pg, 79 pg, 80 pg, 81 pg
  • the effective amount of the lyophilized hCMV immunogenic composition is three doses of 50 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is three doses of 100 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is three doses of 150 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is more than 3 (e.g., 4, 5 or more) doses of 50 pg - 150 pg.
  • the effective amount of the lyophilized hCMV immunogenic composition is more than 3 (e.g., 4, 5 or more) doses of 50 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is more than 3 (e.g., 4, 5 or more) doses of 100 pg. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is more than 3 (e.g., 4, 5 or more) doses of 150 pg.
  • the effective amount of the lyophilized hCMV immunogenic composition relates to the amount of integral mRNA in the composition.
  • integral mRNA refers to intact mRNA transcripts that are capable of producing hCMV antigens and/or inducing an immune response against an antigen in a subject.
  • the amount of integral mRNA in a lyophilized hCMV immunogenic composition is related to the length, rate of degradation, and the length of time from which the immunogenic composition is produced.
  • that dose can be referred to in terms of the total mRNA present (i.e. the total dose) or in terms of the integral mRNA present in the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine).
  • the effective amount of the lyophilized hCMV immunogenic composition is a single dose of 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5- 15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) pentamer components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10- 30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) gB mRNA.
  • 5-35 pmol e.g., 5-35, 5-30, 5-25, 5-20, 5- 15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol
  • 4-50 pmol e.g., 4-50
  • the effective amount of the lyophilized hCMV immunogenic composition is a single dose of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) pentamer components and 15- 45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) gB mRNA.
  • the effective amount of the lyophilized hCMV immunogenic composition is a single dose of 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 pmol (including all values in between) pentamer components and 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 pmol (including all values in between) gB mRNA.
  • an effective amount or dose of the lyophilized hCMV immunogenic composition does not require that the pmoles of each component are equal.
  • a lyophilized hCMV immunogenic composition e.g. mRNA vaccine
  • larger picomolar doses of the mRNAs encoding the integral transmembrane domain containing components such as gH and gB may be required to ensure that these components do not become limiting.
  • the picomolar dose of each of the 6 mRNA may be individually determined because of stability or other biochemical or biophysical requirements.
  • the effective amount of the lyophilized hCMV immunogenic composition is two doses of 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) pentamer components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-30, 10- 20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) gB mRNA.
  • 5-35 pmol e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol
  • 4-50 pmol e.g., 4
  • the effective amount of the lyophilized hCMV immunogenic composition is two doses of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) pentamer components and 15-45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) integral gB mRNA.
  • the effective amount of the lyophilized hCMV immunogenic composition is two doses of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • the effective amount of the lyophilized hCMV immunogenic composition is three doses of 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5- 15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) pentamer components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-
  • the effective amount of the lyophilized hCMV immunogenic composition is three doses of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) pentamer components and 15-45 pmol (e.g., 15-45, 15-30, or 30-45 pmol) integral gB mRNA.
  • the effective amount of the lyophilized hCMV immunogenic composition is three doses of 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 pmol (including all values in between) pentamer components and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
  • the effective amount of the lyophilized hCMV immunogenic composition is more than three (e.g., 4, 5, or more) doses of 5-35 pmol (e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35 pmol) pentamer components and 4-50 pmol (e.g., 4-50, 10-50, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) of integral gB mRNA.
  • 5-35 pmol e.g., 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 25-35, 25-30, or 30-35
  • the effective amount of the lyophilized hCMV immunogenic composition is more than three (e.g., 4, 5, or more) doses of 10-30 pmol (e.g., 10-30, 10-20, or 20-30 pmol) pentamer components and 15-45 pmol (e.g., 15- 45, 15-30, or 30-45 pmol) integral gB mRNA.
  • the effective amount of the lyophilized hCMV immunogenic composition is more than three (e.g., 4, 5, or more) doses of 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 pmol (including all values in between) pentamer components and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
  • the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) contains mRNAs at a gB:gH:gL:UL128:UL130:UL131 A molar ratio of 2:2: 1 : 1 : 1 : 1.
  • one, two, three, or more than three doses (of any of the doses described herein) of the lyophilized hCMV immunogenic composition are administered to a subject.
  • one, two, or three doses (of any of the doses described herein) of the lyophilized hCMV immunogenic composition are administered to a subject.
  • the doses are administered on day 1, around the beginning of month 2 (e.g., day 29), and around the beginning of month 6 (e.g., day 169).
  • a dose of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is administered to a subject on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day
  • day 33 day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day
  • day 45 day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 53, day 54, day 55, day
  • day 69 day 70, day 71, day 72, day 73, day 74, day 75, day 76, day 77, day 78, day 79, day
  • hCMV mRNA vaccines described herein can be in a suitable dosage form, such as a form described herein or known in the art, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous.
  • compositions of the hCMV mRNA vaccine wherein the hCMV mRNA vaccine is in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to an anti- hCMV antigen).
  • an effective amount is a dose of the hCMV mRNA vaccine effective to produce an antigenspecific immune response.
  • methods of inducing an antigen- specific immune response in a subject are also provided herein.
  • an immune response to a vaccine or LNP is the development in a subject of a humoral and/or a cellular immune response to a (one or more) hCMV protein(s) present in the vaccine.
  • a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
  • T-lymphocytes e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
  • CTLs cytolytic T-cells
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes.
  • MHC major histocompatibility complex
  • Another aspect of cellular immunity involves an antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
  • the antigen- specific immune response is characterized by measuring an anti-hCMV antigen antibody titer produced in a subject administered the hCMV mRNA vaccine as provided herein.
  • An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-hCMV antigen) or epitope of an antigen.
  • Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result.
  • Enzyme-linked immunosorbent assay is a common assay for determining antibody titers, for example.
  • an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. An antibody titer may be used to determine the strength of an immune response induced in a subject by the hCMV mRNA vaccine.
  • an anti-hCMV antigen antibody titer produced in a subject is increased by at least 1 log relative to a control.
  • anti-hCMV antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, at least 3 log, at least 4 log, or at least 5 log, or more, relative to a control.
  • the anti-hCMV antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 log relative to a control.
  • the anti-hCMV antigen antibody titer produced in the subject is increased by 1-5 log relative to a control.
  • the anti-hCMV antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1-4, 1-5, 1.5-2, 1.5-2.5, 1.5-3, 1.5-4, 1.5-5, 2-2.5, 2-3, 2-4, 2-5, 2.5-3, 2.5-4, 2.5-5, 3-4. 3-5, or 4-5 log relative to a control.
  • the anti-hCMV antigen antibody titer produced in a subject is increased at least 2 times relative to a control.
  • the anti-hCMV antigen antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control.
  • the anti-hCMV antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control.
  • the anti-hCMV antigen antibody titer produced in a subject is increased 2-10 times relative to a control.
  • the anti-hCMV antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.
  • an antigen-specific immune response is measured as a ratio of geometric mean titer (GMT), referred to as a geometric mean ratio (GMR), of serum neutralizing antibody titers to hCMV.
  • GTT geometric mean titer
  • a geometric mean titer (GMT) is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of the number, where n is the number of subjects with available data.
  • administration of an effective amount of the lyophilized hCMV immunogenic composition e.g., mRNA vaccine elicits serum neutralizing antibody titers against hCMV.
  • administration a single dose e.g., any of the doses described herein, or multiple doses, of the hCMV immunogenic composition (e.g., mRNA vaccine) elicits serum neutralizing antibody titers against hCMV.
  • an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) described herein is sufficient to produce geometric mean titer (GMT) of serum neutralizing anti-CMV antibodies against epithelial cell hCMV infection on day 1, day 29, day 56, day 84, day 168, or day 196 after immunization, and associated GMR of post- baseline/baseline titers.
  • GMT geometric mean titer
  • an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) described herein is sufficient to produce serum neutralizing antibody titers against fibroblast hCMV infection on day 1, day 29, day 56, day 84, day 168, or day 196 after immunization, and GMR of post-baseline/baseline titers.
  • the GMT of serum neutralizing antibodies to hCMV increases in the subject administered the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) by at least 2-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold), relative to baseline.
  • the GMT of serum neutralizing antibodies to hCMV increases in the subject by 2-fold to 10-fold after administering a single dose (e.g., a single dose of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of serum neutralizing antibodies to hCMV increases in the subject by 2-fold to 10-fold after administering two doses (e.g., two doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of serum neutralizing antibodies to hCMV increases in the subject by 2-fold to 10-fold after administering three doses (e.g., three doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • neutralizing antibody (nAb) GMTs against epithelial cell infection are increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11- fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22- fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33- fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44- fold, 45-fold, 46-fold, 47 -fold, 48-fold, 49-fold, 50-fold, or 51-fold over baseline GMTs at a timepoint after administration of a lyophilized hCMV immunogenic composition.
  • the timepoint is after administration of
  • the proportion of human subjects with > 2 fold, > 3-fold, > 4-fold, > 5-fold, > 6-fold, > 7-fold, > 8-fold, > 9-fold, > 10-fold, > 11-fold, > 12-fold, or > 13-fold increases in nAb over baseline against epithelial cell infection is at least 50%, at least 60%, at least 70% at least 80%, or at least 90% at a time point after administration of the lyophilized hCMV immunogenic composition.
  • administration of an effective amount of the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • a single dose e.g., any of the doses described herein, or multiple doses, of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits serum neutralizing antibody titers against hCMV gB protein.
  • the GMT of serum neutralizing antibodies to hCMV gB protein increases in the subject administered the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) by at least 2-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold), relative to baseline.
  • the GMT of serum neutralizing antibodies to hCMV gB protein increases in the subject by 2-fold to 10-fold after administering a single dose (e.g., a single dose of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of serum neutralizing antibodies to hCMV gB protein increases in the subject by 2-fold to 10-fold after administering two doses (e.g., two doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • two doses e.g., two doses of >50 pg, such as 50 pg, 100 pg, or 150 pg
  • the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • the GMT of serum neutralizing antibodies to hCMV gB protein increases in the subject by 2-fold to 10-fold after administering three doses (e.g., three doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • three doses e.g., three doses of >50 pg, such as 50 pg, 100 pg, or 150 pg
  • the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • the proportion of human subjects with > 2-fold increase in nAb over baseline against fibroblast infection is at least 50%, at least 60%, at least 70% at least 80%, or at least 90% at a time point after administration of the lyophilized hCMV immunogenic composition.
  • administration of an effective amount of the lyophilized hCMV immunogenic composition elicits antigen- specific T-cell response against hCMV.
  • administration a single dose e.g., any of the doses described herein, or multiple doses, of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits antigen- specific T-cell response against hCMV.
  • administration of an effective amount of the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • administration of an effective amount of the lyophilized hCMV immunogenic composition elicits antigen- specific T-cell response against hCMV gB protein.
  • administration a single dose (e.g., any of the doses described herein), or multiple doses, of the lyophilized hCMV immunogenic composition elicits antigenspecific T-cell response against hCMV gB protein.
  • administration of an effective amount of the lyophilized hCMV immunogenic composition e.g., mRNA vaccine
  • hCMV pentamer elicits antigen- specific T-cell response against hCMV pentamer.
  • administration a single dose (e.g., any of the doses described herein), or multiple doses, of the lyophilized hCMV immunogenic composition elicits antigen- specific T- cell response against hCMV pentamer.
  • the T-cell response e.g., against hCMV, hCMV gB protein, or hCMV pentamer
  • IFN-y interferon-y
  • a control/baseline in some embodiments, is the anti-hCMV antigen antibody titer produced in a subject who has not been administered the hCMV mRNA vaccine.
  • a control/baseline is an anti-hCMV antigen antibody titer produced in a subject who has a natural hCMV infection, i.e., a subject who is hCMV seropositive prior to being administered the hCMV mRNA vaccine.
  • a control/baseline is an anti- hCMV antigen antibody titer produced in a subject who is hCMV seronegative prior to being administered the hCMV mRNA vaccine.
  • the GMT of serum neutralizing antibodies to hCMV increases in a dose-dependent manner.
  • the GMT of binding antibody response to hCMV pentamer increases in the subject administered the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) by at least 2-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold), relative to baseline.
  • the GMT of binding antibody response to hCMV pentamer increases in the subject by 2-fold to 10-fold after administering a single dose (e.g., a single dose of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of binding antibody response to hCMV pentamer increases in the subject by 2-fold to 10-fold after administering two doses (e.g., two doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of binding antibody response to hCMV increases in the subject by 2-fold to 10-fold after administering three doses (e.g., three doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition e.g., mRNA vaccine), relative to baseline.
  • anti-pentamer binding antibody (bAb) GMTs are increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold over baseline GMTs at a timepoint after administration of an hCMV immunogenic composition.
  • the timepoint is after administration of two doses of the immunogenic composition.
  • the timepoint is after administration of three doses of the lyophilized hCMV immunogenic composition.
  • the proportion of human subjects with > 2-fold, > 3-fold, > 4-fold, > 5-fold, > 6-fold, > 7-fold, > 8-fold, > 9-fold, or > 10-fold increases in anti-pentamer binding antibody (bAb) over baseline is at least 50%, at least 60%, at least 70% at least 80%, or at least 90% at one time point after administration of the lyophilized hCMV immunogenic composition.
  • the GMT of binding antibody response to gB increases in the subject administered the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) by at least 2-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold), relative to baseline.
  • the GMT of binding antibody response to anti-gB increases in the subject by 2- fold to 10-fold after administering a single dose (e.g., a single dose of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of binding antibody response to anti-gB increases in the subject by 2-fold to 10-fold after administering two doses (e.g., two doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • the GMT of binding antibody response to anti-gB increases in the subject by 2-fold to 10-fold after administering three doses (e.g., three doses of >50 pg, such as 50 pg, 100 pg, or 150 pg) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine), relative to baseline.
  • anti-gB binding antibody (Ab) GMTs are increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold over baseline GMTs at a timepoint after administration of a lyophilized hCMV immunogenic composition.
  • the timepoint is after administration of a single dose of the immunogenic composition.
  • the timepoint is after administration of two doses of the immunogenic composition.
  • the timepoint is after administration of three doses of the immunogenic composition.
  • the GMT response reaches a maximum within about 10 days to 2 weeks after a dose is administered.
  • the proportion of human subjects with > 2-fold increase in anti-gB binding antibody (Ab) over baseline is at least 50%, at least 60%, at least 70% at least 80%, or at least 90% at one time point after administration of the lyophilized hCMV immunogenic composition.
  • the ability of the hCMV mRNA vaccine to be effective is measured in a murine model.
  • the hCMV mRNA vaccine may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers.
  • Viral challenge studies may also be used to assess the efficacy of a vaccine.
  • the hCMV mRNA vaccine may be administered to a murine model, the murine model challenged with hCMV, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).
  • T cell response e.g., cytokine response
  • an effective amount of the hCMV mRNA vaccine is a dose that is reduced compared to the standard of care dose of a recombinant hCMV protein vaccine.
  • a “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance.
  • a “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified hCMV protein vaccine, or a live attenuated or inactivated hCMV mRNA vaccine, or a hCMV VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent hCMV, or a hCMV -related condition, while following the standard of care guideline for treating or preventing hCMV, or a hCMV -related condition.
  • the anti-hCMV antigen antibody titer produced in a subject administered an effective amount of the hCMV mRNA vaccine is equivalent to an anti- hCMV antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified hCMV protein vaccine, or a live attenuated or inactivated hCMV mRNA vaccine, or a hCMV VLP vaccine.
  • Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ;201(l 1): 1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas:
  • AR disease attack rate
  • Efficacy (1-RR) x 100.
  • vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun 1 ;201(l 1): 1607-10).
  • Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial.
  • Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine -related factors that influence the ‘real- world’ outcomes of hospitalizations, ambulatory visits, or costs.
  • a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared.
  • Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination:
  • efficacy of the hCMV mRNA vaccine is at least 60% relative to unvaccinated control subjects.
  • efficacy of the hCMV mRNA vaccine may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects.
  • Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host.
  • the effective amount of an hCMV mRNA vaccine is sufficient to provide sterilizing immunity in the subject for at least 1 year.
  • the effective amount of the hCMV mRNA vaccine may be sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years.
  • the effective amount of the hCMV mRNA vaccine is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control.
  • the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.
  • the effective amount of the hCMV mRNA vaccine is sufficient to produce detectable levels of hCMV antigen as measured in serum of the subject at 1-72 hours post administration.
  • an antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti- hCMV antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.
  • the effective amount of the hCMV mRNA vaccine is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the hCMV antigen as measured in serum of the subject at 1-72 hours post administration.
  • the effective amount is sufficient to produce a 1,000-5,000 neutralizing antibody titer produced by neutralizing antibody against the hCMV antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the hCMV antigen as measured in serum of the subject at 1-72 hours post administration.
  • the neutralizing antibody titer is at least 100 NT50.
  • the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50.
  • the neutralizing antibody titer is at least 10,000 NT50.
  • the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL).
  • the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL.
  • the neutralizing antibody titer is at least 10,000 NU/mL.
  • an anti-hCMV antigen antibody titer produced in the subject is increased by at least 1 log relative to a control.
  • an anti-hCMV antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to a control.
  • an anti-hCMV antigen antibody titer produced in the subject is increased at least 2 times relative to a control.
  • an anti-hCMV antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control.
  • a geometric mean which is the nth root of the product of n numbers, is generally used to describe proportional growth.
  • Geometric mean in some embodiments, is used to characterize antibody titer produced in a subject.
  • Example 1 Effect of sucrose concentration on lipid nanoparticle size.
  • Lipid nanoparticles were produced by combining an aqueous solution comprising mRNA and sucrose with an organic solvent comprising lipids, resulting in the formation of LNPs containing mRNA.
  • Aqueous solutions contained varying amounts of sucrose, such that the ratio of sucrose molecules to lipid molecules in the LNP-mRNA compositions varied from 3:1 to 40:1 (FIG. 1).
  • the size of LNPs produced at each sucrose:lipid ratio were measured, and surprisingly, higher sucrose:lipid ratios resulted in the formation of smaller LNPs, as measured by delta diameter (FIGs. 1 and 2C).
  • Example 2 Effect of lyophilization process on stability of lyophilized composition.
  • Lyophilization was used to remove moisture from LNP-mRNA compositions produced by the methods described in Example 1.
  • the lyophilization process consisted of three phases: 1) freezing (solidification) phase, 2) primary drying (sublimation) phase, and 3) secondary drying (desorption) phase, shown in FIG. 4A.
  • freezing phase water present in the composition was solidified by exposing the composition to a cold temperature, -50 °C.
  • the freezing step consisted of either cooling the compositions directly to -50 °C, or holding them at -10 °C for several hours prior to cooling to -50 °C.
  • the compositions were exposed to a slightly higher temperature, -35 °C, and low pressure (75 mTorr), which allowed for sublimation of a majority of the water in the compositions.
  • the primary drying phase was determined to be complete when the reading of the Pirani gauge was similar to the reading of the capacitance manometer (CM), which is commonly used in the art to evaluate the completion of primary drying.
  • CM capacitance manometer
  • the compositions were heated to a higher temperature, either 25 °C or 40 °C, to allow evaporation of remaining water.
  • LNP-mRNA compositions were either briefly cooled to -50 °C, then incubated at -10 °C for 5 hours for annealing, and finally incubated at -50 °C until solidification was complete (“Annealing Step”, FIG. 2A), or directly cooled to -50 °C and incubated for approximately 5 hours (“Normal Freezing”, FIG. 2B), As shown in FIG. 2C, the incorporation of an annealing step into the freezing phase resulted in decreased LNP size relative to a freezing phase that did not have an annealing step. This effect was observed across multiple concentrations of sucrose.
  • FIG. 4A An overview of the parameters of the lyophilization process, including the secondary drying phase, is given by FIG. 4A.
  • compositions were subjected to two distinct lyophilization processes: one in which secondary drying was conducted at 25 °C, and one in which secondary drying was conducted at 40 °C.
  • FIG. 4B secondary drying at a higher temperature resulted in the production of lyophilized LNP-mRNA compositions with moisture contents below 0.5 %w/w, which was not achieved by secondary drying at 25 °C.
  • compositions with lower moisture content had had reduced initial mRNA purity, but the mRNA in these compositions with lower moisture content also degraded more slowly, and after 6 months of storage, the compositions with the lowest moisture content had more intact, pure mRNA than compositions with higher moisture contents.
  • the use of a higher temperature in the secondary drying phase produced markedly more stable LNP- mRNA compositions, offsetting the slight reduction in initial mRNA purity from high secondary drying temperatures.
  • Table 1 Predicted stability of lyophilized LNP-mRNA compositions at 5 °C.
  • Table 2 First-order degradation rate estimates for lyophilized LNP-mRNA compositions at 5 °C.
  • Example 3 In vitro expression of proteins from lyophilized compositions.
  • Lipid nanoparticles containing mRNAs encoding hCMV antigens of the hCMV pentamer which comprises gH, gL, UL128, UL130, and UL131A, were either 1) lyophilized and reconstituted; 2) stored at 4 °C; 3) frozen at -20 °C and thawed, or 4) frozen at -80 °C and thawed.
  • Compositions were diluted to prepare a series of compositions containing varying amounts of mRNA, and then added to Hep3B cells in 24-well plates to transfect cells with mRNA. After transfection, cells were incubated for 18-20 hours to allow for mRNA translation and expression of hCMV pentamer protein expression.
  • Example 4 Immunization with lyophilized compositions.
  • compositions of lipid nanoparticles each containing mRNAs encoding proteins of the hCMV pentamer were lyophilized and reconstituted to prepare LNP-mRNA vaccine compositions (Lyo #1, Lyo #2, Lyo #3). Additionally, a composition containing the same mRNA was frozen and thawed (Frozen Control). Finally, a composition containing the same mRNA was prepared without freezing or lyophilization (Unfrozen).
  • Female BALB/c, 6-8 weeks of age were administered a dose of one of the prepared compositions containing 3 pg of mRNA by intramuscular injection (prime dose, day 0). Three weeks later, mice were administered another 3 pg dose of the same composition (boost dose, day 22).
  • Sera were collected from mice at day 21 (3 weeks post-prime, before boost) and day 36 (2 weeks postboost), and antibodies specific to hCMV pentamer in the serum of each mouse were quantified by ELISA (FIG. 7).
  • the geometric mean antibody titers after the prime dose (bottom number) and boost dose (top number) are shown above the data points.
  • the Lyo #2 group only half the mice received a boost dose, and so some data points reflect antibody titers at day 36, five weeks after the first dose.
  • Lyophilized compositions generated hCMV pentamer- specific antibodies at titers that were approximately equivalent to those generated by frozen compositions (FIG. 7). These results indicate that mRNA in lyophilized compositions is capable of being translated both in vitro and in vivo, with translated proteins being able to exert a therapeutic or prophylactic effect, such as inducing the production of protein- specific antibodies after administration to a subject. Compositions containing lipid nanoparticles and mRNA can thus be lyophilized for prolonged stability and ease of transport, with mRNA retaining the ability to be translated following reconstitution and delivery to cells.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in some embodiments, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in some embodiments, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • Each possibility represents a separate embodiment of the present invention.
  • any of the mRNA sequences described herein may include a 5' UTR and/or a 3' UTR.
  • the UTR sequences may be selected from the following sequences, or other known UTR sequences may be used.
  • any of the mRNA constructs described herein may further comprise a polyA tail and/or cap (e.g., 7mG(5’)ppp(5’)NlmpNp).
  • RNAs and encoded antigen sequences described herein include a signal peptide and/or a peptide tag (e.g., C-terminal His tag), it should be understood that the indicated signal peptide and/or peptide tag may be substituted for a different signal peptide and/or peptide tag, or the signal peptide and/or peptide tag may be omitted.
  • 5' UTR 5' UTR:

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Abstract

Des aspects de la divulgation concernent des méthodes de préparation de compositions lyophilisées comprenant des nanoparticules lipidiques et des acides nucléiques (par exemple, des ARNm codant un ou plusieurs antigènes du cytomégalovirus humain (hCMV). D'autres aspects concernent des compositions lyophilisées présentant de faibles teneurs en humidité et une stabilité améliorée pendant un stockage à long terme.
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