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WO2023212696A1 - Lyophilized human cytomegalovirus vaccines - Google Patents

Lyophilized human cytomegalovirus vaccines 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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WO
WIPO (PCT)
Prior art keywords
mrna
composition
less
lipid
hcmv
Prior art date
Application number
PCT/US2023/066366
Other languages
French (fr)
Inventor
Johnathan GOLDMAN
Kimberly HASSETT
Sarah Sullivan
Xiaohan PENG
Original Assignee
Modernatx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Modernatx, Inc. filed Critical Modernatx, Inc.
Publication of WO2023212696A1 publication Critical patent/WO2023212696A1/en

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Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • A61K39/245Herpetoviridae, e.g. herpes simplex virus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605Excipients; Inactive ingredients
    • A61K9/1617Organic compounds, e.g. phospholipids, fats
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19Particulate 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/20Antivirals for DNA viruses
    • A61P31/22Antivirals for DNA viruses for herpes viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53DNA (RNA) vaccination
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55555Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/70Multivalent vaccine
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/16011Herpesviridae
    • C12N2710/16111Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134Use 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°
  • 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°
  • 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

Aspects of the disclosure relate to methods of preparing lyophilized compositions comprising lipid nanoparticles and nucleic acids (e.g., mRNAs encoding one or more antigens of human cytomegalovirus (hCMV). Other aspects relate to lyophilized compositions with low moisture contents and improved stability during long-term storage.

Description

LYOPHILIZED HUMAN CYTOMEGALOVIRUS VACCINES
RELATED APPLICATION
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/336,525, filed April 29, 2022, the contents of which are incorporated by reference herein in their entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic Sequence Listing (M137870244WO00-SEQ-NTJ.xml; Size: 36,501 bytes; and Date of Creation: April 26, 2023) are herein incorporated by reference in their entirety.
BACKGROUND
Delivery of mRNA present in lipid nanoparticles (LNPs) and subsequent cellular uptake of the mRNA allows desired proteins, such as viral or bacterial antigens, to be expressed in vivo. However, LNPs, and the mRNA inside them, can be degraded by environmental conditions, such as the presence of water or environmental nucleases. This instability raises challenges for extended storage of LNP-mRNA compositions.
SUMMARY
Aspects of the disclosure relate to methods of lyophilizing lipid nanoparticles (LNPs) comprising nucleic acids, such as mRNAs encoding one or more human cytomegalovirus (hCMV) antigens, to produce lyophilized compositions with low moisture contents. 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. Unexpectedly, 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. Furthermore, the inclusion of sucrose as a lyoprotectant unexpectedly resulted in a reduction in LNP size, increasing the efficiency of the LNP-mRNA production process. These features resulted in the preparation of lyophilized compositions of mRNA in LNPs (e.g., LNPs containing mRNAs encoding human cytomegalovirus (hCMV) antigens) with lower moisture contents than were previously possible. 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.
Accordingly, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the one or more hCMV antigens comprises greater than or equal to 1 and less than or equal to 6 hCMV antigens.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
In some embodiments, 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; and the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
In some embodiments, the lipid nanoparticle composition comprises a lyoprotectant.
In some embodiments, the lyoprotectant comprises a sugar.
In some embodiments, the lyoprotectant comprises sucrose.
In some embodiments, the lipid nanoparticle comprises a lipid, and the lipid nanoparticle composition comprises a lyoprotectant mass:lipid mass ratio of at least 5:1.
In some embodiments, 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.
In some embodiments, the composition comprises a buffer.
In some embodiments, the buffer is selected from the group consisting of a Tris buffer, citrate buffer, and phosphate buffer.
In some embodiments, the buffer is a Tris buffer.
In some embodiments, the concentration of the buffer in the composition is about 1 mM to about 100 mM.
In some embodiments, the concentration of the buffer is about 10 mM to about 20 mM.
In some embodiments, the composition has a pH of about 6.5 to about 8.5.
In some embodiments, the composition has a pH of about 7 to about 8.
In some embodiments, the composition has a pH of about 7.4 to about 8.
In some embodiments, the lyophilizing comprises an annealing step. In some embodiments, 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.
In some embodiments, the first temperature is from about -30 °C to about 0 °C.
In some embodiments, 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.
In some embodiments, the second temperature is from about -100 °C to about -30 °C.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the lyophilizing comprises a sublimation step.
In some embodiments, the sublimation is performed at a vacuum pressure of from about 50 mTorr and about 300 mTorr.
In some embodiments, the lyophilizing comprises a desorption step, wherein the desorption step comprises exposing the composition to a desorption temperature.
In some embodiments, the desorption temperature is about 10 °C or higher.
In some embodiments, 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.
In some embodiments, the desorption step comprises exposing a sublimated composition to a desorption temperature of about 40 °C.
In some embodiments, the desorption is conducted for at least 2 hours.
In some embodiments, the desorption is conducted for about 5 hours, about 10 hours, about 15 hours, or about 20 hours.
In some embodiments, 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.
In some embodiments, the desorption is performed at a vacuum pressure of from about 50 mTorr and about 300 mTorr.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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.
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 about 5 °C.
In some embodiments, 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.
In some embodiments, a high performance liquid chromatography analysis of the mRNA in the lyophilized composition produces a chromatogram comprising A peaks, wherein one of the A peaks is a main peak, wherein the main peak measures mRNA that was present in the composition before lyophilization, wherein the area under the curve (AUC) of each peak is calculated by integration, wherein the purity of the mRNA in the lyophilized composition is calculated using the equation % purity =
Figure imgf000007_0001
In some embodiments, the lipid nanoparticle comprises: an ionizable amino lipid.
In some embodiments, the lipid nanoparticle further comprises: a non-cationic lipid; a sterol; and a polyethylene glycol (PEG)-modified lipid.
In some embodiments, 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.
Some aspects of the disclosure relate to 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 polynucleotide comprising an open reading frame encoding a hCMV gB polypeptide, where 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. In some embodiments, 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; and 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 comprising a lipid nanoparticle encapsulating an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the one or more hCMV antigens comprises greater than or equal to 1 and less than or equal to 6 hCMV antigens.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
In some embodiments, 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; and 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 comprising a lipid nanoparticle and an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
In some embodiments, the lyophilized pharmaceutical composition comprises a lyoprotectant.
In some embodiments, the lyoprotectant comprises a sugar.
In some embodiments, the lyoprotectant comprises sucrose.
In some embodiments, the lipid nanoparticle comprises a lipid, and where the lyophilized pharmaceutical composition comprises a lyoprotectant mass:lipid mass ratio of at least 5:1.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
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 about 5 °C.
In some embodiments, 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.
In some embodiments, the lipid nanoparticle comprises: an ionizable amino lipid.
In some embodiments, the lipid nanoparticle further comprises: a non-cationic lipid; a sterol; and a polyethylene glycol (PEG)-modified lipid. In some embodiments, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
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. 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 1st 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. Compositions A and B contained distinct mRNAs.
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 (1st, left group of bars), compositions stored at 4 °C (2nd group), compositions frozen at -20 °C and thawed (3rd group), and compositions frozen at - 80 °C and thawed (4th group). In each 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.
DETAILED DESCRIPTION
Methods for lyophilization
Some aspects of the disclosure relate to methods of lyophilizing nucleic acids, such as mRNA, which can produce enhanced compositions for use as pharmaceuticals. In some embodiments, the nucleic acid is combined with a lipid, e.g., an ionizable lipid (e.g., in a lipid nanoparticle (LNP)). 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.
It has been discovered that the introduction of an annealing step, 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. Furthermore, the inclusion of high levels of sucrose as a lyoprotectant unexpectedly resulted in a reduction in LNP size, increasing the efficiency of the LNP-mRNA production process. The mRNA in the resulting lyophilized compositions thus remains stable during extended storage.
In certain embodiments, 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. In certain embodiments, 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. In some embodiments, the lyophilization is in a freeze dryer.
Thermal treatment
In some embodiments, the lyophilization comprises one or more thermal treatment steps. In some embodiments, the thermal treatment comprises one or more freezing steps (e.g., a first freezing step, a second freezing step, etc.). In some embodiments, the thermal treatment step(s) are performed at atmospheric pressure.
In some embodiments, 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).
In some embodiments 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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 In some embodiments, 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. In some embodiments, 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. In some embodiments, the composition is exposed to each of the temperatures for the same time periods (/'.<?., the first, second, etc. time periods are the same). In some embodiments, 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).
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. Thus, an annealing step enables the use of harsher conditions during primary and/or secondary drying that may otherwise compromise nucleic acid integrity. Additionally, 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.
Sublimation
In some embodiments, the lyophilization comprises a sublimation step (e.g., conducted at temperatures below the compositions critical collapse temperature and performed under a vacuum). In some embodiments, the sublimation occurs in a first drying step. In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, the primary drying phase is conducted at a temperature of from about -35 °C to about -25 °C. In some embodiments, 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.
In some embodiments, 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. When 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.
Desorption
In some embodiments, the lyophilization comprises a desorption step. In some embodiments, the desorption occurs in a secondary drying step. In the secondary drying phase, 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.
In some embodiments, 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. Preferably, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
Lyoprotectants
Some aspects of the disclosure relate to compositions (e.g., lipid nanoparticle compositions) and methods of preparing lyophilized compositions, wherein the composition comprises a lyoprotectant.
In some embodiments, the compositions comprise a lipid nanoparticle comprising a nucleic acid, such as mRNA. In some embodiments, 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. In some embodiments, the lyoprotectant is added to the lipid or lipid nanoparticle before the lipid nanoparticle is combined with the nucleic acid. In some embodiments, the lyoprotectant, lipid, and nucleic acid are combined prior to formation of the lipid nanoparticle. In some embodiments, the lyoprotectant is added to a composition comprising a lipid nanoparticle and a nucleic acid. As used herein, 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. For example, without being bound by theory 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.
In some embodiments, the lyoprotectant is a sugar. As used herein, a sugar is a carbohydrate molecule comprising one or more monosaccharide monomers. Non-limiting examples of sugars include sucrose, trehalose, maltose, lactose, glucose, fructose, and galactose. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, the sucrose is added to the lipid or lipid nanoparticle before the lipid nanoparticle is combined with the nucleic acid. In some embodiments, the sucrose, lipid, and nucleic acid are combined prior to formation of the lipid nanoparticle. In some embodiments, the sucrose is added to a composition comprising a lipid nanoparticle and a nucleic acid.
Buffers and composition components
Some aspects of the disclosure relate to compositions (e.g., lipid nanoparticle compositions) and methods of preparing lyophilized compositions, wherein the composition comprises a buffer and/or other components. In some embodiments, the buffer and/or other components are present in pre-lyophilized compositions. In some embodiments, the buffer and/or other components are present in reconstituted lyophilized compositions (e.g., they can be used to reconstitute a lyophilized composition).
In some embodiments, the compositions comprise a lipid nanoparticle comprising a nucleic acid, such as mRNA, and a buffer. In some embodiments, 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. In some embodiments, the buffer is added to the lipid or lipid nanoparticle before the lipid nanoparticle is combined with the nucleic acid. In some embodiments, the buffer, lipid, and nucleic acid are combined prior to formation of the lipid nanoparticle. In some embodiments, the buffer is added to a composition comprising a lipid nanoparticle and a nucleic acid. As used herein, 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. For example, without being bound by theory a buffer may absorb free hydrogen or hydroxide ions that are released from other components of the composition before, during, or after lyophilization. Absorption of free hydrogen and/or hydroxide ions by 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.
In some embodiments, 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.
In some embodiments, the buffer is selected from the group consisting of a Tris buffer, citrate buffer, phosphate buffer, triethylammonium bicarbonate (TEAB), and histidine buffer. In some embodiments, the buffer is a Tris buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a TEAB buffer. In some embodiments, the buffer is a histidine buffer.
In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the pH is about 8 to about 9. In some embodiments, 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.
In some embodiments, the pH of the composition after lyophilization and reconstitution 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. In some embodiments, 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. In some embodiments, the pH is about 8 to about 9. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the composition (e.g., prior to lyophilization) comprises a salt, such as sodium chloride. In some embodiments, the salt concentration in the composition is from about 0.1 mM to about 300 mM. Preferably, 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. In a reconstitution medium, 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.
In some embodiments, the composition (e.g., prior to lyophilization) comprises a surfactant. Exemplary surfactants include Tween20, Tween80, BrijS200, and PEG-DMG. In some embodiments, 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. In some embodiments, the composition comprises about 0.005wt%, about 0.05wt%, about 0.01wt%, about 0.1 wt%, or about 0.5wt% surfactant. In some embodiments, the composition (e.g., prior to lyophilization) comprises a metal chelator. Exemplary metal chelators include EDTA (ethylenediaminetetraacetic acid) and DTPA (diethylenetriaminepentaacetic acid). In some embodiments, 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.
Lipid compositions
In some embodiments, the nucleic acids are present in a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and/or a lipoplex. In some embodiments, nucleic acids are present in lipid nanoparticle (LNP) compositions. 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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%.
In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid. For example, 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. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid. In some embodiments, 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.
In some embodiments, the lipid nanoparticle comprises 45 - 55 mole percent (mol%) ionizable amino lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.
Ionizable amino lipids
In some embodiments, the ionizable amino lipid is a compound of Formula (Al): its N-oxide, or a salt or isomer thereof,
Figure imgf000021_0001
wherein R’a is R’branched - wherein denotes a point of attachment;
Figure imgf000022_0002
wherein R, R, R, and R are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
R4 is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consisting
Figure imgf000022_0003
wherein
Figure imgf000022_0001
denotes a point of attachment; wherein
R10 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 R5 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H;
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.
In some embodiments of the compounds of Formula (Al), R’a is R’branched; R’branched is denotes a point of attachment; R, R, Ray, and R are each H; R2 and
Figure imgf000022_0004
R3 are each Ci-14 alkyl; R4 is -(CFH2)H; n is 2; each R5 is H; each R6 is H; M and M’ are each - C(O)O-; R’ is a Ci-12 alkyl; 1 is 5; and m is 7.
In some embodiments of the compounds of Formula (Al), R’a is R’branched; R’branched is denotes a point of attachment; R, R, Ray, and R are each H; R2 and
Figure imgf000022_0005
R3 are each Ci-14 alkyl; R4 is -(CH2)nOH; n is 2; each R5 is H; each R6 is H; M and M’ are each -
C(0)0-; R’ is a Ci-12 alkyl; 1 is 3; and m is 7.
In some embodiments of the compounds of Formula (Al), R’a is R’branched; R’branched is denotes a point of attachment; R is C2-12 alkyl; R, Ray, and R are
Figure imgf000023_0001
each H; R2 and R3 are each C 1-14 alkyl alkyl); n2 is 2; R
Figure imgf000023_0002
5 is H; each R6 is H; M and M’ are each -C(O)O-; R' is a Ci-12 alkyl; 1 is 5; and m is 7.
In some embodiments of the compounds of Formula (I), R’a is R’branched; R’branched is
Figure imgf000023_0003
denotes a point of attachment; R, R, and R are each H; R is C2-12 alkyl; R2 and R3 are each Ci-14 alkyl; R4 is -(CH2)nOH; n is 2; each R5 is H; each R6 is H; M and M' are each -C(O)O-; R’ is a Ci-12 alkyl; 1 is 5; and m is 7.
In some embodiments, the compound of Formula (I) is selected from:
Figure imgf000023_0004
In some embodiments, the ionizable amino lipid is a compound of Formula (Ala):
Figure imgf000024_0001
its N-oxide, or a salt or isomer thereof, wherein R’a is R,branched; wherein
R’ branched denotes a point of attachment;
Figure imgf000024_0002
wherein R, Ray, and R are each independently selected from the group consisting of H,
C2-12 alkyl, and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
R4 is selected from the group consisting of -(CFhkOH wherein n is selected from the group consisting
Figure imgf000024_0003
wherein ? denotes a point of attachment; wherein
R10 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 R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H;
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.
In some embodiments, the ionizable amino lipid is a compound of Formula (Alb): its N-oxide, or a salt or isomer thereof,
Figure imgf000024_0004
wherein R’a is R,branched; wherein denotes a point of attachment;
Figure imgf000025_0001
wherein R, R, Ray, and R are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
R4 is -(CH2)nOH, wherein n is selected from the group consisting of 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 R6 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H;
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.
In some embodiments of Formula (Al) or (Alb), R’a is R’branched; R’branched is denotes a point of attachment; R, Ray, and R are each H; R2 and R
Figure imgf000025_0002
3 are each Ci-14 alkyl; R4 is -(CH2)nOH; n is 2; each R5 is H; each R6 is H; M and M’ are each - C(O)O-; R’ is a Ci-12 alkyl; 1 is 5; and m is 7.
In some embodiments of Formula (Al) or (Alb), R’a is R’branched; R’branched is
Figure imgf000025_0003
denotes a point of attachment; R, Ray, and R are each H; R2 and R3 are each Ci-14 alkyl; R4 is -(CH2)nOH; n is 2; each R5 is H; each R6 is H; M and M’ are each - C(O)O-; R’ is a Ci-12 alkyl; 1 is 3; and m is 7.
In some embodiments of Formula (Al) or (Alb), R’a is R’branched; R’branched is denotes a point of attachment; R and R are each H; Ray is C2-12 alkyl;
Figure imgf000025_0004
R2 and R3 are each Ci-14 alkyl; R4 is -(CH2)nOH; n is 2; each R5 is H; each R6 is H; M and M’ are each -C(O)O-; R is a Ci-12 alkyl; 1 is 5; and m is 7.
In some embodiments, the ionizable amino lipid is a compound of Formula (Ale):
Figure imgf000026_0001
its N-oxide, or a salt or isomer thereof, wherein R’a is R,branched; wherein
Figure imgf000026_0002
denotes a point of attachment; wherein R, R, Ray, and R are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
Figure imgf000026_0003
wherein ? denotes a point of attachment; whereinR10 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 R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H;
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.
In some embodiments,
Figure imgf000026_0004
point of attachment; R, Ray, and R are each H; R is C2-12 alkyl; R2 and R3 are each Ci-14 alkyl;
Figure imgf000027_0001
denotes a point of attachment; R10 is NH(CI-6 alkyl); n2 is 2; each R5 is H; each R6 is H; M and M’ are each -C(O)O-; R’ is a Ci-12 alkyl; 1 is 5; and m is
7.
In some embodiments, the compound of Formula (Ale) is:
Figure imgf000027_0002
In some embodiments, the ionizable amino lipid is a compound of Formula (All):
Figure imgf000027_0003
wherein R’a is R’branched Or R’cyclic; wherein
Figure imgf000027_0004
wherein ? denotes a point of attachment;
Ray and R are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of Ray and R is selected from the group consisting of Ci-
12 alkyl and C2-12 alkenyl;
Rby and Rb5 are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of Rby and Rb5 is selected from the group consisting of Ci-
12 alkyl and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl; R4 is selected from the group consisting of -(CFFjnOH wherein n is selected from the group consisting
Figure imgf000028_0001
wherein
Figure imgf000028_0002
denotes a point of attachment; wherein R10 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;
Ya 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.
In some embodiments, the ionizable amino lipid is a compound of Formula (All- a):
Figure imgf000028_0003
wherein R’a is R’branched Or R’cyclic; wherein
Figure imgf000028_0004
wherein ? denotes a point of attachment;
Ray and R are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of Ray and R is selected from the group consisting of Ci-
12 alkyl and C2-12 alkenyl;
Rby and Rb5 are each independently selected from the group consisting of H, Ci-12 alkyl, and C2-12 alkenyl, wherein at least one of Rby and Rb5 is selected from the group consisting of Ci- 12 alkyl and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl; R4 is selected from the group consisting of -(CFFjnOH wherein n is selected from the group consisting
Figure imgf000029_0001
wherein
Figure imgf000029_0002
denotes a point of attachment; wherein R10 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.
In some embodiments, the ionizable amino lipid is a compound of Formula (All-b):
Figure imgf000029_0003
its N-oxide, or a salt or isomer thereof, wherein R’a is R’branched Or R’cyclic; wherein
Figure imgf000029_0004
wherein
Figure imgf000029_0005
denotes a point of attachment;
Ray and Rby are each independently selected from the group consisting of Ci-12 alkyl and
C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and
C2-14 alkenyl;
R4 is selected from the group consisting of -(CFFjnOH wherein n is selected from the group consisting
Figure imgf000029_0006
wherein
Figure imgf000029_0007
denotes a point of attachment; wherein R10 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;
1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. In some embodiments, the ionizable amino lipid is a compound of Formula (AII-c): its N-oxide, or a salt or isomer thereof,
Figure imgf000030_0001
wherein R’a is R’branched Or R’cyclic; wherein
Figure imgf000030_0002
wherein denotes a point of attachment; wherein Ray is selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and C2-14 alkenyl;
R4 is selected from the group consisting of - (CH2)nOH wherein n is selected from the group consisting wherein
Figure imgf000030_0003
denotes a point of attachment; wherein R10 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.
In some embodiments, the ionizable amino lipid is a compound of Formula (All-d): its N-oxide, or a salt or isomer thereof,
Figure imgf000030_0004
wherein R’a is R’branched Or R'cyclic ; wherein wherein denotes a point of attachment;
Figure imgf000030_0005
wherein Ray and Rby are each independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl;
R4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting
Figure imgf000031_0001
wherein
Figure imgf000031_0002
denotes a point of attachment; wherein R10 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;
1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
In some embodiments, the ionizable amino lipid is a compound of Formula (All-e):
Figure imgf000031_0003
its N-oxide, or a salt or isomer thereof, wherein R’a is R’branched Or R’cyclic; wherein
Figure imgf000031_0004
wherein ? denotes a point of attachment; wherein Ray is selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of Ci-14 alkyl and
C2-14 alkenyl;
R4 is -(CH2)nOH 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.
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 independently selected from 4, 5, and 6. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-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.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’b is: R3^^~R2 and R2 and R3 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:
Figure imgf000032_0001
and R2 and R3 are each independently a C6-10 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), R’b is: R3^XR2 and R2 and R3 are each a Cs alkyl.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII-
Figure imgf000032_0002
and R3 are each independently a C6-10 alkyl. In some embodiments of the compound of Formula
Figure imgf000032_0006
R3^^R2 , Ray is a C2-6 alkyl and R2 and R3 are each independently a C6-10 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e),
Figure imgf000032_0003
alkyl, and R2 and R3 are each a
C8 alkyl.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All- d), or (All-e), R’branched is;
Figure imgf000032_0004
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;
Figure imgf000032_0005
each a C2-6 alkyl.
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 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),
(All-b), (AII-c), (All-d), or (All-e), m and 1 are each 5 and each R’ independently is a C2-5 alkyl. In some embodiments of the compound of (All), (All-a), (All-b), (AII-c), (All-d), or
(AII-e), R’branched is; are each
Figure imgf000033_0001
independently selected from 4, 5, and 6, each R’ independently is a Ci- 12 alkyl, and Ray and Rby are each a Ci-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b),
(AII-c), (All-d), or (AII-e), R’branched is;
Figure imgf000033_0002
are each 5, each R’ independently is a C2-5 alkyl, and Ray and Rby are each a C2-6 alkyl.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (AII-e), R’branched is; are each
Figure imgf000033_0003
independently selected from 4, 5, and 6, R’ is a Ci-12 alkyl, Ray is a Ci-12 alkyl and R2 and R3 are each independently a C6-10 alkyl.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (AII-e), R’branched is; are each 5, R’ is a C2-
Figure imgf000033_0004
alkyl, Ray is a C2-6 alkyl, and R2 and R3 are each a C8 alkyl.
In some embodiments of the compound of (All), (All-a), (All-b), (AII-c), (All-d), or wherein R10 is NH(CI-6 alkyl) and n2 is 2. In some
Figure imgf000033_0005
embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (AII-e), R4
Figure imgf000033_0006
wherein R10 is NH(CH3) and n2 is 2.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (AII-e), R’branched is;
Figure imgf000033_0007
independently selected from 4, 5, and 6, each R’ independently is a Ci- 12 alkyl, Ray and Rby are each a Ci-12 alkyl, wherein R10 is NH(CI-6 alkyl), and n2 is 2. In
Figure imgf000034_0007
some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (AII- are each 5, each R’ independently is a C2-5 alkyl, Ray and Rby are each a C2-6 alkyl
Figure imgf000034_0006
wherein R10 is NH(CH3) and n2 is 2.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branched is;
Figure imgf000034_0005
are each independently selected from 4, 5, and 6, R’ is a Ci-12 alkyl, R2 and R3 are each independently a C6-10 alkyl, Ray is a Ci-12 alkyl wherein R10 is NH(CI-6 alkyl) and
Figure imgf000034_0004
n2 is 2. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branched is; are each 5, R’ is a C2-
5 alkyl, R y is a C2-6 alkyl, R2 and R3 are each a Cs alkyl, wherein
Figure imgf000034_0003
R10 is NH(CH3) and n2 is 2.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R4 is -(CH2)nOH 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), R4 is -(CH2)nOH and n is 2.
In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branched is
Figure imgf000034_0001
independently selected from 4, 5, and 6, each R’ independently is a Ci- 12 alkyl, Ray and Rby are each a Ci-12 alkyl, R4 is -(CH2)nOH, 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’branched is:
Figure imgf000034_0002
. m and 1 are each 5, each R’ independently is a C2-5 alkyl, Ray and R
Figure imgf000035_0001
are each a C2-6 alkyl, R4 is -(CH2)nOH, and n is 2.
In some embodiments, the ionizable amino lipid is a compound of Formula (All-f):
Figure imgf000035_0002
wherein R’a is R’branched Or R’cyclic; wherein wherein denotes a point of attachment;
Figure imgf000035_0003
Ray is a C1-12 alkyl;
R2 and R3 are each independently a Ci-14 alkyl;
R4 is -(CH2)nOH 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.
In some embodiments of the compound of Formula (All-f), m and 1 are each 5, and n is 2, 3, or 4.
In some embodiments of the compound of Formula (All-f) R’ is a C2-5 alkyl, Ray is a C2-6 alkyl, and R2 and R3 are each a C6-10 alkyl.
In some embodiments of the compound of Formula (All-f), m and 1 are each 5, n is 2, 3, or 4, R’ is a C2-5 alkyl, Ray is a C2-6 alkyl, and R2 and R3 are each a C6-10 alkyl.
In some embodiments, the ionizable amino lipid is a compound of Formula (All-g):
Figure imgf000035_0004
R’ is a C2-5 alkyl; and R4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting
Figure imgf000036_0001
wherein denotes a point of attachment, R10 is NH(CI-6 alkyl), and n2 is selected
Figure imgf000036_0002
from the group consisting of 1, 2, and 3.
In some embodiments, the ionizable amino lipid is a compound of Formula (All-h): wherein
Figure imgf000036_0003
Ray and Rhy are each independently a C2-6 alkyl; each R’ independently is a C2-5 alkyl; and
R4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting
Figure imgf000036_0004
wherein
Figure imgf000036_0005
denotes a point of attachment, R10 is NH(CI-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.
In some embodiments of the compound of Formula (All-g) or (All-h), R4 is
Figure imgf000036_0006
, wherein
R10 is NH(CH3) and n2 is 2.
In some embodiments of the compound of Formula (All-g) or (All-h), R4 is -(CH2)OH.
In some embodiments, the ionizable amino lipids may be one or more of compounds of Formula (VI):
Figure imgf000036_0007
(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)nQ, - (CH2)nCHQR,
-CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(0)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -0C(0)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(0R)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(0)0R, and each n is independently 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)-, -OC(O)-M”-C(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-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, Ci-13 alkyl or C2-13 alkenyl;
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)2N(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
C3-15 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; and wherein when R4 is -(CH2)nQ, - (CH2)nCHQR, -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.
In some embodiments, another subset of compounds of Formula (VI) includes those in which:
Ri is selected from the group consisting of C5-30 alkyl, Cs-2o 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, -(CH2)nQ, -(CH2)nCHQR, -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(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, mono- or di-alkylamino, and C1-3 alkyl, and each n is independently 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(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, 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)2N(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.
In some embodiments, 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, -(CH2)nQ, -(CH2)nCHQR, -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(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR,
-N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, - N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R4 is - (CH2)nQ in which n is 1 or 2, or (ii) R4 is - (CH2)nCHQR in which n is 1, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to 14- membered heteroaryl or 8- to 14-membered heterocycloalkyl; 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;
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)2N(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.
In some embodiments, 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, -(CH2)nQ, -(CH2)nCHQR, -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(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and - C(=NR9)N(R)2, and each n is independently 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;
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)2N(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.
In some embodiments, 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 -(CH2)nCHQR, 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.
In some embodiments, 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 -(CH2)nQ, -(CH2)nCHQR, -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.
In certain embodiments, a subset of compounds of Formula (VI) includes those of Formula (VI- A):
Figure imgf000043_0001
or its N-oxide, or a salt or isomer 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’; R4 is hydrogen, unsubstituted C1-3 alkyl, or - (CH2)nQ, in which Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, - N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -0C(0)-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. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.
In certain embodiments, a subset of compounds of Formula (VI) includes those of Formula (VI-B):
Figure imgf000043_0002
or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; R4 is hydrogen, unsubstituted C1-3 alkyl, or - (CH2)nQ, in which Q is H, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, - N(R)R8,
-NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; 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. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.
In certain embodiments, a subset of compounds of Formula (VI) includes those of Formula (VII):
Figure imgf000044_0001
or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; Mi is a bond or M’; R4 is hydrogen, unsubstituted C1-3 alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; 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.
In some embodiments, the compounds of Formula (VI) are of Formula (Vila),
Figure imgf000044_0002
or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
In another embodiment, the compounds of Formula (VI) are of Formula (Vllb),
Figure imgf000044_0003
or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
In another embodiment, the compounds of Formula (VI) are of Formula (Vile) or (Vile):
Figure imgf000045_0001
or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.
In another embodiment, the compounds of Formula (VI) are of Formula (Vllf):
Figure imgf000045_0002
(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.
In a further embodiment, the compounds of Formula (VI) are of Formula (Vlld),
Figure imgf000045_0003
(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. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
In some embodiments, an ionizable amino lipid comprises a compound having structure:
Figure imgf000045_0004
In some embodiments, an ionizable amino lipid comprises a compound having structure:
Figure imgf000046_0001
In a further embodiment, the compounds of Formula (VI) are of Formula (Vllg),
Figure imgf000046_0002
(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. For example, M” is C1-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl). For example, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
In some embodiments, 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. Thus, 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.
In some embodiments, the ionizable amino lipids may be one or more of compounds of formula (VIII),
Figure imgf000046_0003
or salts or isomers thereof, wherein
Figure imgf000047_0001
t is 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,
W1 and W2 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;
X1, X2, and X3 are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH2)n-C(O)-, -C(O)-(CH2)n-, -(CH2)n-C(O)O-, -OC(O)-(CH2)n-, -(CH2)n-OC(O)-, -C(O)O-(CH2)n-, -CH(OH)-, -C(S)-, and - CH(SH)-; each Y is independently a C3-6 carbocycle; each R* is independently selected from the group consisting of Ci-12 alkyl and C2-12 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl and a C3-6 carbocycle; each R’ is independently selected from the group consisting of Ci-12 alkyl, C2-12 alkenyl, and H; each R” is independently selected from the group consisting of C3-12 alkyl, C3-12 alkenyl and -R*MR’ ; and n is an integer from 1-6; wherein when ring
Figure imgf000048_0001
then i) at least one of X1, X2, and X3 is not -CH2-; and/or ii) at least one of Ri, R2, R3, R4, and R5 is -R”MR’.
In some embodiments, the compound is of any of formulae (Villa l)-(VIIIa8):
Figure imgf000048_0002
Figure imgf000049_0002
In some embodiments, the ionizable amino lipid is
Figure imgf000049_0003
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. Thus, a lipid may have a positive or partial positive charge at physiological pH.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000049_0004
or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:
R1 is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;
R2 and R3 are each independently optionally substituted C1-C36 alkyl;
R4 and R5 are each independently optionally substituted Ci-Ce alkyl, or R4 and R5 join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;
L1, L2, and L3 are each independently optionally substituted Ci-Cis alkylene;
G1 is a direct bond, -(CH2)nO(C=O)-, -(CH2)n(C=O)O-, or -(C=O)-;
G2 and G3 are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000049_0001
or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: G1 is -N(R3)R4 or -OR5;
R1 is optionally substituted branched, saturated or unsaturated C12-C36 alkyl;
R2 is optionally substituted branched or unbranched, saturated or unsaturated C12- C36 alkyl when L is -C(=O)-; or R2 is optionally substituted branched or unbranched, saturated or unsaturated C4-C36 alkyl when L is C6-C12 alkylene, C6-C12 alkenylene, or C2-C6 alkynylene;
R3 and R4 are each independently H, optionally substituted branched or unbranched, saturated or unsaturated Ci-Ce alkyl; or R3 and R4 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 R3 and R4, together with the nitrogen to which they are attached, join to form a heterocyclyl;
R5 is H or optionally substituted Ci-Ce alkyl;
L is -C(=O)-, C6-C 12 alkylene, C6-C12 alkenylene, or C2-C6 alkynylene; and n is an integer from 1 to 12.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000050_0001
or a pharmaceutically acceptable salt thereof, wherein: each Rla is independently hydrogen, Rlc, or Rld; each Rlb is independently Rlc or Rld; each Rlc is independently -[CH2]2C(O)X1R3; each Rld Is independently -C(O)R4; each R2 is independently -[C(R2a)2]cR2b; each R2a is independently hydrogen or Ci-Ce alkyl;
R2b is -N(LI-B)2; -(OCH2CH2)6OH; or -(OCH2CH2)bOCH3; each R3 and R4 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 X1 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000051_0001
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
X is N, and Y is absent; or X is CR, and Y is NR;
Figure imgf000051_0002
SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, or - NRaC(=O)OR1;
L2 is -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)XR2, -S-SR2, -C(=O)SR2, - SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf; -NRdC(=O)OR2 or a direct bond to R2;
Figure imgf000051_0003
G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene;
G3 is C1-C24 alkylene, C2-C24 alkenylene, C1-C24 heteroalkylene or C2-
C24 heteroalkenylene when X is CR, and Y is NR; and G3 is C1-C24 heteroalkylene or C2- C24 heteroalkenylene when X is N, and Y is absent;
Ra, Rb, Rd and Re are each independently H or C1-C12 alkyl or C1-C12 alkenyl;
Rc and Rf are each independently C1-C12 alkyl or C2-C12 alkenyl; each R is independently H or C1-C12 alkyl;
R1, R2 and R3 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000051_0004
or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
L1 and L2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-s -S-S-, - C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, -NRaC(=O)O- or a direct bond; G1 is C1-C2 alkylene, -(C=0)-, -0(C=0)-, -SC(=O)-, -NRaC(=O)- or a direct bond;
G2 is -C(O)-, -(CO)O-, -C(=O)S-, -C(=O)NRa- or a direct bond;
G3 is Ci-Ce alkylene;
Ra is H or C1-C12 alkyl;
Rla and Rlb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rla is H or C1-C12 alkyl, and Rlb together with the carbon atom to which it is bound is taken together with an adjacent Rlb and the carbon atom to which it is bound to form a carbon-carbon double bond;
R2a and R2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2a is H or C1-C12 alkyl, and R2b together with the carbon atom to which it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R3a and R3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R3a is H or C1-C12 alkyl, and R3b 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;
R4a and R4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4a is H or C1-C12 alkyl, and R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R5 and R6 are each independently H or methyl;
R7 is H or C1-C20 alkyl;
R8 is OH, -N(R9)(C=O)R10, -(C=O)NR9R10, -NR9R10, -(C=O)OR" or -O(C=O)R", provided that G3 is C4-C6 alkylene when R8 is -NR9R10,
R9 and R10 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000052_0001
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
X and X' are each independently N or CR;
Y and Y' are each independently absent, -O(C=O)-, -(C=O)O- or NR, provided that: a) Y is absent when X is N; b) Y' is absent when X' is N; c) Y is -0(C=0)-, -(C=0)0- or NR when X is CR; and d) Y' is -O(C=O)-, -(C=O)O- or NR when X' is CR,
L1 and L1 are each independently -O(C=O)R', -(C=O)OR' , -C(=O)R', -OR1, -S(O)ZR', -S- SR1, -C(=O)SR', -SC(=O)R', -NRaC(=O)R', -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc or -NRaC(=O)OR';
L2 and L2 are each independently -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)ZR2, - S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, - OC(=O)NReRf;-NRdC(=O)OR2 or a direct bond to R2;
G1. G1 , G2 and G2 are each independently C2-Ci2 alkylene or C2-C12 alkenylene;
G is C2-C24 heteroalkylene or C2-C24 heteroalkenylene;
Ra, Rb, Rd and Re are, at each occurrence, independently H, C1-C12 alkyl or C2-C12 alkenyl;
Rc and Rf are, at each occurrence, independently C1-C12 alkyl or C2-C12 alkenyl;
R is, at each occurrence, independently H or C1-C12 alkyl;
R1 and R2 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000053_0001
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
L1 is -O(C=O)R1, -(C=O)OR1, -C(=O)R1, -OR1, -S(O)XR1, -S-SR1, - C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc or - NRaC(=O)OR1;
L2 is -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)XR2, -S-SR2, - C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf;
-NRdC(=O)OR2 or a direct bond to R2;
G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene;
G3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
Ra, Rb, Rd and Re are each independently H or C1-C12 alkyl or C1-C12 alkenyl; Rc and Rf are each independently C1-C12 alkyl or C2-C12 alkenyl;
R1 and R2 are each independently branched C6-C24 alkyl or branched Ce- C24 alkenyl;
R3 is -N(R4)R5;
R4 is C1-C12 alkyl;
R5 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000054_0001
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
L1 is -O(C=O)R1, -(C=O)OR1, -C(=O)R1, -OR1, -S(O)XR1, -S-SR1, -C(=O)SR1, - SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc or - NRaC(=O)OR1;
L2 is -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)XR2, -S-SR2, -C(=O)SR2, - SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf;-NRdC(=O)OR2 or a direct bond to R2;
Gla and G2b are each independently C2-C12 alkylene or C2-C12 alkenylene;
Glb and G2b are each independently C1-C12 alkylene or C2-C12 alkenylene;
G3 is C1-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
Ra, Rb, Rd and Re are each independently H or C1-C12 alkyl or C2-C12 alkenyl;
Rc and Rf are each independently C1-C12 alkyl or C2-C12 alkenyl;
R1 and R2 are each independently branched C6-C24 alkyl or branched Ce- C24 alkenyl;
R3a is -C(=O)N(R4a)R5a or -C(=O)OR6;
R3b is -NR4bC(=O)R5b;
R4a is C1-C12 alkyl;
R4b is H, C1-C12 alkyl or C2-C12 alkenyl;
R5a is H, Ci-C8 alkyl or C2-C8 alkenyl;
R5b is C2-C12 alkyl or C2-C12 alkenyl when R4b is H; or R5b is C1-C12 alkyl or C2- C12 alkenyl when R4b is C1-C12 alkyl or C2-C12 alkenyl;
R6 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000055_0002
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
Figure imgf000055_0001
R is, at each occurrence, independently H or OH;
R1 and R2 are each independently optionally substituted branched, saturated or unsaturated C12-C36 alkyl;
R3 and R4 are each independently H or optionally substituted straight or branched, saturated or unsaturated Ci-Ce alkyl;
R5 is optionally substituted straight or branched, saturated or unsaturated Ci-Ce alkyl; and n is an integer from 2 to 6.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000055_0003
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) , -S-S-, -C(=O)S-, SC(=O)-, -N(Ra)C(=O)-, -C(=O)N(Ra)-, -N(Ra)C(=O)N(Ra)-, -OC(=O)N(Ra)- or - N(Ra)C(=O)O-, and the other of G1 or G2 is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) , -S-S-, -C(=O)S-, -SC(=O)-, -N(Ra)C(=O)-, -C(=O)N(Ra)-, -N(Ra)C(=O)N(Ra)-, - OC(=O)N(Ra)- or -N(Ra)C(=O)O- or a direct bond;
L is, at each occurrence, ~O(C=O)-, wherein ~ represents a covalent bond to X;
X is CRa;
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 ; Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, Ci-
C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxy alkyl, C1-C12 alkoxycarbonyl, Ci-
C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;
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;
R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000056_0002
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1 ; c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000056_0003
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
Figure imgf000056_0001
C(=O) Ra-, , RaC(=O) Ra-, -OC(=O) Ra- or -NRaC(=O)O- or a direct bond;
G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
Ra is H or C1-C12 alkyl;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4 or - R5C(=O)R4;
R4 is C1-C12 alkyl;
R5 is H or Ci-C6 alkyl; and x is 0, 1 or 2.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000057_0002
or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
L1 and L2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, - C(=O)S-, -SC(=O)-, - RaC(=O)-, -C(=O) Ra-, - RaC(=O) Ra-, -OC(=O) Ra-, - RaC(=O)O- or a direct bond;
G1 is C1-C2 alkylene, - (C=O)-, -O(C=O)-, -SC(=O)-, - RaC(=O)- or a direct bond:
Figure imgf000057_0001
direct bond;
G3 is Ci-Ce alkylene;
Ra is H or C1-C12 alkyl;
Rla and Rlb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rla is H or Ci -C 12 alkyl, and Rlb together with the carbon atom to which it is bound is taken together with an adjacent Rlb and the carbon atom to which it is bound to form a carbon-carbon double bond;
R2a and R2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2a is H or C1-C12 alkyl, and R2b together with the carbon atom to which it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R3a and R3b are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R3a is H or C1-C12 alkyl, and R3b 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;
R4a and R4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4a is H or C1-C12 alkyl, and R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R5 and R6 are each independently H or methyl;
R7 is C4-C20 alkyl;
R8 and R9 are each independently C1-C12 alkyl; or R8 and R9, 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.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000058_0001
or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
L1 and L2 are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;
Rla and Rlb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) Rla is H or Ci -C 12 alkyl, and Rlb together with the carbon atom to which it is bound is taken together with an adjacent Rlb and the carbon atom to which it is bound to form a carbon-carbon double bond;
R2a and R2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R2a is H or C1-C12 alkyl, and R2b together with the carbon atom to which it is bound is taken together with an adjacent R2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R3a and R3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R3a is H or C1-C12 alkyl, and R3b together with the carbon atom to which it is bound is taken together with an adjacent R3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R4a and R4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R4a is H or C1-C12 alkyl, and R4b together with the carbon atom to which it is bound is taken together with an adjacent R4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
R5 and R6 are each independently methyl or cyclo alkyl; R7 is, at each occurrence, independently H or C1-C12 alkyl; R8 and R9 are each independently unsubstituted C1-C12 alkyl; or R8 and R9, 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 e is 1 or 2, provided that: at least one of Rla, R2a, R3a or R4a is C1-C12 alkyl, or at least one of L1 or L2 is -O(C=O)- or -(C=O)O-; and
Rla and Rlb are not isopropyl when a is 6 or n-butyl when a is 8.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000059_0001
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, and
R4 and R5 are the same or different, each a lower alkyl.
In some embodiments, the lipid nanoparticle comprises an ionizable lipid having the structure:
Figure imgf000059_0002
or a pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000060_0001
pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000060_0002
or a pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000060_0003
L), or a pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000060_0004
pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000060_0005
(XXII-L), or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000061_0001
pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000061_0002
pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000061_0003
pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000061_0004
(XXVI-L), or a pharmaceutically acceptable salt thereof.
In some embodiments, the lipid nanoparticle comprises a lipid having the structure:
Figure imgf000061_0005
pharmaceutically acceptable salt thereof.
Non-cationic lipids
In certain embodiments, the lipid nanoparticles described herein comprise one or more non-cationic lipids. Non-cationic lipids may be phospholipids. In some embodiments, the lipid nanoparticle comprises 5-25 mol% non-cationic lipid. For example, 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. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.
In some embodiments, 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-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.
In some embodiments, the lipid nanoparticle comprises 5 - 15 mol%, 5 - 10 mol%, or 10 - 15 mol% DSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.
In certain embodiments, 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. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 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. For example, 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.
Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, 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). Under appropriate reaction conditions, 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.
In some embodiments, 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 (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,l,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof. In certain embodiments, a phospholipid is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially is a compound of Formula (IX):
Figure imgf000064_0001
or a salt thereof, wherein: each R1 is independently optionally substituted alkyl; or optionally two R1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R1 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;
A is of the formula:
Figure imgf000064_0002
each instance of L2 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(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN); each instance of R2 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 R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), - OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, - OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or - N(RN)S(O)2O; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;
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:
Figure imgf000065_0001
wherein each instance of R2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.
In some embodiments, the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT/US2018/037922.
In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% noncationic lipid relative to the other lipid components. For example, 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. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% non-cationic lipid.
In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% phospholipid relative to the other lipid components. For example, 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. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% phospholipid lipid.
Structural Lipids
The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term “structural lipid” includes sterols and also to lipids containing sterol moieties.
Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. 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. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.
In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No. 16/493,814. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% structural lipid relative to the other lipid components. For example, 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. In some embodiments, the lipid nanoparticle comprises a molar ratio of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% structural lipid.
In some embodiments, 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. For example, 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. In some embodiments, the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.
In some embodiments, the lipid nanoparticle comprises 35 - 40 mol% cholesterol. For example, the lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.
Polyethylene Glycol (PEG)-Lipids
The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids.
As used herein, the term “PEG-lipid” or “PEG-modified lipid” refers to polyethylene glycol (PEG) -modified lipids. Non-limiting examples of 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. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
In some embodiments, 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) . In some embodiments, 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. In some embodiments, the PEG-modified lipid is PEG- DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and/or PEG-DPG.
In some embodiments, 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. In some embodiments, a PEG moiety, for example an mPEG-NEh, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG-lipid is PEG2k-DMG.
In some embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of 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.
In general, some of the other lipid components (e.g., PEG lipids) 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. For example, a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG- DMG has the following structure:
Figure imgf000067_0001
In some embodiments, 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. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, 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. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment.
In certain embodiments, a PEG lipid is a compound of Formula (X):
Figure imgf000068_0001
or salts thereof, wherein:
R3 is -OR°;
R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive;
L1 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(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);
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;
A is of the formul
Figure imgf000068_0002
each instance of L2 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(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN); each instance of R2 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 R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), - OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O) , OS(O), S(O)O, - OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or - N(RN)S(O)2O; each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;
Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.
In certain embodiments, the compound of Fomula (X) is a PEG-OH lipid (i.e., R3 is - OR°, and R° is hydrogen). In certain embodiments, the compound of Formula (X) is of Formula (X-OH):
Figure imgf000069_0001
or a salt thereof.
In certain embodiments, 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):
Figure imgf000069_0002
or a salts thereof, wherein:
R3 is-OR°;
R° is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive;
R5 is optionally substituted C10-40 alkyl, optionally substituted C10-40 enyl, or optionally substituted C 10-40 alkynyl; and optionally one or more methylene groups of R5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), - NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), - NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), - S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), - N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.
In certain embodiments, the compound of Formula (XI) is of Formula (XI-OH):
Figure imgf000070_0001
or a salt thereof. In some embodiments, r is 40-50.
In yet other embodiments the compound of Formula (XI) is:
Figure imgf000070_0002
or a salt thereof.
In some embodiments, the compound of Formula (XI) is
Figure imgf000070_0003
In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.
In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. US 15/674,872.
In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG lipid relative to the other lipid components. For example, 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. In some embodiments, 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.
In some embodiments, 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%. In some embodiments, the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid. For example, 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%. In some embodiments, 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. In 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).
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid,
10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid,
11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.
In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.
In some embodiments, a LNP comprises an N:P ratio of from about 2:1 to about 30:1.
In some embodiments, a LNP comprises an N:P ratio of about 6:1.
In some embodiments, a LNP comprises an N:P ratio of about 3:1, 4:1, or 5:1.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, a lipid nanoparticle may comprise two or more components (e.g., amino lipid and nucleic acid, PEG-lipid, phospholipid, structural lipid). For instance, 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.
Effective in vivo delivery of nucleic acids represents a continuing medical challenge. Exogenous nucleic acids (i.e., originating from outside of a cell or organism) are readily degraded in the body, e.g., by the immune system. Accordingly, effective delivery of nucleic acids to cells often requires the use of 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. To achieve minimal particle aggregation and pre-delivery stability, many conventional particulate carriers have relied on the presence and/or concentration of certain components (e.g., PEG-lipid). However, it has been discovered that certain components may decrease the stability of encapsulated nucleic acids (e.g., mRNA molecules). The reduced stability may limit the broad applicability of the particulate carriers. As such, there remains a need for methods by which to improve the stability of nucleic acid (e.g., mRNA) encapsulated within lipid nanoparticles.
In some embodiments, 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.
In some embodiments, 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. In certain embodiments, 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. In some embodiments, 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.
In general, an ionizable molecule comprises one or more charged groups. In some embodiments, an ionizable molecule may be positively charged or negatively charged. For instance, an ionizable molecule may be positively charged. For example, an ionizable molecule may comprise an amine group. As used herein, 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. As used herein, 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). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of 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. In general, the charge density of the molecule and/or matrix may be selected as desired.
In some cases, an ionizable molecule (e.g., an amino lipid or ionizable lipid) may include one or more precursor moieties that can be converted to charged moieties. For instance, the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above. As a non-limiting specific example, the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively. Those of ordinary skill in the art will be able to determine whether a given chemical moiety carries a formal electronic charge (for example, by inspection, pH titration, ionic conductivity measurements, etc.), and/or whether a given chemical moiety can be reacted (e.g., hydrolyzed) to form a chemical moiety that carries a formal electronic charge.
The ionizable molecule (e.g., amino lipid or ionizable lipid) may have any suitable molecular weight. In certain embodiments, 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. In some instances, 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. Combinations of the above ranges (e.g., at least about 200 g/mol and less than or equal to about 2,500 g/mol) are also possible. In embodiments in which more than one type of ionizable molecules are present in a particle, each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above.
In some embodiments, 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 or equal to about 60%, greater than or equal to about 62%, greater than or equal to about 65%, or greater than or equal to about 68%. In some instances, 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.). In embodiments in which more than one type of ionizable molecule is present in a particle, 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) 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). Those of ordinary skill in the art would be knowledgeable of techniques to determine the quantity of a component using the above-referenced techniques. For example, 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.
It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given their ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way. According to the disclosures herein, 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.
In some embodiments, 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. As used herein, “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.
In some embodiments 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). In exemplary aspects, the vaccines feature an mRNA or mRNAs encoding antigen(s) derived from infectious diseases.
Thus, the disclosure encompasses infectious disease vaccines. The antigen of the infectious disease vaccine is a viral antigen. In some embodiments, the infectious agent is a human cytomegalovirus (hCMV).
In some embodiments, a disease, disorder, or condition is caused by or associated with a member of the herpesvirus family. In some embodiments, the virus is a human cytomegalovirus (hCMV). In some embodiments, the antigen is a human cytomegalovirus (hCMV) antigen. In some embodiments, 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. In some embodiments, the disease, disorder or condition is a disease or condition caused by or associated with human cytomegalovirus (hCMV).
In some embodiments, a composition disclosed herein does not comprise a pharmaceutical preservative. In other embodiments, a composition disclosed herein does comprise a pharmaceutical preservative. Non-limiting examples of 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. In some embodiments, a composition 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.
In some embodiments, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful. In some embodiments, a composition disclosed herein is administered to a subject enterally. In some embodiments, an enteral administration of the composition is oral. In some embodiments, a composition disclosed herein is administered to the subject parenterally. In some embodiments, 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.
To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease, disorder or condition experienced by a subject. The 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. For example, 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. As is well known in the medical and veterinary arts, 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.
In some embodiments, 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.
In certain embodiments, LNP preparations (e.g., populations or formulations) 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. As used herein “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 (FA) 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. In some embodiments, the compositions are used to deliver a pharmaceutically active agent. In some instances, 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.
Once the compositions have been prepared, they may be combined with pharmaceutically acceptable excipients to form a pharmaceutical composition. As would be appreciated by one of skill in this art, the 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.
Pharmaceutical compositions described herein and for use in accordance with the embodiments described herein may include a pharmaceutically acceptable excipient. As used herein, the term “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; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical 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. In addition to the active ingredients (i.e., the particles), 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. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Injectable preparations, for example, 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, ethanol, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, 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. In such solid dosage forms, 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 monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.
Solid 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.
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. Such 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.
In other embodiments, the compositions are loaded and stored in prefilled syringes and cartridges for patient-friendly autoinjector and infusion pump devices.
Some embodiments relate to kits for use in preparing or administering the compositions described herein. A kit for forming compositions 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. In certain embodiments, 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. For example, the kit may include 1-100 dosage units. In certain embodiments, the kit includes a week supply of dosage units, or a month supply of dosage units. In certain embodiments, 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).
The term “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. Other pharmaceutically acceptable 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, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. 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.
As disclosed herein, the terms “composition” and “formulation” are used interchangeably.
Nucleic acids
Some aspects of the disclosure relate to compositions and methods of preparing lyophilized compositions comprising a nucleic acid in a lipid nanoparticle. In some embodiments, the 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. When present in lipid nanoparticles, nucleic acids, e.g., mRNAs, 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. As used herein, the term “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))). As used herein, the term nucleic acid refers to polyribonucleotides as well as polydeoxyribonucleotides. The term 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) may include a substitution and/or modification. In some embodiments, the substitution and/or modification is in one or more bases and/or sugars. For example, in some embodiments a nucleic acid (e.g., mRNA) 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. Thus, in some embodiments, a substituted or modified nucleic acid (e.g., mRNA) includes a 2'-O-alkylated ribose group. In some embodiments, a modified nucleic acid (e.g., mRNA) 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. Thus, in some embodiments, a nucleic acid (e.g., mRNA) 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. In some embodiments, an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, 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.
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). In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
The terms 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 (e.g., mRNA) 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 (NMP) 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, for example, 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.
It should be understood that the term “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, e.g., in an IVT reaction, include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and 5 -methyluridine triphosphate (m5UTP). In some embodiments, adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and/or uridine diphosphate (UDP) are used.
Examples of 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. Examples of antiviral nucleotide/nucleoside analogs include, but are not limited, to Ganciclovir, Entecavir, Telbivudine, Vidarabine and Cidofovir.
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.
In some embodiments, the nucleic acid is not self-amplifying. Self-amplifying nucleic acids (e.g., self-amplifying RNAs) encode 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. Because a self-amplifying nucleic acid may be replicated in cell to which it is introduced, 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).
In some embodiments, 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.
Chemically unmodified nucleotides
In some embodiments, at least one RNA (e.g., mRNA) of a lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).
Chemical modifications
The lyophilized hCMV immunogenic compositions (e.g., mRNA vaccines) 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. In some embodiments, nucleotides and nucleosides comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
In some embodiments, 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.
In some embodiments, 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.
Hence, nucleic acids (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA 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), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
In some embodiments, 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.
In some embodiments, 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), 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.
In some embodiments, one or more nucleic acids (e.g., RNA nucleic acids, such as mRNA 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. One example of such 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.
In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (ely), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (y). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, 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.
In some embodiments, a mRNA comprises 1-methyl-pseudouridine (mly) substitutions at one or more or all uridine positions of the nucleic acid.
In some embodiments, 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.
In some embodiments, a mRNA comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.
In some embodiments, 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..
In some embodiments, a mRNA comprises uridine at one or more or all uridine positions of the nucleic acid.
In some embodiments, mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, 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. Similarly, 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. For example, 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) may be uniformly modified in a nucleic acid, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a nucleic acid (or in a sequence region thereof) 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 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.
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. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, 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). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the 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 antigens
HCMV includes several surface glycoproteins that are involved in viral attachment and entry into different cell types. The pentameric complex (PC), 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. For example, while entry into fibroblasts requires the gH/gL heterodimer, entry into epithelial and endothelial cells requires the pentameric complex gH/gL/UL128/UL130/ UL131 in addition to gH/gL. Thus, different gH/gL complexes engage distinct entry receptors on epithelial/endothelial cells and fibroblasts. Receptor engagement is followed by membrane fusion, a process mediated by gB and gH/gL. Early antibody studies have supported critical roles for both gB and gH/gL in hCMV entry. gB is essential for entry and cell spread. 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. Thus, the four glycoprotein complexes play a crucial role in viral attachment, binding, fusion and entry into the host cell.
Studies involving the gH/gL/UL128-131A complex have shown that hCMV glycoproteins gB, gH, gL, gM, and gN, as well as UL128, UL130, and UL131A proteins, are immunogenic and involved in the immunostimulatory response in a variety of cell types. Moreover, UL128, UL130, and UL131A genes are relatively conserved among hCMV isolates and therefore represent an attractive target for vaccination. Furthermore, 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.
Without wishing to be bound by any theory, 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 (e.g., gH/gL/UL128/UL130/UL131A) represent major antigenic targets of antiviral immune responses. Some embodiments relate to RNA (e.g., mRNA) vaccines that include polynucleotides encoding an HCMV antigen, in particular an HCMV antigen from one of the HCMV glycoprotein complexes. Some embodiments relate to 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.
The entire contents of International Application No. PCT/US2015/027400 (WO 2015/164674), entitled “Nucleic Acid Vaccines,” International Application No. PCT/US2016/058310 (W02017/070613), entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” International Application No. PCT/US2017/057748 (W02018/075980), entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” International Application No. PCT/US2020/050392 (WO2021/050864), entitled “HUMAN CYTOMEGALOVIRUS VACCINE”, International Application No. PCT/US 2021/047541 (WO2022/046898), entitled “HUMAN CYTOMEGALOVIRUS VACCINE”, US Patent No. 10,064,935, entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” US Patent No. 10,383,937, entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” and US Patent No. 10,064,935, entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” and US Patent No. 10,716,846, entitled “HUMAN CYTOMEGALOVIRUS VACCINE,” are incorporated herein by reference.
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.
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). In some embodiments 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. In some embodiments 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.
In some embodiments, 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). In some embodiments, 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). 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), 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). In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is about 1.5: 1 : 1 : 1 : 1 : 1.5. In some embodiments, the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2
In some embodiments, the hCMV vaccine components comprise the sequences provided in Table 3.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the mRNA components of a hCMV immunogenic composition (e.g., mRNA vaccine) 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.
In accordance with some embodiments of methods of lyophilizing a composition comprising a lipid nanoparticle and an mRNA encoding one or more hCMV antigens, 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. In some embodiments, 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. In some embodiments, 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. NOs: 2 and/or 8); (d) 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); (e) 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); and/or (f) 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). In some embodiments where 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:l. In certain embodiments where 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. 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),
(e), and/or (f), the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
In accordance with some embodiments of lyophilized pharmaceutical compositions comprising a lipid nanoparticle and an mRNA encoding one or more hCMV antigens, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. NOs: 2 and/or 8); (d) 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); (e) 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); and/or (f) 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). In some embodiments where 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:l. In certain embodiments where 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. 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), (e), and/or (f), the molar ratio of (a):(b):(c):(d):(e):(f) is about 2:1:1:1:1:2.
In some embodiments, 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.
It should be understood that the hCMV immunogenic compositions (e.g., mRNA vaccines) may comprise a signal sequence. It should also be understood that 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.
Without wishing to be bound by any theory, the hCMV immunogenic compositions (e.g., mRNA vaccines) described herein, in which molar ratios are used to determine the amounts of each mRNA component, 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. In some embodiments, 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. “Purity,” as used herein, 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.In some embodiments, the RNA (e.g., mRNA) 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. For example, in certain embodiments, the RNA (e.g., mRNA) 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. According to certain embodiments, the RNA (e.g.. mRNA) 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.
The term “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. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) 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). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, 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. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic residues to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the 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.
As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of antigens of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical) of a reference protein, provided that the fragment is immunogenic and confers a protective immune response to the human cytomegalovirus (hCMV). In addition to variants that are identical to the reference protein but are truncated, in some embodiments, 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 entire contents of International Application Nos. PCT/US2015/027400, PCT/US2016/043348, PCT/US2016/043332, PCT/US2016/058327, PCT/US2016/058324, PCT/US2016/058314, PCT/US2016/058310, PCT/US2016/058321, PCT/US2016/058297, PCT/US2016/058319, and PCT/US2016/058314 are incorporated herein by reference.
Lyophilized compositions
In some embodiments, the methods comprise obtaining lyophilized compositions with a moisture content below a certain value. As used herein, “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)).
In some embodiments, 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.
In some embodiments, the methods comprise obtaining lyophilized compositions in which the coefficient of degradation of a nucleic acid in the composition is below a predetermined value. As used herein, a “coefficient of degradation” refers to a parameter of an equation describing the loss of nucleic acid purity over time. As used herein, “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. Water molecules can hydrolyze the phosphodiester bond that bridges a phosphate moiety and sugar moiety in the sugar-phosphate backbone of a nucleic acid, resulting in the production of two separate nucleic acid molecules, neither of which contains an intact sequence encoding the full-length protein encoded by the unhydrolyzed nucleic acid. 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. 27(6): 1083-1092). In HPLC, a sample to be analyzed, such as nucleic acid, is dissolved in a solvent (mobile phase) and passed through a column containing a solid material (stationary phase), with a detector measuring the presence of dissolved sample molecules as the mobile phase is eluted from the column. 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. To calculate the purity of a nucleic acid using HPLC analysis, 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
AUC (main peak) equation % purity =
Loss of nucleic acid purity over time may be described by a differential equation of the dP form — = — P, where P is nucleic acid purity (%), is the coefficient of degradation, and dP/dt is the rate of change in nucleic acid purity. Alternatively, nucleic acid purity over time may be described by an equation of the form P(/) = Poe~ , where P(t) is nucleic acid purity (%) at a given time, /, Po is initial nucleic acid purity at time t=0, e is the base of the natural logarithm, and is the coefficient of degradation. In both equation forms, a positive value of indicates exponential decay, while a negative indicates exponential growth, with larger absolute values of indicating faster decay or growth, respectively. In some embodiments, the coefficient of degradation is expressed in units of month 1. In some embodiments, 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. In some embodiments, the coefficient of degradation is 0.02 month 1 or less. In some embodiments, the coefficient of degradation is 0.01 month 1 or less. In some embodiments, 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. In some embodiments, the coefficient of degradation is 0.004 month 1 or less.
In some embodiments, 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).In some embodiments, 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 length of time for which a composition will comprise at least 50% pure nucleic acid can reliably be predicted by measuring a) the initial purity of the nucleic acid in a composition, and b) the coefficient of degradation of nucleic acid, as described above, then using the equation P(/) = to calculate the value of t at which P(/) = 50% or 0.5. This length of time is given by
Figure imgf000102_0002
the formula t = if expressed as a percentage or t = if p0 is expressed
Figure imgf000102_0003
Figure imgf000102_0001
as a proportion.
In some embodiments, 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. In some embodiments, 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. 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 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.
In some embodiments, substantially no cholesterol crystals (e.g., 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).
Some aspects of the disclosure relate to a lyophilized composition produced by any of the methods provided herein. In some embodiments, the composition is in a solid form.
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 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.
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 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.
In some embodiments of the lyophilized compositions, the composition has a moisture content of less than or equal to 6.0% w/w. In some embodiments of the lyophilized compositions, the PEG-modified lipid is a C18 stearic-OH -PEG. In some embodiments, the composition is stable for at least about 9 months upon storage at about 4°C.
In some embodiments of the lyophilized compositions, 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.
In some embodiments, the PEG-modified lipid is:
Figure imgf000103_0001
In some embodiments, the ionizable amino lipid is:
Figure imgf000103_0002
In some embodiments, 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. By contrast, 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.
Some embodiments of lyophilized pharmaceutical compositions and/or compositions made by lyophilization methods comprise low moisture contents. Without wishing to be bound by theory, it is believed that 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. 44(3):689-700; Blanco et al., J Pharm Biomed Anal. 1997. 16(2): 255-262; Zhou et al., J Pharm Sci. 2003. 92(5): 1058-1065. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, upon reconstitution the lipid nanoparticle 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the reconstitituted lipid nanoparticle has a diameter from 120-200 nm, 120-180 nm, 120-160 nm, 120-150 nm, or 120-140 nm. In some embodiments, 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.
In some embodiments, 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).
In some embodiments, 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. In some embodiments, the coefficient of degradation is at most 0.02 month 1. In some embodiments, the coefficient of degradation is at most 0.01 month 1. In some embodiments, 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. In some embodiments, the coefficient of degradation is between 0.0001 and 0.02 month’ x. In some embodiments, the coefficient of degradation is between 0.001 and 0.01 month’1.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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. For example, in some embodiments, 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. 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 5.0% 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 4.0% 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 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. 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 1.5% 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.
In some embodiments, 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. For example, 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 6.0% 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 5.0% 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 4.0% 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 3.5% 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 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. 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 1.0%. 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.
In some embodiments, 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. In some embodiments, the storage is conducted at a temperature between 2-8 °C. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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,
Figure imgf000108_0001
the lyophilized compositions comprise mRNA with at least 96% relative purity after 1 month of storage between 30-60 °C. In some embodiments, 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.
In some embodiments, 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%. In some embodiments, 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%. In some embodiments, 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%.
In some embodiments, 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. In some embodiments, the lyophilized compositions comprise mRNA with at least 90% relative purity after 12 months of storage between 2-8 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 90% relative purity after 24 months of storage between 2-8°C. In some embodiments, 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.
In some embodiments, 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. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 12 months of storage between 20-30 °C. In some embodiments, the lyophilized compositions comprise mRNA with at least 80% relative purity after 24 months of storage between 20-30 °C. In some embodiments, 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.
In some embodiments, lipid nanoparticles of the lyophilized compositions comprise lipid nanoparticles comprising mRNA with an encapsulation efficiency of 70%. As used herein, “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. In 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.) is calculated by the equation
Figure imgf000110_0001
In some embodiments, 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%.
In some embodiments, 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.
In some embodiments, 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. As used herein, “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 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
Figure imgf000111_0001
Some embodiments comprise reconstituting a lyophilized lipid nanoparticle composition, e.g., in a reconstitution buffer suitable for pharmaceutical administration. In some embodiments, the lyophilized lipid nanoparticle composition is reconstituted in water. In some embodiments, 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
5%, about 10%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about
90% salt solution. In some embodiments, the reconstitution buffer is at a physiological pH (e.g, pH 7-7.5, such as 7.4). Some embodiments comprise a reconstituted lipid nanoparticle composition. In some embodiments, 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°C4 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.
Dosing and administration
In some embodiments, 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) can be used as therapeutic or prophylactic agents. In some aspects, the lyophilized hCMV immunogenic compositions (e.g., mRNA vaccines) are used to provide prophylactic protection from hCMV. In some aspects, the lyophilized hCMV immunogenic compositions (e.g., mRNA vaccines) are used to treat a hCMV infection. In some embodiments, 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.
A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject.
In some embodiments, 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. 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 is provided in aspects. The method 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). In some embodiments, 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). In some embodiments, 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.
In some embodiments, a prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. In some embodiments, 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. In some embodiments, 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.
A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines described herein. For instance, 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. In exemplary embodiments, 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).
In some embodiments, 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 is provided in other aspects. The method 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.
In some embodiments, 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). In some embodiments, 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). In some embodiments, 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).
In other embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In other embodiments, 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. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.
Other aspects 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. In some embodiments, 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.
In some embodiments, 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 (e.g., mRNA vaccine) 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) is typically in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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.
In some embodiments, 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 |ig, 24 pg 25 pg, 26 pg, 27 pg, 28 pg, 29 pg, 30 pg, 31 pg, 32 pg, 33 pg, 34 pg, 35 pg, 36 pg, 37 pg, 38 pg, 39 pg, 40 pg, 41 pg , 42 pg, 43 pg , 44 pg, 45 pg, 46 pg, 47 pg, 48 pg, 49 pg, 50 |ig, 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 |ig, 78 pg, 79 pg, 80 pg, 81 pg , 82 pg , 83 pg , 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, or 500 pg, including all values in between.
As used herein, the dose of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) refers to total pg mRNA in lipid nanoparticles. As used herein, “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.
In some embodiments, 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine including mRNAs encoding gH/gL/UL128/UL130/UL131A/gB), as provided herein, may be as low as 150 pg, administered for example as a single dose.
In some embodiments, the effective amount of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is a single dose of 50-150 pg. For example, 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 pg , 82 pg , 83 pg , 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, or 150 pg, including all values in between. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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.
In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is either 50 pg, 100 pg or 150 pg. In some embodiments, the effective dose is administered as a primary immunization followed by a single boost of the same effective dose. In some embodiments, the effective dose is administered as a primary immunization followed by two sequential booster immunizations of the same effective dose. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the booster immunizations should be at least two weeks apart.
In some embodiments, the effective amount of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is two doses of 50-150 pg. For example, 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 , 82 pg , 83 pg , 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, or 150 pg, including all values in between. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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.
In some embodiments, the effective amount of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is three doses of 50-150 pg. For example, 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, 82 pg, 83 pg, 84 pg, 85 pg, 86 pg, 87 pg, 88 pg, 89 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, or 150 pg, including all values in between. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. 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. 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.
In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) relates to the amount of integral mRNA in the composition. As used herein, “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 (e.g., mRNA vaccine) is related to the length, rate of degradation, and the length of time from which the immunogenic composition is produced. When the effective amount is determined from clinical results, 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).
In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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.
In certain embodiments, an effective amount or dose of the lyophilized hCMV immunogenic composition (e.g. mRNA vaccine) does not require that the pmoles of each component are equal. For example, 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. In addition, in some embodiments the picomolar dose of each of the 6 mRNA may be individually determined because of stability or other biochemical or biophysical requirements.
In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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,
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.
In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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-
30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, or 40-50 pmol) integral gB mRNA. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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,
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) integral gB mRNA.
In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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. In some embodiments, the effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) 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,
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) integral gB mRNA.
In some embodiments, in certain of the integral mRNA doses described herein, 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.
In some embodiments, one, two, three, or more than three doses (of any of the doses described herein) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) are administered to a subject. In some embodiments, one, two, or three doses (of any of the doses described herein) of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) are administered to a subject. In some embodiments, 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).
In some embodiments, 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
32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day
44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 53, day 54, day 55, day
56, day 57, day 58, day 59, day 60, day 61, day 62, day 63, day 64, day 65, day 66, day 67, day
68, day 69, day 70, day 71, day 72, day 73, day 74, day 75, day 76, day 77, day 78, day 79, day
80, day 81, day 82, day 83, day 84, day 85, day 86, day 87, day 88, day 89, day 90, day 91, day
92, day 93, day 94, day 95, day 96, day 97, day 98, day 99, day 100, day 101, day 102, day 103, day 104, day 105, day 106, day 107, day 108, day 109, day 110, day 111, day 112, day 113, day 114, day 115, day 116, day 117, day 118, day 119, day 120, day 121, day 122, day 123, day 124, day 125, day 126, day 127, day 128, day 129, day 130, day 131, day 132, day 133, day 134, day 135, day 136, day 137, day 138, day 139, day 140, day 141, day 142, day 143, day 144, day 145, day 146, day 147, day 148, day 149, day 150, day 151, day 152, day 153, day 154, day 155, day 156, day 157, day 158, day 159, day 160, day 161, day 162, day 163, day 164, day 165, day 166, day 167, day 168, day 169, day 170, day 171, day 172, day 173, day 174, day 175, day 176, day 177, day 178, day 179, day 180, day 181, day 182, day 183, day 184, day 185, day 186, day 187, day 188, day 189, day 190, day 191, day 192, day 193, day 194, day 195, day 196, day 197, day 198, day 199. In some embodiments, a dose of lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) is administered to a subject after day 199.
The 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).
Vaccine efficacy
Some aspects of the disclosure provide 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. Also provided herein are methods of inducing an antigen- specific immune response in a subject.
As used 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. As used herein, 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. One important aspect of cellular immunity involves an antigen- specific response by cytolytic T-cells (CTLs). 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. 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. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells. In some embodiments, 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 (ELISA) is a common assay for determining antibody titers, for example.
In some embodiments, 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.
In some embodiments, an anti-hCMV antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, 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. In some embodiments, 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. In some embodiments, the anti-hCMV antigen antibody titer produced in the subject is increased by 1-5 log relative to a control. For example, 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.
In some embodiments, the anti-hCMV antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, 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. In some embodiments, 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. In some embodiments, the anti-hCMV antigen antibody titer produced in a subject is increased 2-10 times relative to a control. For example, 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.
In some embodiments, 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. 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.
In some embodiments, administration of an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits serum neutralizing antibody titers against hCMV. In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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. In some embodiments, the timepoint is after administration of two doses of the immunogenic composition. In some embodiments, the timepoint is after administration of three doses of the lyophilized hCMV immunogenic composition.
In some embodiments, 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.
In some embodiments, administration of an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits serum neutralizing antibody titers against hCMV gB protein. In some embodiments, administration of 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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.
In some embodiments, administration of an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits antigen- specific T-cell response against hCMV. In some embodiments, 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. In some embodiments, administration of an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits antigen- specific T-cell response against hCMV gB protein. In some embodiments, 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 antigenspecific T-cell response against hCMV gB protein. In some embodiments, administration of an effective amount of the lyophilized hCMV immunogenic composition (e.g., mRNA vaccine) elicits antigen- specific T-cell response against hCMV pentamer. In some embodiments, 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 pentamer. In some embodiments, the T-cell response (e.g., against hCMV, hCMV gB protein, or hCMV pentamer) comprises interferon-y (IFN-y) secretion.
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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the GMT of serum neutralizing antibodies to hCMV increases in a dose-dependent manner.
In some embodiments, the GMT of binding antibody response to hCMV pentamer (antipentamer antibody titer) 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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. In some embodiments, the timepoint is after administration of two doses of the immunogenic composition. In some embodiments, the timepoint is after administration of three doses of the lyophilized hCMV immunogenic composition.
In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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. In some embodiments, the timepoint is after administration of a single dose of the immunogenic composition. In some embodiments, the timepoint is after administration of two doses of the immunogenic composition. In some embodiments, the timepoint is after administration of three doses of the immunogenic composition. In some embodiments the GMT response reaches a maximum within about 10 days to 2 weeks after a dose is administered.
In some embodiments, 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.
In some embodiments, the ability of the hCMV mRNA vaccine to be effective is measured in a murine model. For example, 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. For example, 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)).
In some embodiments, 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.
In some embodiments, 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:
Efficacy = (ARU - ARV)/ARU x 100; and
Efficacy = (1-RR) x 100. Likewise, 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. For example, 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:
Effectiveness = (1 - OR) x 100.
In some embodiments, efficacy of the hCMV mRNA vaccine is at least 60% relative to unvaccinated control subjects. For example, 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. Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host. In some embodiments, the effective amount of an hCMV mRNA vaccine is sufficient to provide sterilizing immunity in the subject for at least 1 year. For example, 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. In some embodiments, 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. For example, 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.
Detectable Antigen. In some embodiments, 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.
Titer. 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, the neutralizing antibody titer is at least 100 NT50. For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50.
In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL.
In some embodiments, an anti-hCMV antigen antibody titer produced in the subject is increased by at least 1 log relative to a control. For example, 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.
In some embodiments, an anti-hCMV antigen antibody titer produced in the subject is increased at least 2 times relative to a control. For example, 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.
In some embodiments, 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.
EXAMPLES
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. In the 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. In the primary drying phase, 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. In the secondary drying phase, the compositions were heated to a higher temperature, either 25 °C or 40 °C, to allow evaporation of remaining water.
Annealing step
During the freezing phase, 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.
Secondary drying phase
Following completion of the primary drying phase, as determined by the Pirani profile, residual water was removed from the composition by a secondary drying phase. An overview of the parameters of the lyophilization process, including the secondary drying phase, is given by FIG. 4A. To test the effect of temperature in the secondary drying phase, 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. As shown in 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. This reduction in moisture content was achieved without substantially compromising the integrity of the mRNA, as the 40 °C secondary drying phase only marginally affected the particle size, encapsulation efficiency, mRNA purity, and in vitro potency of lyophilized LNP-mRNA compositions (FIGs. 4C-4F). In each pair of bars in FIGs. 4C-4F, the left bar represents 25 °C drying and the right bar represents 40 °C drying.
Stability of lyophilized compositions
The effect of moisture content on the stability of mRNA in lyophilized LNP-mRNA compositions was evaluated by measuring the mRNA purity over time in compositions with varying moisture contents. From these measurements, the rate constant of mRNA degradation during storage of the LNP-mRNA compositions at 5 °C was regressed (FIG. 3). This analysis revealed that mRNA degrades faster in compositions with higher moisture contents.
The purity of mRNA in lyophilized compositions was also measured during storage at 25 °C for several months (FIG. 5A). 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. Thus, 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. From these analyses of mRNA stability based on moisture content, the degradation constants of lyophilized LNP-mRNA compositions with various moisture contents during storage at 5 °C were estimated (Table 1).
Table 1: Predicted stability of lyophilized LNP-mRNA compositions at 5 °C.
Figure imgf000130_0001
In a second experiment, multiple lots of lyophilized LNP-mRNA compositions were stored for several months at 5 °C, and the purity of mRNAs in the compositions was monitored over 12, 18, or 24 months (FIGs. 5B-5C). Each lot included an LNP-mRNA composition containing an mRNA encoding an hCMV glycoprotein B (gB), and another LNP-mRNA composition containing an mRNA encoding hCMV glycoprotein H (gH). Lyophilized compositions contained moisture contents of 0.20-0.65 %w/w. Observations of mRNA purity over 12-24 months of storage were used to estimate the first-order degradation constants for each mRNA in lyophilized LNP-mRNA compositions, shown below in Table 2. Based on a starting mRNA purity of 80%, the predicted shelf life of mRNAs (duration of time during which mRNA in lyophilized LNP-mRNA compositions remains at least 50% pure) was also estimated.
Table 2: First-order degradation rate estimates for lyophilized LNP-mRNA compositions at 5 °C.
Figure imgf000131_0001
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. 18-20 hours post-transfection, cells were harvested, stained with an antibody specific to hCMV pentamer, and the intensity of hCMV pentamer expression was measured by flow cytometry. (FIG. 6). mRNA in lyophilized and reconstituted compositions was efficiently expressed after delivery to cells, with lyophilized compositions resulting in greater hCMV pentamer production than compositions stored at 4 °C or -20 °C.
Example 4: Immunization with lyophilized compositions.
Three distinct 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). For each group, the geometric mean antibody titers after the prime dose (bottom number) and boost dose (top number) are shown above the data points. In 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.
EQUIVALENTS AND SCOPE
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, 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. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, 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. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or 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.
It should be understood that, unless clearly indicated to the contrary, the disclosure of numerical values and ranges of numerical values in the specification includes both i) the exact value(s) or range specified, and ii) values that are “about” the value(s) or ranges specified (e.g., values or ranges falling within a reasonable range (e.g., about 10% similar)) as would be understood by a person of ordinary skill in the art.
It should also be understood that, unless clearly indicated to the contrary, in any methods disclosed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are disclosed.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
SEQUENCES
It should be understood that 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. It should also be understood that any of the mRNA constructs described herein may further comprise a polyA tail and/or cap (e.g., 7mG(5’)ppp(5’)NlmpNp). Further, while many of the mRNAs 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:
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 13)
3' UTR: UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCA CCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 14)
Table 3 - hCMV mRNA and antigen sequences
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001

Claims

CLAIMS What is claimed is:
1. A method of preparing a lyophilized composition, the method comprising lyophilizing a composition that comprises a lipid nanoparticle encapsulating an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
2. The method of claim 1, wherein 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.
3. The method of claim 1 or 2, wherein 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.
4. The method of any one of claims 1-3, wherein the one or more hCMV antigens comprises greater than or equal to 1 and less than or equal to 6 hCMV antigens.
5. The method of any one of claims 1-4, wherein 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.
6. The method of claim 5, wherein 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.
7. The method of claim 6, wherein the molar ratio of (a):(b):(c):(d):(e):(f) is about 2:1:1:1:1:2.
8. The method of any one of claims 5-7, wherein 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; and the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
9. The method of any one of claims 1-8, wherein the composition comprises a lyoprotectant.
10. The method of claim 9, wherein the lyoprotectant comprises a sugar.
11. The method of claim 9 or 10, wherein the lyoprotectant comprises sucrose.
12. The method of any one of claims 9-11, wherein the lipid nanoparticle comprises one or more lipids, and where the lipid nanoparticle composition comprises a lyoprotectant mass:lipid mass ratio of at least 5:1.
13. The method of claim 12, wherein 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.
14. The method of any one of claims 1-13, wherein the composition comprises a buffer.
15. The method of claim 14, wherein the buffer is selected from the group consisting of a Tris buffer, citrate buffer, phosphate buffer, triethylammonium bicarbonate (TEAB), and histidine buffer.
16. The method of claim 15, wherein the buffer is a Tris buffer.
17. The method of any one of claims 14-16, wherein the concentration of the buffer in the composition is about 1 mM to about 100 mM.
18. The method of claim 17, wherein the concentration of the buffer is about 10 mM to about 30 mM.
19. The method of any one of claims 1-18, wherein the composition has a pH of about 6.5 to about 8.5.
20. The method of claim 19, wherein the composition has a pH of about 7 to about 8.
21. The method of claim 20, wherein the composition has a pH of about 7.4 to about 8.
22. The method of any one of claims 1-21, wherein the lyophilizing comprises an annealing step.
23. The method of claim 22, wherein 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.
24. The method of claim 23, wherein the first temperature is from about -30 °C to about 0 °C.
25. The method of claim 24, wherein 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.
26. The method of any one of claims 23-25, wherein the second temperature is from about - 100 °C to about -30 °C.
27. The method of claim 26, wherein 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.
28. The method of any one of claims 23-27, further comprising 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.
29. The method of claim 28, wherein 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.
30. The method of any one of claims 22-29, wherein 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.
31. The method of any one of claims 23-30, wherein 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.
32. The method of any one of claims 1-31, wherein the lyophilizing comprises a sublimation step.
33. The method of claim 32, wherein the sublimation is performed at a vacuum pressure of from about 50 mTorr and about 300 mTorr.
34. The method of any one of claims 1-33, wherein the lyophilizing comprises a desorption step, wherein the desorption step comprises exposing the composition to a desorption temperature.
35. The method of claim 34, wherein the desorption temperature is about 10 °C or higher.
36. The method of claim 35, wherein 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.
37. The method of any one of claims 34-36, wherein the desorption step comprises exposing a sublimated composition to a desorption temperature of about 40 °C.
38. The method of any one of claims 34-37, wherein the desorption is conducted for at least 2 hours.
39. The method of claim 38, wherein the desorption is conducted for about 5 hours, about 10 hours, about 15 hours, or about 20 hours.
40. The method of any one of claims 34-39, wherein the desorption is performed at a vacuum pressure of from about 50 mTorr and about 300 mTorr.
41. The method of any one of claims 1-40, wherein 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.
42. The method of any one of claims 1-41, wherein 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.
43. The method of claim 42, wherein 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.
44. The method of any one of claims 1-43, wherein 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.
45. The method of claim 44, wherein the storage is conducted at a temperature between about 2 °C and about 8 °C.
46. The method of claim 45, wherein the storage is conducted at about 5 °C.
47. The method of any one of claims 44-46, wherein 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.
48. The method of any one of claims 1-47, wherein the lipid nanoparticle comprises: an ionizable amino lipid.
49. The method of claim 48, wherein the lipid nanoparticle further comprises: a non-cationic lipid; a sterol; and a polyethylene glycol (PEG)-modified lipid.
50. The method of claim 49, wherein 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.
51. A lyophilized composition produced by a method of any one of claims 1-50.
52. 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 polynucleotide comprising an open reading frame encoding a hCMV gB polypeptide, wherein: 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.
53. The lyophilized pharmaceutical composition of claim 52, wherein the molar ratio of (a):(b):(c):(d):(e):(f) is about 2: 1:1: 1:1:2.
54. The lyophilized pharmaceutical composition of claim 52 or 53, wherein 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; and the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
55. A lyophilized pharmaceutical composition comprising a lipid nanoparticle encapsulating an mRNA encoding one or more human cytomegalovirus (hCMV) antigens.
56. The lyophilized pharmaceutical composition of claim 55, wherein 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.
57. The lyophilized pharmaceutical composition of claim 55 or 56, wherein 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.
58. The lyophilized pharmaceutical composition of any one of claims 55-57, wherein the one or more hCMV antigens comprises greater than or equal to 1 and less than or equal to 6 hCMV antigens.
59. The lyophilized pharmaceutical composition of any one of claims 55-58, wherein 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.
60. The lyophilized pharmaceutical composition of claim 59, wherein 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.
61. The method of claim 59 or 60, wherein the molar ratio of (a):(b):(c):(d):(e):(f) is about 2:1:1:1:1:2.
62. The lyophilized pharmaceutical composition of any one of claims 59-61, wherein 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; and the mRNA of (f) comprises a nucleotide sequence with at least 90% sequence identity to SEQ ID NO: 1 or 7.
63. The lyophilized pharmaceutical composition of any one of claims 52-62, wherein the lyophilized pharmaceutical composition comprises a lyoprotectant.
64. The lyophilized pharmaceutical composition of claim 63, wherein the lyoprotectant comprises a sugar.
65. The lyophilized pharmaceutical composition of claim 63 or 64, wherein the lyoprotectant comprises sucrose.
66. The lyophilized pharmaceutical composition of any one of claims 63-65, wherein the lipid nanoparticle comprises one or more lipids, and where the lyophilized pharmaceutical composition comprises a lyoprotectant massdipid mass ratio of at least 5:1.
67. The lyophilized pharmaceutical composition of claim 66, wherein the lyoprotectant massdipid mass ratio is about 6:1 to about 40:1, optionally wherein the lyoprotectant massdipid 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.
68. The lyophilized pharmaceutical composition of any one of claims 52-67, wherein 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.
69. The lyophilized pharmaceutical composition of any one of claims 52-68, wherein 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.
70. The lyophilized pharmaceutical composition of claim 69, wherein 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.
71. The lyophilized pharmaceutical composition of any one of claims 52-70, wherein 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.
72. The lyophilized pharmaceutical composition of claim 71, wherein the storage is conducted at a temperature between about 2 °C and about 8 °C.
73. The lyophilized pharmaceutical composition of claim 72, wherein the storage is conducted at about 5 °C.
74. The lyophilized pharmaceutical composition of any one of claims 71-73, wherein 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.
75. The lyophilized pharmaceutical composition of any one of claims 52-74, wherein the lipid nanoparticle comprises: an ionizable amino lipid.
76. The lyophilized pharmaceutical composition of any one of claims 52-75, wherein the lipid nanoparticle further comprises: a non-cationic lipid; a sterol; and a polyethylene glycol (PEG)-modified lipid.
77. The lyophilized pharmaceutical composition of claim 76, wherein 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.
78. A method comprising reconstituting the lyophilized pharmaceutical composition of any one of claims 51-77.
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