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WO2025117816A1 - Compositions de nanoparticules lipidiques et leurs utilisations - Google Patents

Compositions de nanoparticules lipidiques et leurs utilisations Download PDF

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Publication number
WO2025117816A1
WO2025117816A1 PCT/US2024/057850 US2024057850W WO2025117816A1 WO 2025117816 A1 WO2025117816 A1 WO 2025117816A1 US 2024057850 W US2024057850 W US 2024057850W WO 2025117816 A1 WO2025117816 A1 WO 2025117816A1
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Prior art keywords
syndrome
composition
disease
molar percentage
lipid
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Inventor
Arunan Kaliyaperumal
Min Chen
Daniella ISHIMARU
Joseph S. Cefalu
Mirko HENNIG
Sakya Sing Mohapatra
Jialu WANG
Karpagam SRINIVASAN
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Recode Therapeutics Inc
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Recode Therapeutics Inc
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Publication of WO2025117816A1 publication Critical patent/WO2025117816A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • lipid nanoparticle compositions for specific delivery of antigens and polynucleotides encoding antigens.
  • the present disclosure provides lipid nanoparticle compositions, as wel as pharmaceutical compositions, and methods of using them as a vaccine.
  • the disclosure provides a lipid nanoparticle (LNP) composition
  • LNP lipid nanoparticle
  • the LNP composition comprises a polynucleotide encoding an antigen and the polynucleotide is an mRNA.
  • the polynucleotide is a chemicaly modified mRNA.
  • the polynucleotide encoding an antigen and the polynucleotide is a DNA.
  • the mRNA or DNA encodes antigen shown in Table 3, Table 4, and Table 5.
  • An aspect of the present disclosure is a lipid nanoparticle (LNP) composition comprising: a pathogen-associated antigen or a polynucleotide encoding a pathogen-associated antigen; and a selective organ targeting (SORT) lipid.
  • SORT selective organ targeting
  • Another aspect of the present disclosure is a lipid nanoparticle (LNP) composition comprising: a pathogen-associated antigen or a polynucleotide encoding a pathogen-associated antigen; and a permanently cationic lipid.
  • the LNP composition further comprises a selective organ targeting (SORT) lipid; a helper lipid; a sterol; and/or a polyethylene glycol-conjugated lipid (PEG-lipid).
  • the LNP composition further comprises a selective organ targeting (SORT) lipid.
  • SORT selective organ targeting
  • a further aspect of the present disclosure is a lipid nanoparticle (LNP) composition comprising: a pathogen-associated antigen or a polynucleotide encoding a pathogen-associated antigen; and an ionizable dendrimer lipid.
  • the LNP composition further comprises a selective organ targeting (SORT) lipid; a helper lipid; a sterol; and/or a polyethylene glycol-conjugated lipid (PEG-lipid).
  • SORT selective organ targeting
  • the ionizable dendrimer lipid has the formula D-IV.
  • the LNP composition further comprises a selective organ targeting (SORT) lipid.
  • An additional aspect of the present disclosure is a lipid nanoparticle (LNP) composition
  • LNP lipid nanoparticle
  • the LNP composition comprises a polynucleotide encoding a pathogen-associated antigen, e.g., the polynucleotide is chemicaly modified.
  • the polynucleotide is an mRNA.
  • the polynucleotide is a DNA.
  • the polynucleotide encodes a pathogen-associated antigen shown in Table 3, Table 4, or Table 5.
  • the polynucleotide encodes a pathogen-associated antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an antigen shown in Table 3, Table 4, or Table 5.
  • the polynucleotide encodes an influenza virus antigen, a coronavirus antigen, an RSV antigen, a PIV3 antigen, or an hMPV antigen.
  • the disclosure provides a lipid nanoparticle (LNP) composition
  • a lipid nanoparticle (LNP) composition comprising a cancer antigen or a polynucleotide encoding a cancer antigen, a helper lipid, a sterol, and/or a polyethylene glycol-conjugated lipid (PEG-lipid), an ionizable cationic lipid, and a selective organ targeting (SORT) lipid.
  • the LNP composition comprises a polynucleotide encoding a cancer antigen and the polynucleotide is an mRNA.
  • the polynucleotide is a chemicaly modified mRNA.
  • the polynucleotide encoding a cancer antigen and the polynucleotide is a DNA.
  • the mRNA or DNA encodes an antigen shown in Table 3, Table 4, and Table 5.
  • An aspect of the present disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; and a selective organ targeting (SORT) lipid.
  • SORT selective organ targeting
  • Another aspect of the present disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; and a permanently cationic lipid.
  • the LNP composition further comprises a selective organ targeting (SORT) lipid; a helper lipid; a sterol; and/or a polyethylene glycol-conjugated lipid (PEG-lipid).
  • the LNP composition further comprises a selective organ targeting (SORT) lipid.
  • a further aspect of the present disclosure is a lipid nanoparticle (LNP) composition comprising: a cancer antigen or a polynucleotide encoding a cancer antigen; and an ionizable dendrimer lipid.
  • the LNP composition further comprises a selective organ targeting (SORT) lipid; a helper lipid; a sterol; and/or a polyethylene glycol-conjugated lipid (PEG-lipid).
  • SORT selective organ targeting
  • the ionizable dendrimer lipid has the formula D- IV.
  • the LNP composition further comprises a selective organ targeting (SORT) lipid.
  • An additional aspect of the present disclosure is a lipid nanoparticle (LNP) composition
  • LNP lipid nanoparticle
  • the LNP composition comprises a polynucleotide encoding a cancer antigen, e.g., the polynucleotide is chemicaly modified.
  • the polynucleotide is an mRNA.
  • the polynucleotide is a DNA.
  • the polynucleotide encodes a cancer antigen shown in Table 6, Table 7, or Table 8.
  • the polynucleotide encodes a cancer antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an antigen shown in Table 6, Table 7, or Table 8.
  • the polynucleotide encodes a lung cancer antigen, a prostate cancer antigen, or a neoantigen.
  • the SORT lipid is a permanently cationic lipid, e.g., the SORT lipid is 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP).
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • the SORT lipid is dimethyldioctadecylammonium (DDAB) or the SORT lipid is ethylphosphocholine.
  • the ethylphosphocholine is 1,2-dipalmitoyl-sn-glycero-3–ethylphosphocholine (16:0 EPC) or is 1,2-dimyristoyl-sn-glycero-3–ethylphosphocholine (14:0 EPC).
  • the DOTAP is present in the composition at a molar percentage from about 30% to about 50%.
  • the ethylphosphocholine is present in the composition at a molar percentage from about 30% to about 50%.
  • the SORT lipid is 1,2-dioleoyl-3-dimethylammonium- propane (DODAP).
  • the DODAP is present in the composition at a molar percentage from about 5% to about 30%.
  • the SORT lipid is 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA).
  • the 18:1 PA is present in the composition at a molar percentage from about 5% to about 30%.
  • the SORT lipid is present in the composition at a molar percentage from about 5% to about 50%.
  • the ionizable cationic lipid is a dendrimer lipid.
  • the ionizable cationic lipid is a dendrimer lipid according to Formula (I) or Formula (X).
  • the dendrimer is: (4A3-SC7) or is (5A2-SC8).
  • the ionizable cationic lipid is present in the composition at a molar percentage from about 10% to about 25%.
  • the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the helper lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • the helper lipid is present in the LNP composition at a molar percentage from about 7.5% to about 30%.
  • the sterol is cholesterol or is sitosterol.
  • the cholesterol is present in the LNP composition at a molar percentage from about 20% to about 50%.
  • the PEG-lipid is present in the LNP composition at a molar percentage from about 0.5% to about 10%.
  • the LNP composition comprises mRNA at a lipid:mRNA ratio (weight/weight) between about 5:1 and about 40:1.
  • the lipid:mRNA ratio (weight/weight) is about 40:1, is about 36:1, or is about 30:1.
  • the LNP composition comprises: DOPE at a molar percentage of about 5 to about 25%; cholesterol at a molar percentage of about 20 to about 50%; DMG- PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and 16:0 EPC at a molar percentage of about 30 to about 40%.
  • the LNP composition comprises: DOPE at a molar percentage of about 5 to about 25%; cholesterol at a molar percentage of about 20 to about 50%; DMG- PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and DODAP at a molar percentage of about 10 to about 30%.
  • the LNP composition comprises: DOPE at a molar percentage of about 5 to about 25%; cholesterol at a molar percentage of about 20 to about 50%; DMG-PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and 18:1 PA at a molar percentage of about 5 to about 20%.
  • the LNP composition comprises: DOPE at a molar percentage of about 5 to about 25%; cholesterol at a molar percentage of about 20 to about 50%; DMG-PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and DOTAP at a molar percentage of about 30 to about 50%.
  • the LNP composition comprises: DOPE at a molar percentage of about 15%; cholesterol at a molar percentage of about 30%; DMG-PEG at a molar percentage of about 3%; ionizable cationic lipid at a molar percentage of about 15%; and 16:0 EPC at a molar percentage of about 30%.
  • the LNP composition comprises: DOPE at a molar percentage of about 20%; cholesterol at a molar percentage of about 40%; DMG-PEG at a molar percentage of about 3%; ionizable cationic lipid at a molar percentage of about 15%; and DODAP at a molar percentage of about 15%.
  • the LNP composition comprises: DOPE at a molar percentage of about 20%; cholesterol at a molar percentage of about 40%; DMG-PEG at a molar percentage of about 5%; ionizable cationic lipid at a molar percentage of about 20%; and 18:1 PA at a molar percentage of about 5%.
  • the LNP composition comprises: DOPE at a molar percentage of about 10%; cholesterol at a molar percentage of about 20%; DMG-PEG at a molar percentage of about 3%; ionizable cationic lipid at a molar percentage of about 10%; and DOTAP at a molar percentage of about 50%.
  • the ionizable cationic lipid is a dendrimer lipid, e.g., 4A3-SC7.
  • the LNP composition comprises Tris buffer, optionaly at a pH from about 6 to about 9; the LNP composition comprises sucrose, optionaly at a concentration (weight/weight) or at a molar percentage of from about 5% to about 15%; the LNP composition comprises citrate bufer, optionaly at a pH from about 4 to about 6; the LNP composition comprises: (a) 15 mM Tris bufer, optionaly at a pH from about 6 to about 9; and/or (b) sucrose at a concentration (weight/weight) or at a molar percentage of from about 5% to about 15%; the LNP composition comprises 10 mM citrate bufer, optionaly at a pH from about 4 to about 6.
  • the present disclosure provides, a pharmaceutical composition comprising a therapeuticaly effective amount of the LNP composition of any herein aspect or embodiment.
  • a pharmaceutical composition comprising the LNP composition of any herein aspect or embodiment and a pharmaceuticaly acceptable excipient.
  • a vaccine comprising the LNP composition of any herein aspect or embodiment or the pharmaceutical composition of any herein aspect or embodiment.
  • the vaccine comprises an adjuvant, e.g., the adjuvant comprises squalene.
  • the present disclosure provides a method of delivering an LNP composition to a subject in need thereof, the method comprising administering to the subject in need thereof the LNP composition of any herein aspect or embodiment, the pharmaceutical composition of any herein aspect or embodiment, and/or the vaccine of any herein aspect or embodiment.
  • the present disclosure provides a method of preventing an infectious disease caused by a pathogen in a subject in need thereof, the method comprising administering to the subject in need thereof the LNP composition of any herein aspect or embodiment, the pharmaceutical composition of any herein aspect or embodiment, and/or the vaccine of any herein aspect or embodiment.
  • the present disclosure provides a method of immunizing a subject in need thereof against an infection by a pathogen and/or a disease or disorder caused by a pathogen, the method comprising administering the vaccine of any herein aspect or embodiment to the subject.
  • the pathogen is a virus, optionaly wherein the virus comprises influenza, coronavirus, RSV, PIV3, hMPV, or MeV.
  • the pathogen is a bacterium.
  • administering to the subject in need thereof comprises intravenous administration, intrathecal administration, intramuscular administration, intradermal administration, subcutaneous administration, or intranasal administration.
  • the method results in an immune response in the subject in need thereof, optionaly wherein the immune response is greater than the immune response after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in an antibody response in the subject in need thereof, optionaly wherein the antibody response is greater than the antibody response after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in T cel proliferation in the subject in need thereof, optionaly wherein the T cel proliferation is greater than the T cel proliferation after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the T cel is a T helper cel or a cytotoxic T cel.
  • the method results in T cel activation in the subject in need thereof, optionaly wherein the T cel activation is greater than the T cel activation after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the T cel is a T helper cel or a cytotoxic T cel.
  • the method results in a cytokine response in the subject in need thereof.
  • An aspect of the present disclosure is a use of any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine for the manufacture of a medicament for vaccinating a subject in need against an infection by a pathogen or for the manufacture of a medicament for treating a disease or disorder caused by a pathogen.
  • Another aspect of the present disclosure is any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine for use in the manufacture of a medicament for vaccinating a subject in need against an infection by a pathogen or for use in the manufacture of a medicament for treating a disease or disorder caused by a pathogen.
  • a further aspect of the present disclosure is any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine for use in vaccinating a subject in need against an infection by a pathogen or for use in treating a disease or disorder caused by a pathogen.
  • the SORT lipid is a permanently cationic lipid.
  • the SORT lipid is 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP).
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • the SORT lipid is dimethyldioctadecylammonium (DDAB).
  • the permanently cationic lipid is ethylphosphocholine.
  • the ethylphosphocholine is 1,2-dipalmitoyl-sn-glycero-3–ethylphosphocholine (16:0 EPC). In some embodiments, the ethylphosphocholine is 1,2-dimyristoyl-sn-glycero-3– ethylphosphocholine (14:0 EPC). In some embodiments, the SORT lipid is 1,2-dioleoyl-3- dimethylammonium-propane (DODAP). In some embodiments, the SORT lipid is 1,2- dioleoyl-sn-glycero-3-phosphate (18:1 PA).
  • DODAP 1,2-dioleoyl-3- dimethylammonium-propane
  • the SORT lipid is 1,2- dioleoyl-sn-glycero-3-phosphate (18:1 PA).
  • the SORT lipid is present in the LNP composition at a molar percentage from about 5% to about 50%.
  • the DOTAP is present in the LNP composition at a molar percentage from about 30% to about 50%.
  • the ethylphosphocholine is present in the LNP composition at a molar percentage from about 30% to about 50%.
  • the DODAP is present in the LNP composition at a molar percentage from about 5% to about 30%.
  • the 18: PA is present in the LNP composition at a molar percentage from about 5% to about 30%.
  • the method results in immune response in the subject, optionaly wherein the immune response is greater than the immune response after administration of a Composition 2H. In some embodiments, the method results in antibody response in the subject, optionaly wherein the antibody response is greater than the antibody response after administration of a Composition 2H. In some embodiments, the method results in T cel proliferation in the subject, optionaly wherein the T cel proliferation is greater than the T cel proliferation after administration of a Composition 2H. In some embodiments, the method results in T cel activation in the subject, optionaly wherein the T cel activation is greater than the T cel activation after administration of a Composition 2H. In some embodiments, the T cel is T helper and cytotoxic T cel.
  • the method results in cytokine response in the subject.
  • the present disclosure provides a method of preventing cancer in a subject in need thereof, the method comprising administering to the subject in need thereof the LNP composition of any herein aspect or embodiment, the pharmaceutical composition of any herein aspect or embodiment, and/or the vaccine of any herein aspect or embodiment.
  • the present disclosure provides a method of treating a cancer or reducing the severity of a cancer in a subject in need thereof, the method comprising administering to the subject in need thereof the LNP composition of any herein aspect or embodiment, the pharmaceutical composition of any herein aspect or embodiment, and/or the vaccine of any herein aspect or embodiment.
  • the present disclosure provides method of inhibiting the growth, proliferation, progression, and/or metastasis of cancer cels in a subject in need thereof, the method comprising administering to the subject in need thereof the LNP composition of any herein aspect or embodiment, the pharmaceutical composition of any herein aspect or embodiment, and/or the vaccine of any herein aspect or embodiment.
  • the present disclosure provides a method of immunizing a subject in need thereof against a cancer and/or a cancerous disease or disorder, the method comprising administering the vaccine of any herein aspect or embodiment to the subject.
  • administering to the subject in need thereof comprises intravenous administration, intrathecal administration, intramuscular administration, intradermal administration, subcutaneous administration, or intranasal administration.
  • the method results in an immune response in the subject in need thereof, optionaly wherein the immune response is greater than the immune response after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in an antibody response in the subject in need thereof, optionaly wherein the antibody response is greater than the antibody response after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in T cel proliferation in the subject in need thereof, optionaly wherein the T cel proliferation is greater than the T cel proliferation after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the T cel is a T helper cel or a cytotoxic T cel.
  • the method results in T cel activation in the subject in need thereof, optionaly wherein the T cel activation is greater than the T cel activation after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the T cel is a T helper cel or a cytotoxic T cel.
  • the cancer is one or more of acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder cancer, bladder urothelial carcinoma (BLCA), brain cancer, breast cancer, breast invasive carcinoma (BRCA), Burkit lymphoma, cervical squamous cel carcinoma and endocervical adenocarcinoma (CESC), Chronic lymphocytic Leukaemia (CLL), colon adenocarcinoma (COAD), colon cancer, colorectal cancer (CRC), Difuse large B-cel lymphoma (DLBCL), Ewing's sarcoma, glioblastoma multiforme (GBM), glioma, head and neck carcinomas, head and neck squamous cel carcinoma (HNSC), hepatoma, human carcinomas (including colorectal, gastric, renal, and ovarian cancers), kidney chromophobe (KICH), kidney renal clear cel carcinoma (KIRC), kidney renal
  • An aspect of the present disclosure is a use of any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine for the manufacture of a medicament for vaccinating a subject in need against a cancer and/or a cancerous disease or disorder or for the manufacture of a medicament for preventing a cancer; for treating a cancer or for reducing the severity of a cancer; and/or for inhibiting the growth, proliferation, progression, and/or metastasis of cancer cels.
  • Another aspect of the present disclosure is any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine for use in the manufacture of a medicament for vaccinating a subject in need against a cancer and/or a cancerous disease or disorder or for use in the manufacture of a medicament for preventing a cancer; for treating a cancer or for reducing the severity of a cancer; and/or for inhibiting the growth, proliferation, progression, and/or metastasis of cancer cels.
  • a further aspect of the present disclosure is any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine for use in vaccinating a subject in need against a cancer and/or a cancerous disease or disorder or for use in preventing a cancer; in treating a cancer or for reducing the severity of a cancer; and/or in inhibiting the growth, proliferation, progression, and/or metastasis of cancer cels.
  • a kit comprising any herein disclosed LNP composition, any herein disclosed pharmaceutical composition, or any herein disclosed vaccine and instructions for use thereof.
  • the method results in immune response in the subject, optionaly wherein the immune response is greater than the immune response after administration of a Composition 2H. In some embodiments, the method results in antibody response in the subject, optionaly wherein the antibody response is greater than the antibody response after administration of a Composition 2H. In some embodiments, the method results in T cel proliferation in the subject, optionaly wherein the T cel proliferation is greater than the T cel proliferation after administration of a Composition 2H. In some embodiments, the method results in T cel activation in the subject, optionaly wherein the T cel activation is greater than the T cel activation after administration of a Composition 2H. In some embodiments, the T cel is T helper and cytotoxic T cel.
  • the method results in cytokine response in the subject.
  • lipid nanoparticle compositions for specific delivery to the central nervous system CNS.
  • the disclosure provides a lipid nanoparticle (LNP) composition comprising a payload, an ionizable cationic lipid, a selective organ targeting (SORT) lipid, and/or a helper lipid, a sterol, and/or a polyethylene glycol-conjugated lipid (PEG-lipid), wherein the composition delivers the payload to a cel in the central nervous system (CNS) of a subject.
  • LNP lipid nanoparticle
  • SORT selective organ targeting
  • PEG-lipid polyethylene glycol-conjugated lipid
  • the payload comprises a polynucleotide, optionaly an mRNA, shRNA, or microRNA.
  • the mRNA encodes a polypeptide or protein selected from the group shown in Table 9, Table 10, and/or SEQ ID NOs: 201-207.
  • the mRNA encodes a gene-editing system or a component thereof.
  • the payload comprises a polypeptide or a protein.
  • the ionizable cationic lipid is a dendrimer lipid.
  • the ionizable cationic lipid is a dendrimer lipid according to Formula (I) or Formula (X).
  • the ionizable cationic lipid is 4A3-SC7. In some embodiments, the ionizable cationic lipid is 5A2-SC8. In some embodiments, the ionizable cationic lipid is present in the composition at a molar percentage from about 10% to about 35%. [0088] In some embodiments, the SORT lipid selected from the group shown in Table 16A and Table 16B. In some embodiments, the SORT lipid is present in the composition at a molar percentage from about 5% to about 65%. In some embodiments, the SORT lipid is present in the composition at a molar percentage from about 5% to about 35%.
  • the helper lipid is 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE). In some embodiments, the helper lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC). In some embodiments, the helper lipid is present in the composition at a molar percentage from about 5% to about 35%.
  • the sterol is cholesterol. In some embodiments, the sterol is sitosterol. In some embodiments, the cholesterol is present in the composition at a molar percentage from about 20% to about 50%.
  • the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%.
  • the composition comprises mRNA at a lipid:mRNA ratio (weight/weight) between 5:1 and 40:1.
  • the composition comprises 4A3-SC7 at a molar percentage of about 5 to about 30%, SORT lipid at a molar percentage of about 5 to about 30%, DOPE at a molar percentage of about 8 to about 23%, cholesterol at a molar percentage of about 15 to about 46%, and/or DMG-PEG at a molar percentage of about 0.5 to about 10%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 13 to about 20%, SORT at a molar percentage of about 10 to about 30%, DOPE at a molar percentage of about 13 to about 20%, cholesterol at a molar percentage of about 30 to about 40%, and/or DMG-PEG at a molar percentage of about 3 to about 5%, wherein the SORT lipid is an ethylphosphocholine.
  • the composition comprises 4A3-SC7 at a molar percentage of about 13 to about 20%, SORT at a molar percentage of about 5 to about 30%, DOPE at a molar percentage of about 13 to about 20%, cholesterol at a molar percentage of about 30 to about 40%, and/or DMG-PEG at a molar percentage of about 3 to about 5%, wherein the SORT lipid is an anionic lipid.
  • the composition comprises 4A3-SC7 at a molar percentage of about 19%, 14:0 EPC at a molar percentage of about 20%, DOPE at a molar percentage of about 19%, cholesterol at a molar percentage of about 38%, and/or DMG-PEG at a molar percentage of about 4%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 17%, 16:0 EPC at a molar percentage of about 30%, DOPE at a molar percentage of about 17%, cholesterol at a molar percentage of about 33%, and/or DMG-PEG at a molar percentage of about 3%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 17%, 14:0 EPC at a molar percentage of about 30%, DOPE at a molar percentage of about 17%, cholesterol at a molar percentage of about 33%, and/or DMG-PEG at a molar percentage of about 3%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 23%, 18:1 PA at a molar percentage of about 5%, DOPE at a molar percentage of about 23%, cholesterol at a molar percentage of about 45%, and/or DMG-PEG at a molar percentage of about 5%.
  • the composition is capable of delivering mRNA to a cel in the central nervous system (CNS) of a subject in an amount effete to increase expression and/or function of a gene encoded by the mRNA.
  • the cel is an endothelial cel.
  • the cel is a pericyte.
  • the cel is a smooth muscle cel (SMA).
  • SMA smooth muscle cel
  • the composition comprises a payload wherein the payload is a messenger RNA (mRNA) and wherein the method results in delivery of the payload to the CNS in an amount effective to increase expression and/or function of a gene encoded by the mRNA.
  • mRNA messenger RNA
  • the method results in expression of a polypeptide in a CNS of the subject. In some embodiments, the method results in expression of the polypeptide in the CNS of the subject between 1 and 72 hours after administration of the composition to the subject.
  • the CNS disease is acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, atention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold- Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosaminuria
  • ADHD tention deficit hyperactivity disorder
  • the disclosure provides a method of delivering a payload to a cel in a CNS of a subject, wherein the method comprises administering to the subject, by intrathecal injection, the composition described herein.
  • the disclosure provides a kit comprising the composition described herein.
  • the disclosure provides a pharmaceutical composition comprising the composition described herein and a pharmaceuticaly acceptable excipient and/or diluent.
  • the disclosure provides use of the composition described herein for treatment of a CNS disease.
  • the disclosure provides use of the composition described herein for treatment of a CNS disease by intrathecal administration.
  • the disclosure provides the composition described herein or the pharmaceutical composition described herein for use in the treatment of a CNS disease in a subject in need thereof.
  • the CNS disease is acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, atention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid
  • a CNS disease is Abulia, Achromatopsia, acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, atention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Agraphia, Aicardi syndrome, Aicardi- Goutieres syndrome disorder, Akinetopsia, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Amnesia, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold-Chiari malformation, aromatic L-amino acid decarboxy
  • ADHD tention deficit hyperactivity
  • FIG.1 illustrates a schematic of the in vivo mouse study design.
  • FIG.2A shows a trace of fragment analyzer data of uncapped H1N1 HA mRNA (left panel) and H1N1 HA protein expression (right panel).
  • FIG.2B shows exemplary LNP compositions and their characterization.
  • FIG.3A to FIG.3C show anti-H1N1 HA IgG/M antibody titer analysis.
  • FIG.3A shows a graph of ELISA data of ⁇ -H1N1 HA IgG/M antibody levels by Compositions as indicated.
  • FIG.3B shows ELISA data of ⁇ -H1N1 HA IgG/M antibody levels by dose.
  • FIG. 3C shows ELISA data of ⁇ -H1N1 HA IgG/M antibody levels at micro-dosing levels (0.03 ⁇ g/dose and 0.1 ⁇ g/dose) dose.
  • FIG.4A to FIG.4C show graphs for T cel proliferation analysis at day 14.
  • FIG.4A shows proliferation analysis of al live cels.
  • FIG.4B shows proliferation analysis of T helper cels.
  • FIG.4C shows proliferation analysis of cytotoxic T cels.
  • FIG.5A to FIG.5C show graphs for T cel proliferation analysis at day 28.
  • FIG.5A shows proliferation analysis of al live cels.
  • FIG.5B shows proliferation analysis of T helper cels.
  • FIG.5C shows proliferation analysis of cytotoxic T cels.
  • FIG.6A to FIG.6C show graphs for T cel activation analysis at day 14.
  • FIG.6A shows activation analysis of al T cels.
  • FIG.6B shows activation analysis of T helper cels.
  • FIG.6C shows activation analysis of cytotoxic T cels.
  • FIG.7A to FIG.7C show graphs for T cel activation analysis at day 28.
  • FIG.7A shows activation analysis of al T cels.
  • FIG.7B shows activation analysis of T helper cels.
  • FIG.7C shows activation analysis of cytotoxic T cels.
  • FIG.8A to FIG.8D show heatmaps for cytokine expression in mouse serum after treatment with H1N1 HA mRNA LNPs.
  • FIG.8A shows a cytokine heatmap analysis at day 14.
  • FIG.8B shows a cytokine heatmap analysis at day 28.
  • FIG.8C shows a cytokine heatmap analysis by micro-dosing at day 14.
  • FIG.8D shows a cytokine heatmap analysis by micro- dosing at day 28.
  • FIG.9A to FIG.9B show anti-H1N1 HA IgG/M antibody titer analysis.
  • FIG.9A shows a graph of ELISA data of ⁇ -H1N1 HA IgG/M antibody levels by Compositions as indicated.
  • FIG.9B shows ELISA data of ⁇ -H1N1 HA IgG/M antibody levels by dose (0.03 ⁇ g/dose, 1 ⁇ g/dose, and 10 ⁇ g/dose) dose.
  • FIG.10A to FIG.10B show heatmaps for cytokine expression in cultured splenocytes after treatment with H1N1 HA mRNA LNPs.
  • FIG.10A shows a cytokine heatmap analysis at day 7.
  • FIG.10B shows a cytokine heatmap analysis at day 28.
  • FIG.11A to FIG.11B show heatmaps for cytokine expression in cultured lymph nodes after treatment with H1N1 HA mRNA LNPs.
  • FIG.11A shows a cytokine heatmap analysis at day 7.
  • FIG.11B shows a cytokine heatmap analysis at day 28.
  • FIG.12A to FIG.12C show antibody analysis and cytokine analysis of RSV mRNA LNP-treated mice.
  • FIG.12A shows graph of ELISA data of ⁇ -RSV antibody levels by Compositions as indicated.
  • FIG.12A shows a heatmap for cytokine expression in cultured splenocytes after treatment with H1N1 HA mRNA LNPs.
  • FIG.12B shows a cytokine heatmap analysis in cultured splenocytes at day 7 and day 28 after treatment with RSV mRNA LNPs.
  • FIG.12C shows a cytokine heatmap analysis in cultured lymph nodes at day 7 and day 28 after treatment with RSV mRNA LNPs.
  • FIG.13 (top) shows the number of differentialy expressed genes (DEGs) in innate cels.
  • FIG.13 (botom) shows the number of diferentialy expressed genes (DEGs) in adaptive immune cels.
  • FIG.14A to FIG.14K show volcano plots for various immune cels on day 1.
  • FIG. 14A shows a volcano plot in Na ⁇ ve CD8 T cels.
  • FIG.14B shows a volcano plot in Na ⁇ ve CD4 T cels.
  • FIG.14C shows a volcano plot in B cels.
  • FIG.14D shows a volcano plot in T-reg cels.
  • FIG.14E shows a volcano plot in natural kiler (NK) cels.
  • FIG.14F shows a volcano plot in monocyte-derived macrophage els.
  • FIG.14G shows a volcano plot in pDC1 cels.
  • FIG. 14H shows a volcano plot in cDC1 cels.
  • FIG.14I shows a volcano plot in mDC cels.
  • FIG. 14A shows a volcano plot in Na ⁇ ve CD8 T cels.
  • FIG.14B shows a volcano plot in Na ⁇ ve CD4 T cels.
  • FIG.14C shows a volcano plot in B cel
  • FIG.15A to FIG.15G show comparison results of single-cel RNA sequence dosed with vaccine-injected lymph node cels shown by uniform manifold approximation and projection (UMAP).
  • FIG.15A shows a comparison of Composition 2H and bufer.
  • FIG.15B shows comparison Composition 2B and bufer.
  • FIG.15C shows a comparison of Composition X/pH4 and bufer.
  • FIG.15D shows a comparison Composition X/pH6.5 and bufer.
  • FIG.15E shows a comparison Composition X/pH4, Composition X/pH6.5, and buffer.
  • FIG.15F shows a comparison Composition 2B, Composition 2H, and bufer.
  • FIG.15G shows a comparison Composition X/pH4, Composition X/pH6.5, Composition 2H, and bufer.
  • FIG.16A to FIG.16B show conditional survival rate of mice chalenged with H1N1 PR8 at 100 PFU at 28-Day.
  • FIG.16A shows comparison results of conditional survival rate of mice dosed with either Composition 2B, Composition X, or Composition 2H.
  • FIG.16B shows conditional survival rate of mice dosed with either Composition 2B (top), Composition X (middle), or Composition 2H (botom).
  • FIG.17A to FIG.17B show conditional survival rate of mice chalenged with H1N1 PR8 at 60 PFU at 84-Day.
  • FIG.17A shows comparison results of conditional survival rate of mice dosed with either Composition 2B, Composition X, or Composition 2H.
  • FIG.17B shows conditional survival rate of mice dosed with either Composition 2B (top), Composition X (middle), or Composition 2H (botom).
  • FIG.18A to FIG.18C show conditional survival rate of mice chalenged with H1N1 PR8 at 60 PFU at 84-Day.
  • FIG.18A shows conditional survival rate of mice dosed with either Composition V, Composition 2B, Composition X, or Composition 2H.
  • FIG.18B shows comparison results of conditional survival rate of mice dosed with either Composition V, Composition 2B, or Composition X to Composition 2H.
  • FIG.18C shows comparison results of conditional survival rate of mice dosed with either Composition V, Composition 2B, Composition X, or Composition 2H.
  • FIG.19A to FIG.19C show body weight change of mice chalenged with H1N1 PR8 at 60 PFU at 84-Day.
  • FIG.19A shows body weight change of mice dosed with either Composition V, Composition 2B, Composition X, or Composition 2H.
  • FIG.19B shows comparison results of body weight change of mice dosed with either Composition V, Composition 2B, or Composition X to Composition 2H.
  • FIG.19C shows comparison results of body weight change of mice dosed with either Composition V, Composition 2B, Composition X, or Composition 2H.
  • FIG.20 shows exemplary LNP compositions and its characterization.
  • FIGs.21A-21F show brain in vivo imaging (IVIS®) dosed with LNP composition.
  • FIG. 21A shows an in vivo imaging (IVIS®) of a whole brain pre-MAV removal, a whole brain post- MAV removal, and sectioned brain dosed with Composition A-treated mice.
  • FIG.21B shows an in vivo imaging (IVIS®) of a whole brain pre-MAV removal, a whole brain post-MAV removal, and sectioned brain dosed with Composition W-treated mice.
  • FIG.21C shows an in vivo imaging (IVIS®) of a whole brain pre-MAV removal, a whole brain post-MAV removal, and sectioned brain dosed with Composition B-treated mice.
  • FIG.21D shows an in vivo imaging (IVIS®) of a whole brain pre-MAV removal, a whole brain post-MAV removal, and sectioned brain dosed with Composition G-treated mice.
  • FIG.21E shows an in vivo imaging (IVIS®) of a whole brain pre-MAV removal, a whole brain post-MAV removal, and sectioned brain dosed with Composition I-treated mice.
  • FIG.21F shows quantitative results of in vivo imaging (IVIS®).
  • FIGs.22A-22C show an in vivo imaging (IVIS®) dosed with LNP composition.
  • FIG. 22A shows whole body in vivo imaging (IVIS®) of mice dosed with various LNP composition.
  • FIG.22B shows organ in vivo imaging (IVIS®) of mice dosed with various LNP composition.
  • FIG.22C shows quantitative results of in vivo imaging (IVIS®).
  • FIG.23 shows immunofluorescent staining of tdTomato mice brain dosed with Composition A.
  • FIGs.24A-24B show immunofluorescent staining of tdTomato mice brain dosed with Composition A.
  • FIG.24A shows endothelial staining of tdTomato mouse (Paxinos & Franklin Figure 15).
  • FIG.24B shows endothelial staining of tdTomato mouse (Paxinos & Franklin Figure 18).
  • FIGs.25A-25B show immunofluorescent staining of tdTomato mice brain dosed with Composition A.
  • FIG.25A shows neuron staining (NeuN) of tdTomato mouse (Paxinos & Franklin Figure 59-59).
  • FIG.25B shows neuron staining (NeuN) of tdTomato mouse (Paxinos & Franklin Figure 59-59).
  • FIG.26 shows astrocytes staining (GFAP) of tdTomato mouse dosed with Composition A (Paxinos & Franklin Figure 1, olfactory bulbs).
  • GFAP astrocytes staining
  • FIG.27 shows neuron staining (NeuN) of tdTomato mouse dosed with Composition A (Paxinos & Franklin Figure 43, cortex/thalamus/hypothalamus; top) and astrocytes staining (GFAP) of tdTomato mouse dosed with Composition A (Paxinos & Franklin Figure 67, cerebelum/rhombencephalon; botom).
  • FIG.28 shows exemplary LNP compositions and its characterization.
  • FIGs.29A–29C show immunofluorescent staining of tdTomato mice brain dosed with either Composition 2F, Composition 2I, or Composition 2B.
  • FIG.29A shows CD31 staining of tdTomato mouse dosed with Composition 2I (Paxinos & Franklin Figure 58).
  • FIG.29B CD31 staining of tdTomato mouse dosed with Composition 2B (Paxinos & Franklin Figure 43).
  • FIG.29C shows CD31 staining of tdTomato mouse dosed with either Composition 2F, Composition 2I, or Composition 2B (Paxinos & Franklin Figure 43 or 58).
  • FIGs.30A–30C show immunofluorescent staining of tdTomato mice brain dosed with either Composition 2F, Composition 2I, or Composition 2B.
  • FIG.30A shows PDGFR ⁇ staining of tdTomato mouse dosed with Composition 2I (Paxinos & Franklin Figure 58).
  • FIG. 30B shows PDGFR ⁇ staining of tdTomato mouse dosed with Composition 2B (Paxinos & Franklin Figure 43).
  • FIG.30C shows PDGFR ⁇ staining of tdTomato mouse dosed with either Composition 2F, Composition 2I, or Composition 2B (Paxinos & Franklin Figure 43 or 58).
  • FIGs.31A–31C show immunofluorescent staining of tdTomato mice brain dosed with either Composition 2F, Composition 2I, or Composition 2B.
  • FIG.31A shows SMA and PDGFR ⁇ staining of tdTomato mouse dosed with Composition 2I (Paxinos & Franklin Figure 58).
  • FIG.31B shows SMA and PDGFR ⁇ staining of tdTomato mouse dosed with Composition 2B (Paxinos & Franklin Figure 43).
  • FIG.31C shows SMA staining of tdTomato mouse dosed with either Composition 2F, Composition 2I, or Composition 2B (Paxinos & Franklin Figure 43 or 58).
  • lipid nanoparticle (LNP) compositions comprising an antigen, such as a pathogen-associated antigen, or a polynucleotide encoding an antigen, such as a pathogen- associated antigen.
  • the LNP composition comprises an antigen from a pathogen involved in an infectious disease.
  • pharmaceutical compositions comprising the LNP compositions and vaccines comprising the LNP compositions.
  • methods of use of LNP compositions, pharmaceutical compositions, and vaccines comprising LNP compositions are also provided herein.
  • lipid nanoparticle (LNP) compositions comprising a cancer antigen or a polynucleotide encoding a cancer antigen.
  • the LNP composition comprises a cancer antigen.
  • the disclosure also provides for pharmaceutical compositions comprising the LNP compositions and vaccines comprising the LNP compositions. Also provided herein are methods of use of LNP compositions, pharmaceutical compositions, and vaccines comprising LNP compositions.
  • the disclosure relates to the surprising discovery by the present inventors that LNP compositions described herein generate at least as strong or stronger adaptive immune responses in vivo as a reference LNP composition used to deliver the same antigen-encoding polynucleotide (e.g., an LNP composition employing SM-102 as the ionizable cationic lipid and/or lacking a SORT lipid).
  • LNP compositions described herein generate immune responses with the same or lower cytokine response compared with a reference LNP composition.
  • LNP compositions described herein generate immune responses with the same or higher cytokine response compared with a reference LNP composition.
  • LNP compositions comprising a polypeptide or polynucleotides encoding polypeptides, such as a a gene related to CNS disease, or a polynucleotide encoding a gene editor for editing a gene related to CNS disease, or polynucleotides encoding polypeptides, such as a encoding a gene related to CNS disease, or a polynucleotide encoding a gene editor for editing a gene related to CNS disease.
  • the disclosure also provides for pharmaceutical compositions comprising the LNP compositions. Also provided herein are methods of use of the LNP compositions, and pharmaceutical compositions. [0149] In some aspects, the disclosure relates to the surprising discovery by the present inventors that LNP compositions described herein generate at least as strong or stronger adaptive immune responses in vivo as a reference LNP composition used to deliver the same polypeptide-encoding polynucleotide (e.g., an LNP composition employing SM-102 as the ionizable cationic lipid and/or lacking a SORT lipid). In some aspects, the disclosure relates to the surprising discovery by the present inventors that LNP compositions described herein generate immune responses with the same or lower cytokine response compared with a reference LNP composition.
  • a reference LNP composition used to deliver the same polypeptide-encoding polynucleotide
  • LNP compositions described herein generate immune responses with the same or lower cytokine response compared with a reference LNP composition.
  • the disclosure relates to the surprising discovery by the present inventors that LNP compositions described herein generate immune responses with the same or higher cytokine response compared with a reference LNP composition.
  • Other advantages of disclosed embodiments wil be apparent from the description of embodiments that folow.
  • Other advantages of disclosed embodiments wil be apparent from the description of embodiments that folow.
  • the term “about” means a range of values including the specified value, which a person of ordinary skil in the art would consider reasonably similar to the specified value. For example, about means within a standard deviation using measurements generaly acceptable in the art. For example, about means a range extending to +/- 10%, +/- 5%, +/- 3%, or +/- 1% of the specified value.
  • the term “at least” folowed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.
  • the term “at most” folowed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined).
  • “at most 4” means 4 or less than 4
  • “at most 40%” means 40% or less than 40%.
  • a range is given as “(a first number) to (a second number)” or “(a first number)- (a second number)” this means a range whose lower limit is the first number and whose upper limit is the second number.
  • 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.
  • lipid nanoparticle refers to a carier or vehicle, formed by one or more lipid components, for payload (e.g., nucleic acid, protein, peptide, polypeptide, polynucleotide, or oligonucleotide) delivery in the context of pharmaceutical development.
  • payload e.g., nucleic acid, protein, peptide, polypeptide, polynucleotide, or oligonucleotide
  • Lipid nanoparticle can have one or more lipids with at least one dimension on the order of nanometers (e.g., 1-1000 nm).
  • lipid nanoparticle compositions for delivery are composed of one or more lipids, such as, but not limited to, a synthetic ionizable or cationic lipid, a phospholipid, a structural lipid, and a polyethylene glycol (PEG) lipid. These compositions may also include other lipids.
  • at least one therapeutic agent e.g., mRNA
  • lipid nanoparticles comprise at least one therapeutic agent (e.g., mRNA) that is either organized within inverse lipid miceles and encased within a lipid monolayer envelop or intercalated between adjacent lipid bilayers.
  • the morphology of lipid nanoparticles is not like a traditional liposome, which are characterized by a lipid bilayer surounding an aqueous core.
  • lipid nanoparticles are substantialy non-toxic.
  • the therapeutic agent e.g., mRNA
  • the therapeutic agent is resistant in aqueous solution to degradation by intracellular or intercellular enzymes.
  • SORT lipid refers to a lipid that when included in a lipid nanoparticle (LNP) composition enables the LNP to selectively and predictably target an organ, a cel type, or a tissue (for example as described in Cheng et al. Nature 15:313-320 (2020); Wang et al. Nat. Protoc.18(1):265-291; and U.S. Pat. Pub. No. US 2022/0071916 A1 and US 2021/0259980 A1, the entire contents of which is incorporated herein by reference).
  • addition of a specific SORT lipid to an LNP may redirect (e.g., re-target) the LNP from the liver to the lung.
  • SORT lipids include, but are not limited to, permanently cationic lipids, anionic lipids, zwiterionic lipids, and ionizable cationic lipids.
  • anionic SORT lipids generaly favor delivery to the spleen, at least when administered intravenously; ionizable cationic SORT lipids generaly favor delivery to the liver; permanently cationic SORT lipids generaly favor delivery to the lungs; and zwiterionic SORT lipids favor delivery to the spleen.
  • the term “ionizable cationic lipid” refers to lipid and lipid-like molecules having at least one pKa in the range of about 4.5-8, such that, without being bound by theory, they may facilitate release of LNP payloads upon uptake into the endosomal compartment of a cel.
  • the ionizable cationic lipid may maintain a neutral charge in pH above the pKa of the lipid, while it becomes positively charged in the pH lower than pKa to facilitate membrane fusion and subsequent cytosolic release.
  • Ilustrative ionizable cationic lipids have one or more nitrogen atoms having pKa’s in the range of about 4.5-8, such are tertiary amine groups.
  • the term “permanently cationic lipid” refers to lipid or lipid-like molecules that are positively charged in physiologicaly relevant solutions, regardless of a pH (positively charged without pKa or with a pKa greater than 8).
  • Ilustrative permanently cationic lipids may include a quaternary ammonium group, and lack a negatively charged phosphate group.
  • a permanently cationic lipid may act as a SORT lipid by raising the apparent pKa of an LNP, as described, e.g., in Diliard et al. PNAS USA. 118(52):e2109256118 (2021), the entire contents of which is incorporated herein by reference.
  • anionic lipid refers to a lipid that is negatively charged at physiological pH.
  • these lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), ethylphosphocholines, and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerols cardiolipins
  • diacylphosphatidylserines diacylphosphatidic acids
  • N-dodecanoyl phosphatidylethanolamines N-succinyl phosphatidylethanolamines
  • phospholipid refers to lipids that comprise a phosphate group.
  • the lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more faty acid moieties.
  • sterol refers to a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring of a gonane ringsystem.
  • “Cholesterol” is a sterol that has a structure of four fused hydrocarbon rings (gonane ringsystem) with a polar hydroxyl group at one end and an eight-carbon branched aliphatic tail at the other end.
  • the structure of the tetracyclic ring of cholesterol contributes to the fluidity of the cel membrane, as the molecule is in a trans conformation making al but the side chain of cholesterol rigid and planar.
  • Cholesterol influences the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers, for example in LNPs.
  • “sterol” can be cholesterol or sitosterol.
  • the term “PEG-lipid” refers to a lipid modified with a polyethylene glycol unit.
  • the PEG-lipid comprises dimyristoyl glycerol (DMG).
  • the PEG-lipid comprises 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine (DSPE).
  • DMG dimyristoyl glycerol
  • DSPE 1,2-distearoyl-sn-glycero-3- phosphorylethanolamine
  • N/P ratio refers to a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide payload.
  • the term “apparent pKa” refers to the overal dissociation constant of al titratable groups in the lipid nanoparticles.
  • Apparent pKa is an experimentaly determined value of molecules or nanoparticles.
  • Apparent pKa can be expressed as the pH at which the number of ionized (protonated) and deionized groups are equal in a system.
  • the surface charge and ionic interaction of assembled nanomaterials in nanoparticles can be estimated according to apparent pKa.
  • the apparent pKa of a nanoparticle can be the result of the average ratio of al the ionized to deionized groups in the nanoparticles.
  • apparent pKa is not the intrinsic pKa value of any individual molecule.
  • the apparent pKa of nanoparticles can be measured by various techniques.
  • lipid:RNA ratio refers to an amount (e.g., nanogram or miligram) of lipid for each amount (i.e., nanogram or miligram) of mRNA drug substance. Without being bound by theory, the lipid:RNA ratio may influence the encapsulation eficiency of lipid nanoparticles.
  • molar percentage is the number of moles of one ingredient in a given mixture divided by the total number of moles in the given mixture ⁇ 100%.
  • encapsulation refers to the process of confining a payload within an LNP described herein.
  • encapsulation refers to confining an mRNA molecule within an LNP described herein.
  • encapsulation eficiency refers to the fraction of a payload that is encapsulated within or otherwise coupled with a lipid nanoparticle composition when LNPs are formed. Encapsulation eficiency may be determined by comparing the amount of input payload to the amount of payload in a sample of LNPs, or by comparing the amount of payload in the LNPs to the free excess payload in the sample.
  • a fluorescence detection assay e.g., RiboGreenTM
  • RiboGreenTM a fluorescence detection assay
  • the term “payload” refers to a bioactive molecule or bioactive molecules, such as a smal molecule, biomolecule, nucleic acid (e.g., DNA, RNA, siRNA, and shRNA), protein, or peptide, which is comprised in an LNP composition.
  • the payload can be bound covalently or non-covalently to the LNP, encapsulated in the LNP, coupled to the LNP, or complexed with the LNP within the LNP composition.
  • antigen or “immunogen” refers to its plain and ordinary meaning of a molecule, compound, or composition that induces an immune response, celular or humoral, e.g., cytotoxic T lymphocyte (CTL) response, a B cel response (for example, production of antibodies that specificaly bind an epitope in the antigen), an NK cel response or any combinations thereof, when administered to or expressed in an immunocompetent subject.
  • Antigens can include peptides or polypeptides (including glycoproteins).
  • an antigen comprises a peptide, a polypeptide, or polypeptide complex that elicits an immune response.
  • an antigen can include one or more immunogenic epitopes associated with a viral pathogen or with a cancer antigen.
  • An antigen can be a ful length naturaly occuring protein or a fragment of a naturaly occuring protein.
  • An antigen can be a ful-length modified proteins or a fragment of a modified protein, where a modified protein difers from a naturaly-occuring protein by one or more amino acid, one or more associated compound (e.g., glycosylation, acetylation, methylation, or phosphorylation), and the like.
  • antigen is not limited to the portion of the polypeptide or polypeptide complex that contains antigenic epitopes.
  • pathogen-associated antigen is an antigen which is derived from or homologous to or similar to an antigen present in or on a pathogenic organism, e.g., an infectious microorganism. As with other antigens, a pathogen-associated antigen elicits an immune response in a subject who is contacted with the pathogen-associated antigen.
  • a pathogen-associated antigen may be identical to an antigen associated with a natural, i.e., wild-type, pathogen; alternately, the pathogen-associated antigen may be engineered or modified to difer from the coresponding antigen associated with the natural pathogen.
  • the term “cancer antigen” or “tumor-specific antigen” is an antigen which is derived from or homologous to or similar to an antigen present in or on a cancer cel or in or on a tumor comprising cancerous cels. As with other antigens, a cancer antigen elicits an immune response in a subject who is contacted with the cancer antigen.
  • a cancer antigen may be identical to an antigen associated with a naturaly obtained (e.g., biopsied) cancer cel, a cancer cel present in a subject, or a cultured cancerous cel; alternately, the cancer antigen may be engineered or modified to difer from the corresponding antigen associated with the natural cancer cel.
  • a naturaly obtained e.g., biopsied
  • cancer antigen may be engineered or modified to difer from the corresponding antigen associated with the natural cancer cel.
  • tumor- specific antigen and the like are synonymous.
  • An “epitope” refers to the part of an antigen that is recognized by the immune system, specificaly by antibodies, or T cel receptor.
  • nucleic acid As used herein the term “immunogenic” is the ability to stimulate an immune response, e.g., via T cels, B cels, or both.
  • nucleic acid molecule As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribosomal RNA
  • a ribozyme cDNA
  • a recombinant polynucleotide a branched polynucleotide
  • plasmid a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.
  • Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
  • nucleotides or nucleosides are chemicaly and/or structuraly modified the nucleotides or nucleosides may be refered to as “modified nucleotides” or “modified nucleosides”.
  • a polynucleotide or polynucleside comprising modified nucleotides or modified nucleosides may be refered to as a “modified polynucleotide” or as a “modified polynucleside.”
  • Modified nucleotides or modified nucleosides may reduce an innate immune response relative to unmodified nucleotides and unmodified nucleosides. See, e.g., Karikó K, Buckstein M, Ni H, Weissman D “Suppression of RNA recognition by Tol-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity.” 2005 Aug;23(2):165-75 and Dr. Karikó and Dr.
  • identity refers to the extend to which two optimaly aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can readily be calculated by known methods, including, but not limited to, those described in Needleman and Wunsch, J. Mol. Biol.48:443 (1970).
  • polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence.
  • the term “percent sequence identity”, “percent identity”, or “identical to” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimaly aligned.
  • percent identity can refer to the percentage of identical amino acids in an amino acid sequence.
  • RNA messenger RNA
  • mRNA messenger RNA
  • mRNA can be purified from natural sources, produced using recombinant expression systems and optionaly purified, chemicaly synthesized, etc. Where appropriate, e.g., in the case of chemicaly synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemicaly modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated. [0179] As used herein, the term “shRNA” or “short hairpin RNA” refers to a short sequence of RNA, which can make a tight hairpin turn and can be used to silence gene expression.
  • microRNA refers to noncoding RNA consisting of about 22 ribonucleotides which regulates gene expression in the post transcriptional stage by silencing messenger RNA by base-pairing with a complementary sequence in its targeted mRNA.
  • gene-editing system refers to a DNA or RNA editing system that comprises one or more guide RNA elements and one or more RNA-guided endonuclease elements.
  • the guide RNA element comprises a target RNA comprising a nucleotide sequence substantialy complementary to a nucleotide sequence at the one or more target genomic regions or a nucleic acid comprising a nucleotide sequence(s) encoding the target RNA.
  • the RNA-guided endonuclease element comprises an endonuclease that is guided or brought to a target genomic region(s) by a guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding such endonuclease.
  • polypeptide refers to a polymer of amino acid residues and optionaly one or more post-translational modifications (e.g., glycosylation, acetylation, methylation, and phosphorylation) and/or other modifications.
  • isolated when applied to a polynucleotide or polypeptide, denotes that the polynucleotide or polypeptide is essentialy free of other celular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution.
  • the term “variant” refers to a polypeptide or polynucleotide having one or more insertions, deletions, or amino acid substitutions relative to a reference polypeptide or polynucleotide.
  • the terms “subject” refers to a living organism to which any of the compositions as described herein may be administered. The subject may be suffering from or be at risk for a disease or condition that can be treated by administration of an aerosolized pharmaceutical composition as provided herein.
  • Non-limiting examples of subjects include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non- mammalian animals.
  • the subject is human.
  • therapeuticy effete amount refers to an amount of the therapeutic agent sufficient to treat a disease, a disorder, or a condition.
  • a therapeuticaly effective amount is the dosage or concentration of the mRNA (e.g., CFTR or PCD mRNA) capable of eradicating, inhibiting, preventing, slowing down the progression of al or part of e.g., CF or PCD respiratory symptoms or some combination thereof.
  • mRNA e.g., CFTR or PCD mRNA
  • a therapeuticaly effective amount is the dosage or concentration of the mRNA (e.g., which encodes a cancer antigen) capable of eradicating, inhibiting, preventing, slowing down the growth, proliferation, progression, and/or metastasis of cancer or some combination thereof.
  • a therapeuticaly effete amount wil show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as “- fold” increase or decrease.
  • a therapeuticaly effete amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
  • the “therapeuticaly effete amount” can vary depending, for example, but not limited to, on the compound, the disease, or the condition and/or symptoms thereof, severity of the disease or the condition and/or symptoms thereof, the age, weight, and/or health of the subject to be treated, and the judgment of the prescribing physician.
  • a therapeutically effete amount may also refer to an amount of a pathogen-associated antigen, of a cancer antigen, or a polynucleotide encoding such an antigen, such as a pathogen-associated antigen, sufficient to immunize a subject against the pathogen.
  • An appropriate amount in any given instance can be ascertained by those skiled in the art or capable of determination by routine experimentation.
  • the term “delivering” means causing, through chemical or biophysical properties of a composition (e.g., an LNP composition), a payload (e.g., a polynucleotide) to pass from a site of administration to a subject to a target organ, target tissue, or target cel.
  • selective delivering refers delivery to a target organ, tissue, or cel at a greater rate or in a great amount than to a reference, non-target organ, tissue, or cel, or that a greater fraction of total amount administered to a subject is delivered to a target organ, tissue, or cel by the composition than by a reference composition.
  • selective delivery may mean that at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the total amount administered is delivered to the target organ, tissue, or cel.
  • selective delivery is determined by comparing the fraction of an LNP composition or payload that is delivery to a target organ (e.g., the lung) by an LNP composition comprises a selected lipid (e.g., SORT lipid) compared to a reference LNP composition in which the selected lipid is replaced by a control lipid.
  • a selected lipid e.g., SORT lipid
  • prevention refers to inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or al of the pathology or symptomatology of the disease, and/or slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or al of the pathology or symptomatology of the disease.
  • the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.
  • administering refers to providing a composition to a subject in a manner that permits the composition to have its intended effect.
  • Administration for vaccination or post- exposure prophylaxis may be performed by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.
  • “Co-administer” means that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies.
  • compositions provided herein can be administered alone or can be co-administered to the subject. Co-administration is meant to include simultaneous or sequential administration of the compounds individualy or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).
  • pharmaceuticalaly acceptable excipients and “pharmaceuticaly acceptable carier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject or patient and can mean excipients approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • pharmaceuticaly acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, filers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, faty acid esters, hydroxymethycelluloseose, polyvinyl pyrolidine, and colors, and the like.
  • compositions can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • emulsifiers salts for influencing osmotic pressure, buffers, coloring,
  • expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western bloting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).
  • “Treating” or “treatment” as used herein (and as wel-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, aleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or paliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • treatment as used herein includes any cure, amelioration, or prevention of a disease, e.g., a cancer.
  • Treatment may prevent the disease from occuring; inhibit the disease’s spread, e.g., metastasis of cancer cels or expansion of a tumor; relieve the disease’s symptoms, fuly or partialy remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
  • the term “vaccine” refers to a composition that can provide actively acquired immunity to a pathogen, or to a cancer cel, or cancer, in general.
  • a vaccine typicaly contains one or more agents that can induce an immune response in a subject against a pathogen, i.e., a target pathogen or disease.
  • the immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure.
  • Vaccines can be prophylactic (e.g., preventing or ameliorating the effectss of a future infection by a pathogen) or therapeutic (e.g., treating an infection by a pathogen).
  • the administration of vaccines is refered to as vaccination.
  • a vaccine composition can provide a nucleic acid, e.g., mRNA that encodes antigenic e.g., polypeptides (e.g., pathogen-associated antigenic polypeptides) to a subject.
  • the nucleic acid that is delivered via the vaccine composition in the subject can be expressed as an antigen (e.g., an antigenic protein) and cause the subject to acquire immunity against the antigenic molecules (e.g., pathogen-associated antigenic molecules).
  • the vaccine composition can provide mRNA encoding antigenic proteins (e.g., pathogen-associated antigenic proteins) that are associated with a certain pathogen, e.g., one or more peptides that are known to be expressed in the pathogen (e.g., pathogenic bacterium or virus).
  • pathogen refers to a bacterium, virus, fungus, protozoa, or any organism or an infectious agent that can cause disease.
  • virus or “virus particle” are used according to its plain ordinary meaning within Virology and refers to a virion including the viral genome (e.g., DNA, RNA, single strand, double strand), viral capsid and associated proteins, and in the case of enveloped viruses (e.g., herpesvirus), an envelope including lipids and optionaly components of host cel membranes, and/or viral proteins.
  • adjuvant refers to a pharmaceuticaly acceptable substance that enhances the immune response to an antigen (e.g., a pathogen-associated antigen) when co-administered with the antigen or administered before, during, or after administration of the antigen to a subject.
  • the LNP polynucleotide e.g., mRNA
  • the term “protective immune response” refers to an immune response that prevents and/or reduces the severity of infection with a pathogen when the subject is later chalenged with the pathogen, or reduces the severity of a cancerous cel growth, or to an immune response that generates a level of immune response that corelates with protection.
  • vaccination may generate a protective immune response if it results in production, in the plasma or serum, of the subject (e.g., human, pet, or agricultural animal), of neutralizing antibodies that protect the subject against subsequent infection or the emergence of proliferation of a cancerous cel or tumor, and/or are present in a quantity observed to confer protection upon test subjects.
  • the terms “immunization” and “immunizing” refer to administering a composition to a subject in an amount sufficient to elicit, after one or more administering steps, a desired immune response. Immunization may comprise between one and ten, or more administrations (e.g., injections) of the composition, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more administrations.
  • the first administration may elicit no detectable immune response as generaly each subsequent administration wil boost the immune response generated by prior administrations.
  • immunizing includes post-exposure prophylaxis.
  • cytokine as used herein is meant a generic term for proteins released by one cel population that act on another cel as intercellular mediators. For example, as described in Penichet et al., J Immunol Methods 248:91-101 (2001).
  • Cytokines include, but are not limited to IFN ⁇ , IL-10, IL-12p70, IL-1 ⁇ , IL-2, IL-4, IL-5, IL-6, KC/GRO, TNF- ⁇ , IL-16, IL-17A, IL- 17C, IL-21, IL-22, IL-23, IL-17E/IL-25, IL-15, IL-17F, IL-27P28/IL-30, IL-31, IL-33, IL- 17A/F, Il-9, OP-10, MCP-1, MIP-1 ⁇ , MIP-2, MIP-3 ⁇ . Cytokine expression can be detected by methods know in the art, for example with cytokine specific enzyme linked immunosorbent assay (ELISA). II.
  • ELISA cytokine specific enzyme linked immunosorbent assay
  • LNP compositions useful in the delivery of a payload for example an antigen (e.g., a pathogen-associated antigen) or a polynucleotide encoding an antigen to a host cel.
  • the LNPs are formulated to target diferent host cels in vitro or in vivo and the payload is then released in the host cel.
  • Payloads [0203]
  • the present disclosure contemplates delivery by an LNP composition of various payloads capable as acting directly as an antigen (e.g., a pathogen-associated antigen, or cancer antigen) or indirectly by encoding an antigen.
  • the payload may be an antigen associated with a pathogen, or an antigenic fragment thereof, or it may be a polynucleotide encoding such an antigen or antigenic fragment.
  • An antigen may also be a tumor-specific antigen, or an antigenic fragment thereof, or it may be a polynucleotide encoding such an antigen or antigenic fragment.
  • payloads comprise therapeutic polypeptides or polynucleotides encoding polypeptides.
  • the payload may be a polynucleotide encoding a gene related to CNS disease, or a polynucleotide encoding a gene editor for editing a gene related to CNS disease. A.
  • antigens include, but are not limited to, synthetic, recombinant, foreign, or homologous antigens, and antigenic materials may include but are not limited to proteins, peptides, polypeptides, lipids, glycolipids, carbohydrates, RNA, and DNA.
  • antigen in the LNP composition or an antigen expressed from a nucleic acid in an LNP composition comprises an antigen associated with a pathogen, i.e., a microbial or viral antigen.
  • Influenza virus is an enveloped RNA virus. Typical influenza epidemics cause increases in incidence of pneumonia and lower respiratory disease. Influenza leads to an estimated 9 milion – 41 milion ilnesses, 140,000 – 710,000 hospitalizations and 12,000 – 52,000 deaths per year in the USA.
  • influenza virus A, B, and C There are three types of influenza virus, A, B, and C. Influenza virus A and B are further classified based on the viral surface proteins hemagglutinin (HA) and neuraminidase (NA). Curently, 18 HA and 11 NA subtypes are identified.
  • HA hemagglutinin
  • NA neuraminidase
  • influenza virus antigen is from influenza A, an influenza B, or an influenza C virus.
  • influenza A virus may be selected from influenza A viruses characterized by a hemagglutinin (HA) selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18.
  • HA hemagglutinin
  • H8 hemagglutinin
  • H10 hemagglutinin
  • H11 hemagglutinin
  • H12 H13
  • H14 H15
  • H16 H17
  • H18 neuraminidase
  • the strain of influenza virus may be any strain of influenza virus.
  • a strain of influenza virus for use as provided herein includes A/California/07/2009 (H1N1), A/Michigan/45/2015 (H1N1), A/Netherlands/602/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2), A/Vietnam/1203/2004 (H5N1), and A/Vietnam/1194/2004 (H5N1).
  • an influenza virus antigen is an antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to HA protein, NA protein, NP (nucleoprotein) protein, M1 (matrix protein 1) protein, M2 (matrix protein 2) protein, non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2), or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to antigenic fragments thereof.
  • SARS-CoV-2 is a single, positive-strand RNA virus which can cause severe respiratory disease in humans.
  • the SARS CoV-2 viral spike (S) protein binds to angiotensin-converting enzyme 2 (ACE2), which is the entry receptor utilized by SARS-CoV- 2.
  • ACE2 angiotensin-converting enzyme 2
  • the spike (S) protein of coronaviruses is a major surface protein and is a target for neutralizing antibodies in infected subjects or patients. Therefore, it is considered a potential protective antigen for vaccine design.
  • the SARS-CoV N protein contains two distinct RNA- binding domains (the N-terminal domain [NTD] and the C-terminal domain [CTD]) linked by a poorly structured linkage region (LKR) containing a serine/arginine-rich (SR-rich) domain (SRD). Due to the positive amino acids, SARS-CoV N-NTD and N-CTD have been reported to bind with viral RNA genome. LKR is able to improve oligomerization.
  • a SARS-CoV-2 antigen has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SARS-CoV-2 S protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • a wel-characterized antigenic fragment of the S protein is its receptor binding domain (RBD).
  • RSD Respiratory Syncytial Virus
  • RSV is a negative-sense, single-stranded RNA virus which is the most common cause of bronchiolitis.
  • the virus is present in at least two antigenic subgroup (Group A and Group B). Proteins encoded by RSV are shown in Table 1. Table 1. Exemplary proteins encoded by RSV [0215] Two RSV surface glycoproteins, which is the atachment glycoprotein (G) and the fusion (F) glycoprotein, mediate atachment with and atachment to cels of the respiratory epithelium. G protein tethers and stabilizes the virus particle at the surface of bronchial epithelial cels, while F protein interacts with celular glycosaminoglycans to mediate fusion and delivery of the RSV virion contents into the host cel.
  • G atachment glycoprotein
  • F fusion glycoprotein
  • an RSV antigen comprises an F protein, G protein, SH protein, L protein, P protein, N protein, M2 protein, M protein, NS1 protein, and NS2 protein.
  • an RSV antigen is from an antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with RSV F protein or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • an RSV antigen is from an antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to RSV F protein or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • hMPV Human metapneumovirus
  • hMPV In addition to F protein, hMPV also encodes Glycoprotein (G), Matrix protein (M), phosphoprotein (P), Nucleoprotein (N), SH protein.
  • an hMPV antigen comprises an F protein, G protein, SH protein, P protein, N protein, and M protein.
  • the present disclosure is not limited by a particular strain of hMPV. Exemplary strains of hMPV comprise, but are not limited to A1, A2, B1, B2, CAN98-75, CAN97-83, hMPV isolate TN/92-4, hMPV isolate NL/1/99, or hMPV isolate PER/CFO0497/2010/B.
  • an hMPV antigen comprises an hMPV antigenic polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to hMPV F protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • an hMPV antigen comprises an hMPV antigenic polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to hMPV G protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • Human Parainfluenza virus type 3 (PIV3).
  • PIV is a negative-sense, single-stranded RNA virus which causes a variety of respiratory ilnesses. It is a major cause of ubiquitous acute respiratory infections of infancy and early childhood.
  • the PIV3 genome encodes two envelope glycoproteins, the hemagglutinin- neuraminidase (HN) and the fusion protein (F), a matrix protein (M), a nucleocapsid protein (N), and several nonstructural proteins including the viral replicase (L).
  • HN protein is necessary for atachment and cel entry while PIV3 F protein facilitates the viral fusion and cel entry.
  • a PIV3 antigen comprises a polynucleotide encoding F protein, HN protein, L protein, N protein, and M protein.
  • the present disclosure is not limited by a particular strain of PIV3.
  • the strain of PIV3 may be any strain of PIV3.
  • a PIV3 antigen comprises a PIV3 antigenic polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to PIV3 F protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • a PIV3 antigen comprises a PIV3 antigenic polypeptides having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to PIV3 HN protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • a MERS-CoV antigen comprises a MERS-CoV S protein, S1 subunit protein, S2 subunit protein, E protein, N protein, and M protein.
  • MERS-CoV The present disclosure is not limited by a particular strain of MERS-CoV.
  • the strain of MERS-CoV may be any strain of MERS-CoV.
  • Non-limiting examples of strains of MERS- CoV for use as provide herein include Riyadh_14_2013, 2cEMC/2012, and Hasa_1_2013.
  • Measles virus is a single-stranded RNA virus.
  • Fusion protein (F) executes membrane fusion, after receptor binding by the hemagglutinin (HA) protein.
  • a MeV antigen comprises a MeV F protein, HA protein, P protein, V protein, and C protein.
  • a MeV antigen comprises a MeV antigenic polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to MeV F protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • a MeV antigen comprises a MeV antigenic polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to MeV HA protein, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • the present disclosure is not limited by a particular strain of MeV.
  • the strain of MeV may be any strain of MeV.
  • Non-limiting examples of strains of MeV for use as provide herein include B3/B3.1, C2, D4, D6, D7, D8, G3, H1, Moraten, Rubeovax, Mvi/New Jersey.USA/45.05, MVi/Texas.USA/4.07, AIK-C, MVi/New York.USA/26.09, MVi/California.USA/8.04, and MVi/Pennsylvania.USA/20.09.
  • Streptococci antigens comprise a Group A carbohydrate or a modified group A carbohydrate.
  • Tuberculosis is an infectious disease usualy caused by Mycobacterium tuberculosis that can cause infection in lungs or other tissues.
  • the Bacile Calmete-Guerin (BCG) vaccine is one of the most widely used of al current vaccines in some countries. However, it does not prevent primary infection and reactivation of latent pulmonary infection. Therefore, the impact of BCG vaccination of transmission of TB is limited.
  • Tuberculosis antigens comprise an antigen listed in Table 5 or a polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigen listed in Table 5, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an antigenic fragment thereof.
  • the antigen comprises the H1N1 polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NO: 1.
  • the antigen comprises the influenza virus antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 2-26.
  • the antigen comprises the SARS-CoV-2 antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 27-29.
  • the antigen comprises the RSV antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 30-33.
  • the antigen comprises the PIV antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 34-35.
  • the antigen comprises the MERS-CoV antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 36-39.
  • the antigen comprises the measles virus antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 40-43.
  • the antigen comprises the tuberculosis antigen polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the polypeptide sequence encoded by SEQ ID NOs: 44-56. In some embodiments, the antigen comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 57-96. [0234] Exemplary viral antigen polynucleotide sequences are shown in Table 3. It is understood that T is T in DNA and T is U in RNA polynucleotide sequences. [0235] Table 3 shows the list of viral antigens. Table 3. Viral antigens
  • NSCLC non-smal cel lung cancers
  • 5T4 is trophoblast glycoprotein, which is expressed at high levels on the placenta and also on a wide range of human carcinomas including colorectal, gastric, renal, and ovarian cancers but rarely on normal tissues.
  • a 5T4 antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 101.
  • Survivin (baculoviral IAP repeat-containing 5) is highly expressed in most human cancer cels of epithelial and hematopoietic origin, and overexpression is associated with cancer progression, poor prognosis, resistance, and short patient survival.
  • a survivin antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 102.
  • a 5T4 antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 103.
  • NY-ESO-1 a cancer/testis antigen, has been reported the mRNA expression in various human tumors (melanoma, ovarian cancer, breast cancer, thyroid cancer, prostate cancer, bladder cancer, colon cancer, Burkit lymphoma, glioma, and hepatoma).
  • NY-ESO-1-specific antibody responses and/or specific CD8 and CD4 T cel responses directed against a broad range of NY-ESO-1 epitopes were induced by vaccination.
  • a NY-ESO-1 antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 104.
  • MAGE-C1 and MAGE-C2 are not expressed in a panel of normal tissues tested with the exception of testis. Among tumoral samples, they are frequently expressed in seminomas, melanomas, and bladder carcinomas. It is also expressed in a significant fraction of head and neck carcinomas, breast carcinomas, non-smal lung carcinomas, prostate adenocarcinomas and sarcomas.
  • a MAGE-C1 antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 105.
  • a MAGE-C2 antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 106.
  • Prostate cancer Prostate-specific antigen PSA
  • PSA Prostate-specific antigen
  • PSA is an androgen-regulated kalikrein-like, serine protease that is produced exclusively by the epithelial cels of al types of prostatic tissue, benign and malignant. PSA is the most widely used tumor marker for screening, diagnosing, and monitoring prostate cancer today. In particular, several immunoassays for the detection of serum PSA are in widespread clinical use.
  • a PSA antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 107.
  • Prostate-specific membrane antigen PSMA
  • Prostate-specific membrane antigen PSMA
  • Folate hydrolase 1 FOLH1
  • PSMA is a type I transmembrane glycoprotein, wherein PSMA expression is largely restricted to prostate tissues, but detectable levels of PSMA mRNA have been observed in brain, salivary gland, smal intestine, and renal cel carcinoma.
  • PSMA is highly expressed in most primary and metastatic prostate cancers. Particularly, PSMA is highly expressed in prostate cancer cels and nonprostatic solid tumor neovasculature and is a target for anticancer imaging and therapeutic agents.
  • PSMA acts as a glutamate carboxypeptidase (GCPI) on smal molecule substrates, including folate, the anticancer drug methotrexate, and the neuropeptide N-acetyl-L-aspartyl- L-glutamate.
  • GCPI glutamate carboxypeptidase
  • PSMA expression has been shown to correlate with disease progression, with highest levels expressed in hormone-refractory and metastatic disease.
  • PSMA is considered a biomarker for prostate cancer and is under intense investigation for use as an imaging and therapeutic target.
  • a PSMA antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 108.
  • Prostate stem cel antigen PSCA
  • PSCA Prostate stem cel antigen
  • PIN prostatic intraepithelial neoplasia
  • the PSCA gene shows 30% homology to stem cel antigen-2, a member of the Thy-l/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cel surface antigens.
  • GPI glycosylphosphatidylinositol
  • PSCA may be used as a prostate cancer marker to discriminate between malignant prostate cancers, normal prostate glands and non-malignant neoplasias.
  • a PSCA antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 109.
  • STEAP Six transmembrane epithelial antigens of the prostate (STEAP) [0251] Six transmembrane epithelial antigens of the prostate (STEAP) is a novel cel surface protein and is expressed predominantly in human prostate tissue and in other common malignancies including prostate, bladder, colon, and ovarian carcinomas, and in Ewing's sarcoma, suggesting that it could function as an almost universal tumor antigen. Particularly, STEAP is highly expressed in primary prostate cancer, with restricted expression in normal tissues. [0252] In some embodiments, a STEAP antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 110.
  • Prostatic acid phosphatase is an enzyme, which is secreted by epithelial cels of the prostate gland and catalyzes the conversion of orthophosphoric monoester to alcohol and orthophosphate. > 95% of normal adult prostate tissue samples, including normal tissue adjacent to tumor, as wel as >95% of primary adenocarcinomas, strongly express PAP. PAP expression can be detected in some normal human tissues besides the prostate (e.g. kidney, lung, testis, colon, and pancreas) but at a level approximately 1-2 orders of magnitude less, and PAP has generaly been considered a tissue-specific prostate antigen, highly expressed in both normal and malignant prostate cels.
  • PAP Prostatic acid phosphatase
  • a PAP antigen having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with SEQ ID NO: 111.
  • Mucin 1 (MUC1) is a large mucinous glycoprotein that is normaly expressed on the luminar surface of glandular epithelia.
  • MUC1 Its function in normal epithelia is to lubricate and to protect epithelial cels.
  • the expression of MUC1 is often increased, no longer restricted to a luminal surface and characterized by aberant glycosylation in many human malignancies, including prostate cancer.
  • MUC1 is expressed in about 60% of primary prostate cancers and 90% of lymph node metastases.
  • 86% of MUC1-positive primary prostate tumors were Gleason grade ⁇ 7, supporting an association with more aggressive disease. Both over- and underexpression of MUC-1 have been found to increase the risk of prostate cancer progression.
  • MUC1 has been shown to be immunogenic and has been described to induce specific immune responses comprising CD8+ CTLs and IgM antibodies in patients.
  • Vaccination against MUC1 using diferent vaccination approaches was associated with trends for clinical benefit in phase I trials in patients with advanced non-smal cel lung cancer and appeared wel tolerated. Vaccination against MUC1 in prostate cancer using the same vaccines was associated with a prolongation in PSA doubling time in some patients.
  • antigens, antigenic proteins or antigenic peptides as defined above which are encoded by the at least one mRNA of the composition according to the present invention, may comprise fragments or variants of those sequences.
  • Such fragments or variants may typicaly comprise a sequence having a sequence homology with one of the above mentioned antigens, antigenic proteins or antigenic peptides or sequences or their encoding nucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, equaly more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, to the entire wild-type sequence, either on nucleic acid level or on amino acid level.
  • Table 6 shows the exemplary polypeptide sequence of cancer antigens Table 6. Polypeptide sequence for antigen
  • Neoantigens are a class of tumor-specific antigen that arises from tumor-specific mutations which alter the amino acid sequence of genome-encoded proteins. These mutation- generated polypeptides play an important role in immune response against cancer cels by marking cancers as foreign to T cels.
  • the antigen in the LNP composition comprises a tumor- specificantigen, i.e., a neoantigen.
  • Tumors include, but are not limited to, adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cel carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), Chronic lymphocytic Leukaemia (CLL), colorectal cancer (CRC), Difuse large B-cel lymphoma (DLBCL), glioblastoma multiforme (GBM), head and neck squamous cel carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cel carcinoma (KIRC), kidney renal papilary cel carcinoma (KIRP), acute myeloid leukemia (LAML), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cel carcinoma (LUSC), multiple myeloma (MM), ovarian serous cystadenocarcinoma (OV),
  • the cancer or tumor antigen is one of the folowing antigens: CD2, CD 19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4- IBB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EGFR (e.g., EGFR-L858R), EpCAM, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, glucocorticoid
  • the antigen e.g., a pathogen-associated antigen, or cancer antigen
  • the antigen is greater than 9 amino acids in length, greater than 10 amino acids in length, greater than 11 amino acids in length, greater than 12 amino acids in length, greater than 13 amino acids in length, greater than 14 amino acids in length, greater than 15 amino acids in length, greater than 16 amino acids in length, greater than 17 amino acids in length, greater than 18 amino acids in length, greater than 19 amino acids in length, greater than 20 amino acids in length, greater than 25 amino acids in length, greater than 30 amino acids in length, greater than 35 amino acids in length, greater than 40 amino acids in length, greater than 45 amino acids in length, greater than 50 amino acids, greater than 100 amino acids, greater than 200 amino acids, greater than 300 amino acids, greater than 400 amino acids, greater than 500 amino acids, greater than 600 amino acids, greater than 700 amino acids, greater than 800 amino acids, greater than 900 amino acids, greater than 1000 amino acids, greater than 1500 amino acids, or greater than 2000 amino acids in length.
  • the nucleic acids or polynucleotides encoding for an antigen include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino- LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), or hybrids or combinations thereof.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose
  • the polynucleotide comprises a messenger RNA (mRNA), short hairpin (shRNA), or microRNA.
  • the polynucleotide comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine
  • the polynucleotides may encode at least one antigen (e.g., a pathogen-associated antigen, or a cancer antigen) or a fragment thereof.
  • the antigen may be a peptide, polypeptide, or protein.
  • the polynucleotide is greater than 30 nucleotides in length, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length.
  • the polynucleotide comprises about 50 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides.
  • the polynucleotide comprises about 50 to about 100 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides.
  • the polynucleotide comprises about 100 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 5000 nucleotides.
  • the polynucleotide comprises about 1000 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 2000 nucleotides. [0271] In some embodiments, the mRNA molecule is about 50 nucleotides in length. In some embodiments, the mRNA molecule is about 100 nucleotides in length. In some embodiments, the mRNA molecule is about 200 nucleotides in length. In some embodiments, the mRNA molecule is about 300 nucleotides in length. In some embodiments, the mRNA molecule is about 400 nucleotides in length. In some embodiments, the mRNA molecule is about 500 nucleotides in length.
  • the mRNA molecule is about 600 nucleotides in length. In some embodiments, the mRNA molecule is about 700 nucleotides in length. In some embodiments, the mRNA molecule is about 800 nucleotides in length. In some embodiments, the mRNA molecule is about 900 nucleotides in length. In some embodiments, the mRNA molecule is about 1000 nucleotides in length. In some embodiments, the mRNA molecule is about 2000 nucleotides in length. In some embodiments, the mRNA molecule is about 3000 nucleotides in length. In some embodiments, the mRNA molecule is about 4000 nucleotides in length.
  • the mRNA molecule is about 5000 nucleotides in length.
  • the mRNA comprises a viral polynucleotide shown in Table 3.
  • the mRNA comprises a bacterial polynucleotide shown in Table 5.
  • the mRNA encodes a viral antigen shown in Table 4.
  • the mRNA encoding the H1N1 comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1.
  • the mRNA encoding the influenza virus antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 2- 26.
  • the mRNA encoding the SARS-CoV-2 antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 27- 29.
  • the mRNA encoding the RSV antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 30- 33.
  • the mRNA encoding the PIV antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 34- 35.
  • the mRNA encoding the MERS-CoV antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 36- 39.
  • the mRNA encoding the measles virus antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 40- 43.
  • the mRNA encoding the tuberculosis antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 44- 56.
  • the mRNA encodes a polypeptide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 57-96.
  • the mRNA comprises a lung cancer polynucleotide shown in Table 8.
  • the mRNA comprises a prostate cancer polynucleotide shown in Table 9.
  • the mRNA encodes a neoantigen shown in Table 10.
  • the mRNA encoding the 5T4 antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 113- 116.
  • the mRNA encoding the survivin antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 117- 120.
  • the mRNA encoding the NY ESO-1 antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 121- 124.
  • the mRNA encoding the MAGE-C1 antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 125- 129.
  • the mRNA encoding the MAGE-C2 antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 130- 133.
  • the mRNA encoding the PSA (Prostate-Specific Antigen) antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 136-138.
  • the mRNA encoding the PSMA (Prostate-Specific Membrane Antigen) antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 139-143.
  • the mRNA encoding the PSCA (Prostate Stem Cel Antigen) antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 144-148.
  • the mRNA encoding the Six transmembrane epithelial antigens of the prostate (STEAP) antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 149-153.
  • the mRNA encoding the PAP (Prostatic Acid Phosphatase) antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 154-157.
  • the mRNA encoding the MUC1 (Mucin 1) antigen comprises a polynucleotide sequence which is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NOs: 158- 161. Table 8.
  • polynucleotide sequence encodes for one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., pathogen-associated antigens, or cancer antigens). In some embodiments, the polynucleotide encodes one or more, two or more, three or more polynucleotides encoding antigens from diferent pathogens. [0295] Examples of pathogens further include, but are not limited to, infectious fungi that infect mammals, and more particularly humans. C.
  • Central nervous system disease also known as central nervous system disorders, are neurological disorders that afect the structure or function of the brain or spinal cord that form the central nervous system (CNS).
  • a CNS disease is acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, atention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid
  • ADHD tention deficit hyperactivity disorder
  • Adie's pupil A
  • Alzheimer’s disease is the most common type of dementia. It is a progressive disease beginning with mild memory loss and possibly leading to loss of the ability to cary on a conversation and respond to the environment. It involves parts of the brain that control thought, memory, and language.
  • Amyotrophic lateral sclerosis (ALS) [0298] Amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease, is a neurological disease that affects motor neurons. ALS causes loss of muscle control.
  • Huntington’s disease is an inherited disorder that causes the progressive degeneration of nerve cels in the brain. Huntington’s disease causes changes in the central area of the brain, which afect movement, mood and thinking skils.
  • Parkinson’s disease is a brain disorder that causes unintended or uncontrolable movements, such as shaking, stifness, and dificulty with balance and coordination. Lewy bodies, unusual clumps of the protein ⁇ -synuclein, is observed in patient’s brain cels.
  • Spinal muscular atrophy (SMA) [0301] Spinal muscular atrophy (SMA) is a motor neuron disease involving the loss of motor neurons in the spinal cord. SMA is a genetic disease affecting the central nervous system, peripheral nervous system, and voluntary muscle movement (skeletal muscle).
  • genes involved in CNS diseases include, but are not limited to, 3R tau, 4R tau, AARS, ABCD1, ACOX1, ADGRV1, ADRA2B, AGA, AGER, ALDH7A1, ALG13, ALS2, ANG, ANXA11, APP, ARHGEF9, ARSA, ARSB, ARV1, ASAH1, ASPA, ATN1, ATP10A, ATP13A2, ATXN1, ATXN2, ATXN3, BAX, BCL-2, BDNF, BICD2, C9orf72, CACNA1A, CACNA1H, CACNB4, CASR, CCNF, CDKL5, CERS1, CFAP410, CHCHD10, CHD2, CHMP2B, CHRNA2, CHRNA4, CHRNA7, CHRNB2, CLCN2a, CLN1, CLN2, CLN3, CLN5, CLN6, CLN8, CNTN2, CPA6, CSTB, CTNS, CTSA, CTSD, DAO,
  • the payload comprises a polynucleotide, optionaly an mRNA, shRNA, or microRNA.
  • the mRNA encodes a polypeptide or protein selected from the group shown in Table 9, Table 10, and/or SEQ ID NOs: 201-207.
  • the mRNA encodes a gene-editing system or a component thereof.
  • the payload comprises a polypeptide or a protein.
  • the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject. Table 9. Ilustrative sequence of genes related to CNS disease
  • the SOD1 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 201.
  • the SMN-1 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 202.
  • the Frataxin polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 203.
  • the Frataxin polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 204. In some embodiments, the Frataxin polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 205. In some embodiments, the GAD-65 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 206.
  • the GAD-67 polypeptide comprises a polypeptide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 207.
  • Polynucleotides [0306]
  • the lipid composition described herein comprises one or more polynucleotides.
  • the polynucleotides encode for one or more polypeptides described herein.
  • nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ - LNA having a 2′-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • the present disclosure is not limited to the specific polynucleotides disclosed herein.
  • the present disclosure is not limited in scope to any particular source, sequence, or type of polynucleotides, however, as one of ordinary skil in the art could readily identify related homologs in various other sources of the polynucleotides including polynucleotides from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). It is contemplated that the polynucleotides used in the present disclosure can comprise a sequence based upon a naturaly-occuring sequence.
  • sequences that have at least about 50%, usualy at least about 60%, more usualy about 70%, most usualy about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the naturaly-occuring sequence.
  • the polynucleotide is a complementary sequence to a naturaly occuring sequence, or complementary to at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and 100%. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated herein.
  • the polynucleotide used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism.
  • the polynucleotide comprises complementary DNA (cDNA).
  • cDNA complementary DNA
  • a cDNA plus a natural intron or an intron derived from another gene such engineered molecules are sometime refered to as “mini –genes”.
  • mini –genes is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • cDNA primarily contains coding sequences of the coresponding protein. There may be times when the ful or partial genomic sequence is prefered, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.
  • the polynucleotide comprises one or more segments comprising a smal interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro- ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, deoxyribonucleic acid (DNA), a double stranded deoxyribonucleic acid (dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a single stranded ribonucleic acid
  • the polynucleotide encodes at least one of the therapeutic agent (or prophylactic agent) described herein.
  • the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length.
  • the mRNA is about 50 nucleotides in length. In some embodiments, the mRNA molecule is about 100 nucleotides in length. In some embodiments, the mRNA molecule is about 200 nucleotides in length. In some embodiments, the mRNA molecule is about 300 nucleotides in length. In some embodiments, the mRNA molecule is about 400 nucleotides in length. In some embodiments, the mRNA molecule is about 500 nucleotides in length. In some embodiments, the mRNA molecule is about 600 nucleotides in length. In some embodiments, the mRNA molecule is about 700 nucleotides in length.
  • the mRNA molecule is about 800 nucleotides in length. In some embodiments, the mRNA molecule is about 900 nucleotides in length. In some embodiments, the mRNA molecule is about 1000 nucleotides in length. In some embodiments, the mRNA molecule is about 2000 nucleotides in length. In some embodiments, the mRNA molecule is about 3000 nucleotides in length. In some embodiments, the mRNA molecule is about 4000 nucleotides in length. In some embodiments, the mRNA molecule is about 5000 nucleotides in length. [0313] In some embodiments, the polynucleotide comprises about 50 to about 100000 nucleotides.
  • the polynucleotide comprises about 50 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 100 nucleotides.
  • the polynucleotide comprises about 100 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 200 nucleotides.
  • the polynucleotide comprises about 500 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 2500 nucleotides.
  • the polynucleotide comprises about 1000 to about 2000 nucleotides.
  • the LNP composition comprises mRNA at a lipid:mRNA (weight/weight) ratio is between 5:1 and 40:1.
  • the LNP comprises mRNA at a lipid:mRNA ratio between 10:1 and 40:1, between 15:1 and 40:1, between 20:1 and 40:1, between 25:1 and 40:1, between 30:1 and 40:1, between 35:1 and 40:1, between 20:1 and 35:1, between 25:1 and 35:1, between 30:1 and 35:1, between 20:1 and 30:1, between 25:1 and 30:1, between 20:1 and 25:1, between 25:1 and 30:1, between 25:1 and 35:1, between 20:1 and 36:1, between 25:1 and 36:1, between 5:1 and 45:1, between 20:1 and 40:1, between 25:1 and 40:1, between 35:1 and 40:1, or between 30:1 and 40:1.
  • the LNP comprises mRNA at a lipid:mRNA ratio of 30:1. In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio of 40:1. [0315] In some embodiments, the mRNA encodes a gene or a portion of a gene related to CNS disease shown in Table 10. Table 10. Ilustrative genes related to CNS
  • the CNS disease is Alzheimer's disease and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of 3R tau, 4R tau, AGER, APP, BAX, BCL-2, CHRNA7, DRD2, GFAP, GRIA1, GRIA2, GRIK1, GRIN1, IL-1, SLC1A1, SYP, and SYT1.
  • the CNS disease is amyotrophic lateral sclerosis (ALS) and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of ALS2, ANG, ANXA11, ATXN2, C9orf72, CHMP2B, CFAP410, CHCHD10, CCNF, DAO, DCTN1, EPHA4, ERBB4, FIG4, FUS, GLE, GLT8D1, HNRNPA1, MATR3, NEFH, NEK1, OPTN, PFN1, PON1, PON2, PON3, PPARGC1A, PRPH, SETX, SIGMAR1, SMN1, SOD1, SPG11, SQSTM1, TAF15, TARDBP, TBK1, TREM2, UBQLN2, UNC13A, VAPB and VCP.
  • ALS2 amyotrophic lateral sclerosis
  • the CNS disease is Huntington's disease and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of ATN1, ATXN1, ATXN2, ATXN3, FTL, HTT, IT15, JPH3, PRNP, SLC2A3, TBP, TITF-1 and XBP1.
  • the CNS disease is Parkinson's disease and the at least one transgene comprises a mRNA comprises cDNA of a gene selected from the group comprising or consisting of ATP13A2, BDNF, EGLN1, GBA, GSTM1, LRRK2, NR4A2, NTRK2, PARK2, PARK7, PINK1, PRKN, S106 ⁇ , SKP1, SNCA, VPS35 and UCH-L1.
  • a mRNA comprises cDNA of a gene selected from the group comprising or consisting of ATP13A2, BDNF, EGLN1, GBA, GSTM1, LRRK2, NR4A2, NTRK2, PARK2, PARK7, PINK1, PRKN, S106 ⁇ , SKP1, SNCA, VPS35 and UCH-L1.
  • the polynucleotide comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine, 4-thio-l-methyl-pseudouridine, 2- thio-1-methyl-pseud
  • a polynucleotide of the disclosure comprises a modified pyrimidine, such as a modified uridine.
  • a uridine analogue is selected from pseudouridine ( ⁇ ), 1-methylpseudouridine (mlP), 2-thiouridine (s2U), 5-methyluridine (m5U), 5-methoxyuridine (mo5U), 4-thiouridine (s4U), 5-bromouridine (Br5U), 2'O-methyluridine (U2'm), 2'-amino-2'-deoxyuridine (U2'NH2), 2'-azido-2'-deoxyuridine (U2'N3), and 2'-fluoro- 2'-deoxyuridine (U2'F).
  • a polynucleotide such as a nucleic acid construct, a vector, or a polyribonucleotide of the disclosure can comprise one or more untranslated regions.
  • An untranslated region can comprise any number of modified or unmodified nucleotides.
  • Untranslated regions (UTRs) of a gene are transcribed but not translated into a polypeptide.
  • UTRs Untranslated regions
  • an untranslated sequence can increase the stability of the polynucleotide and the eficiency of translation.
  • the regulatory features of a UTR can be incorporated into the modified mRNA molecules of the present disclosure, for instance, to increase the stability of the molecule.
  • a 5' UTR can comprise a Kozak sequence which is involved in the process by which the ribosome initiates translation of many genes.
  • Kozak sequences can have the consensus GCC(R)CCAUGG, where R is a purine (adenine or guanine) that is located three bases upstream of the start codon (AUG).5 ' UTRs may form secondary structures which are involved in binding of translation elongation factor.
  • mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferin, alpha fetoprotein, erythropoietin, or Factor VII
  • 5' UTR from muscle proteins MyoD, Myosin, Myoglobin, Myogenin, Herculin
  • endothelial cels Te- 1, CD36
  • myeloid cels C/EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, i-NOS
  • leukocytes CD45, CD18
  • adipose tissue CD36, GLUT4, ACRP30, adiponectin
  • SP-A/B/C/D lung epithelial cels
  • Non-UTR sequences can be incorporated into the 5' (or 3' UTR) UTRs of the polynucleotides of the present disclosure.
  • the 5' and/or 3' UTRs can provide stability and/or translation eficiency of polynucleotides.
  • introns or portions of intron sequences can be incorporated into the flanking regions of a polynucleotide. Incorporation of intronic sequences can also increase the rate of translation of the polynucleotide.
  • 3' UTRs may have stretches of Adenosines and Uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover.
  • AU rich elements can be separated into classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class I AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and T F- ⁇ . Class II ARES are less wel defined. These U rich regions do not contain an AUUUA motif c-Jun and Myogenin are two wel-studied examples of this class.
  • AREs Proteins binding to the AREs may destabilize the messenger RNA (mRNA), whereas members of the ELAV family, such as HuR, may increase the stability of mRNA.
  • HuR may bind to AREs of al the three classes.
  • Engineering the HuR specific binding sites into the 3 ' UTR of polynucleotide molecules can lead to HuR binding and thus, stabilization of the message in vivo.
  • Engineering of 3' UTR AU rich elements (AREs) can be used to modulate the stability of a polynucleotide.
  • One or more copies of an ARE can be engineered into a polynucleotide to modulate the stability of a polynucleotide.
  • AREs can be identified, removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • Transfection experiments can be conducted in relevant cel lines, using polynucleotides and protein production can be assayed at various time points post-transfection.
  • cels can be transfected with diferent ARE- engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days post-transfection.
  • a polynucleotides such as a nucleic acid construct, a vector, a polyribonucleotide, or compositions of the disclosure can comprise an engineered 5' cap structure, or a 5 '-cap can be added to a polynucleotide intracellularly.
  • the 5 'cap structure of an mRNA can be involved in binding to the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cel and translation competency through the association of CBP with poly(A) binding protein to form the mature pseudo-circular mRNA species.
  • CBP mRNA Cap Binding Protein
  • the 5 'cap structure can also be involved in nuclear export, increases in mRNA stability, and in assisting the removal of 5' proximal introns during mRNA splicing.
  • a polynucleotides such as a nucleic acid construct, a vector, or a polynucleotide can be 5 '-end capped generating a 5 '-GpppN-3 ' -triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule.
  • the cap-structure can comprise a modified or unmodified 7- methylguanosine linked to the first nucleotide via a 5 '-5 ' triphosphate bridge.
  • This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue (Cap-0 structure).
  • a cap can comprise further modifications, including the methylation of the 2' hydroxy-groups of the first 2 ribose sugars of the 5' end of the mRNA.
  • an eukaryotic cap-1 has a methylated 2'-hydroxy group on the first ribose sugar
  • a cap-2 has methylated 2 '-hydroxy groups on the first two ribose sugars.
  • the 5' cap can be chemicaly similar to the 3 ' end of an RNA molecule (the 5 ' carbon of the cap ribose is bonded, and the free 3'-hydroxyls on both 5'- and 3 '- ends of the capped transcripts.
  • Such double modification can provide significant resistance to 5' exonucleases.
  • Non-limiting examples of 5 ' cap structures that can be used with a polynucleotide include, but are not limited to, m7G(5')ppp(5')N (Cap-0), m7G(5')ppp(5')N1mpNp (Cap-1), and m7G(5')-ppp(5 ')N1mpN2mp (Cap-2).
  • Modifications to the modified mRNA of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life while facilitating translation. Because cap structure hydrolysis requires cleavage of 5'-ppp- 5' triphosphate linkages, modified nucleotides may be used during the capping reaction.
  • a Vaccinia Capping Enzyme from New England Biolabs may be used with guanosine a-thiophosphate nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.
  • Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides. Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the mRNA on the 2'-hydroxyl group of the sugar ring.
  • 5' terminal caps may include endogenous caps or cap analogues.
  • a 5' terminal cap may comprise a guanine analogue.
  • Useful guanine analogues include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.
  • an untranslated region can comprise any number of nucleotides.
  • An untranslated region can comprise a length of about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length.
  • An untranslated region can comprise a length of for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length.
  • a polynucleotide of the disclosure can comprise a polyA sequence.
  • a polyA sequence (e.g., polyA tail) can comprise any number of nucleotides.
  • a polyA sequence can comprise a length of about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length.
  • a polyA sequence is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length.
  • a polyA sequence can comprise a length of for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs
  • a polyA sequence can comprise a length of at most 100 bases or base pairs, 90 bases or base pairs, 80 bases or base pairs, 70 bases or base pairs, 60 bases or base pairs, 50 bases or base pairs, 40 bases or base pairs, 30 bases or base pairs, 20 bases or base pairs, 10 bases or base pairs, or 5 bases or base pairs.
  • the LNPs of the present disclosure can comprise one or more components for gene editing, such as, but not limited to, a guide RNA, a tracr RNA, a sgRNA, an mRNA encoding a gene or base editing protein, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease (e.g., Cas9), a DNA template for gene editing, or a combination thereof.
  • the payload of the LNPs can be suitable for a genome editing technique.
  • the genome editing technique can be CRISPR or TALEN.
  • the LNPs can comprise one or more mRNAs, which can encode a gene editing or base editing protein.
  • the LNPs can comprises both a gene- or base- editing protein encoding mRNA and one or more guide RNAs.
  • the LNPs can comprise at least one nucleic acid suitable for a genome editing technique, such as a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), and a DNA repair template.
  • CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it can be a nickase instead of making double-strand cuts or so that it can bind the sequence specified by the guide RNA but has no enzymatic activity.
  • the base editing protein can be a fusion protein comprising a deaminase domain and a sequence-specific DNA binding domain, such as an inactive CRISPR nuclease.
  • Gene Editing Methods [0332]
  • the presently described LNPs or pharmaceutical composition can comprise a payload of any conventional gene editing methods.
  • gene editing components can be selectively delivered to the cels of target organ.
  • the target organ can be lungs.
  • the cels of target organ can be lung cels.
  • the cels can be ciliated cels, goblet cels, secretory cels, club cels, basal cels or ionocytes.
  • the gene editing can be targeted editing. Targeted editing can be achieved either through a nuclease-independent approach or through a nuclease- dependent approach.
  • the nuclease-independent targeted editing such as base-editing and/or prime editing, can involve precise modifications to DNA sequences without creating double-strand breaks.
  • Homologous recombination can be guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the cels of target organ.
  • Base editing can alow for the conversion of one DNA base pair into another at a specific target site.
  • the nuclease can be a fusion of a deaminase enzyme to a modified Cas9 protein (dCas9) or other engineered Cas variants.
  • base editing can change C (cytosine) to T (thymine) or A (adenine) to G (guanine) in the endogenous DNA.
  • a guide RNA can be designed to target the specific genomic location of interest in the cels of target organ.
  • Prime editing can alow for more complex and precise DNA modifications, including insertions, deletions, and al 12 possible base-to-base conversions (A, C, G, T) without double-strand breaks.
  • a prime editing guide RNA which can consist of a guide sequence and a template for the desired edit, can be designed.
  • the prime editor protein (PE2) which can combine a reverse transcriptase and a Cas9 variant, can be guided to the target site by the prime editing guide RNA.
  • the Cas9 variant can generate a single-strand break (nick) in the DNA.
  • the reverse transcriptase then can use the prime editing guide RNA’s template sequence to copy the desired changes into the nicked strand of DNA. Subsequently, the celular repair machinery of the cels of target organ can repair the nick, incorporating the edited sequence, via homology-directed repair (HDR).
  • HDR homology-directed repair
  • the nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare- cuting nucleases (e.g., endonucleases).
  • DSBs double strand breaks
  • Such nuclease-dependent targeted editing can also utilize DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which can occur in response to DSBs.
  • NHEJ non-homologous end joining
  • DNA repair by NHEJ can lead to random insertions or deletions (indels) of a smal number of endogenous nucleotides.
  • repair can also occur by a homology directed repair (HDR).
  • HDR homology directed repair
  • a nuclease of the nuclease-dependent targeted editing can include, but not limited to, CRISPR-Cas9, CRISPR-Cas12 (Cpf1), CRISPR-Cas13, C2c2, C2c6, NgAgo, and/or TALEN.
  • CRISPR-Cas9 CRISPR-Cas9
  • CRISPR-Cas12 Cpf1
  • CRISPR-Cas13 C2c2, C2c6, NgAgo
  • TALEN TALEN
  • CRISPR-Cas9 Gene Editing System
  • DICE dual integrase cassete exchange
  • the CRISPR-Cas9 system is a naturaly occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform used for gene editing.
  • CRISPR is a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cel, for example, by viruses that have infected or atacked the prokaryote. These fragments of DNA can be used by the prokaryote to detect and destroy similar foreign DNA upon re- introduction, for example, from similar viruses during subsequent atacks.
  • RNA molecules comprising the spacer sequence, which can associate with and target Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA.
  • CRISPR-associated proteins able to recognize and cut the foreign, exogenous DNA.
  • CRISPR/Cas systems Numerous types and classes of CRISPR/Cas systems have been described in e.g., Koonin et al., Curr Opin Microbiol 37:67-78 (2017).
  • crRNA can drive sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typicaly with about 20 nucleotide sequence in the target DNA. Changing the sequence of the 5’ 20 nucleotides in the crRNA can alow targeting of the CRISPR-Cas9 complex to specific loci.
  • the CRISPR-Cas9 complex can only bind DNA sequences that contain a sequence match to the first 20 nucleotides of the crRNA, if the target sequence is folowed by a specific short DNA motif (with the sequence NGG) refered to as a protospacer adjacent motif (PAM).
  • tracrRNA can hybridize with the 3’ end of crRNA to form an RNA-duplex structure that can be bound by the Cas9 endonuclease to form the catalyticaly active CRISPR-Cas9 complex, which can then cleave the target DNA.
  • NHEJ non- homologous end joining
  • HDR homology-directed repair
  • NHEJ can be eror-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications can typicaly be less than 20 nucleotides.
  • the resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes.
  • HDR can use a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cels and can occur at a relatively low frequency in most cel types.
  • Cas9 endonuclease can be used in a CRISPR method for geneticaly engineering cels of the target organ of the LNPs described herein.
  • Cas9 enzyme can be from Streptococcus pyogenes, although other Cas9 homologs can also be used.
  • the Cas9 enzyme can be wild- type Cas9.
  • the Cas9 enzyme can be a modified version of Cas9 (e.g., evolved versions of Cas9, or Cas9 orthologues or variants).
  • Cas9 can be substituted with another RNA-guided endonuclease, such as Cpf1 (class I CRISPR/Cas system).
  • CRISPR/Cas system can comprise components derived from a Type-I, Type-I, or Type-II system.
  • the CRISPR/Cas system can comprise components derived from Class 1 and Class 2 CRISPR/Cas systems, having Types I to V or Types I, V, and VI, respectively (Makarova et al., Nat Rev Microbiol 13(11):722-36 (2015); Shmakov et al., Mol Cel 60:385-397 (2015).
  • Class 2 CRISPR/Cas systems can have single protein effectors.
  • Cas proteins of Types I, V, and VI can be single-protein, RNA-guided endonucleases, herein caled Class 2 Cas nucleases.
  • Class 2 Cas nucleases can include, for example, but not limited to, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins.
  • the Cpf1 nuclease is homologous to Cas9 and contains a RuvC-like nuclease domain.
  • the Cas nuclease can be from a Type-I CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR/Cas9 system).
  • the Cas nuclease can be from a Class 2 CRISPR/Cas system (a single-protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein).
  • the Cas9 and Cpf1 family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, which is further explained infra.
  • a Cas nuclease can comprise more than one nuclease domain.
  • a Cas9 nuclease can comprise at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9).
  • the Cas9 nuclease can introduce a DSB in the target sequence.
  • the Cas9 nuclease can be modified to contain only one functional nuclease domain.
  • the Cas9 nuclease can be modified such that one of the nuclease domains can be mutated or fuly or partialy deleted to reduce its nucleic acid cleavage activity.
  • the Cas9 nuclease can be modified to contain no functional RuvC-like nuclease domain.
  • the Cas9 nuclease can be modified to contain no functional HNH-like nuclease domain.
  • the Cas9 nuclease in which only one of the nuclease domains can be functional, can be a nickase that can introduce a single-stranded break (nick) into the target sequence.
  • a conserved amino acid within a Cas9 nuclease domain can be substituted to reduce or alter a nuclease activity.
  • the Cas nuclease nickase can comprise an amino acid substitution in the RuvC-like nuclease domain.
  • Exemplary amino acid substitutions in the RuvC-like nuclease domain can include D10A (based on the S. pyogenes Cas9 nuclease).
  • the nickase can comprise an amino acid substitution in the HNH-like nuclease domain.
  • Exemplary amino acid substitutions in the HNH-like nuclease domain can include, but not limited to, E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 nuclease).
  • the Cas nuclease can be from a Type-I CRISPR/Cas system.
  • the Cas nuclease can be a component of the Cascade complex of a Type-I CRISPR/Cas system.
  • the Cas nuclease can be a Cas3 nuclease.
  • the Cas nuclease can be derived from a Type-II CRISPR/Cas system. In some embodiments, the Cas nuclease can be derived from Type-IV CRISPR/Cas system. In some embodiments, the Cas nuclease can be derived from a Type-V CRISPR/Cas system. In some embodiments, the Cas nuclease can be derived from a Type-VI CRISPR/Cas system. [0351] A Type I CRISPR/Cas system can utilize a large effector complex known as Cascade (CRISPR-associated complex for antiviral defense) for target binding and interference.
  • Cascade CRISPR-associated complex for antiviral defense
  • the Cascade complex can contain multiple Cas proteins, including Cas3, which can be responsible for the destruction of the target DNA.
  • a Type I CRISPR/Cas system particularly the CRISPR-Cas9 system, can utilize a single Cas9 protein, guided by a synthetic guide RNA (sgRNA), to introduce double-strand breaks in target DNA for subsequent repair or modification.
  • a Type II CRISPR/Cas system can utilize a Csm (CRISPR-Cas subtype multiprotein) or Cmr (CRISPR-Cas subtype ribonucleoprotein) complex for interference.
  • Type II CRISPR/Cas system can target RNA molecules in addition to DNA.
  • a Type V CRISPR/Cas system including Cpf1 (also known as Cas12) and C2c2 (also known as Cas13), can utilize a single effector protein to perform interference.
  • a Type VI CRISPR/Cas system can utilize a single Cas protein, such as C2c2 (also known as Cas13), to target and cleave RNA molecules, making it useful for RNA editing and manipulation.
  • Guide RNAs gRNAs
  • the CRISPR technology can involves the use of a genome- targeting nucleic acid that can direct one or more endonucleases to a specific target sequence within a target gene for gene editing at the specific target sequence.
  • the genome-targeting nucleic acid can be an RNA.
  • a genome-targeting RNA is refered to as a “guide RNA” or “gRNA” herein.
  • a guide RNA can comprise at least one spacer sequence that can hybridize to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence.
  • the gRNA can also comprise a second RNA caled the tracrRNA sequence.
  • the CRISPR repeat sequence and tracrRNA sequence can hybridize to each other to form a duplex.
  • the crRNA can form a duplex.
  • the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide can form a complex.
  • the genome- targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide.
  • the genome-targeting nucleic acid can thus direct the activity of the site-directed polypeptide.
  • each guide RNA can be designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science 337:816-821 (2012); Deltcheva et al., Nature 471:602-607 (2011).
  • the genome-targeting nucleic acid e.g., gRNA
  • the first strand can comprise in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, and a minimum CRISPR repeat sequence.
  • the second strand can comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence.
  • the genome-targeting nucleic acid e.g., gRNA
  • sgRNA single-molecule guide RNA
  • sgRNA in a Type I system can comprise, in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension can comprise elements that can contribute additional functionality (e.g., stability) to the guide RNA.
  • the single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension can comprise one or more hairpins.
  • a single-molecule guide RNA in a Type V system can comprise, in the 5’ to 3’ direction, a minimum CRISPR repeat sequence and a spacer sequence.
  • a spacer sequence in a gRNA is a sequence (e.g., a 20-nucleotide sequence) that can define the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest (e.g., DNAI1 or CFTR).
  • the spacer sequence can range from 15 to 30 nucleotides.
  • the spacer sequence can contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • a spacer sequence can contain 20 nucleotides.
  • the target sequence is in a target gene (e.g., DNAI1 or CFTR) that can be adjacent to a PAM sequence and can be the sequence to be modified by an RNA-guided nuclease (e.g., Cas9).
  • the target sequence is on the PAM strand in a target nucleic acid, which is a double-stranded molecule containing the PAM strand and a complementary non-PAM strand.
  • a target gene e.g., DNAI1 or CFTR
  • Cas9 RNA-guided nuclease
  • the target sequence is on the PAM strand in a target nucleic acid, which is a double-stranded molecule containing the PAM strand and a complementary non-PAM strand.
  • the gRNA spacer sequence can hybridize to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest.
  • the gRNA spacer sequence can be the RNA equivalent of the target sequence.
  • the spacer of a gRNA can interact with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the spacer thus can vary depending on the target sequence of the target nucleic acid of interest.
  • the spacer sequence can be designed to hybridize to a region of the target nucleic acid that is located 5’ of a PAM recognizable by a Cas9 enzyme used in the system.
  • the spacer can perfectly match the target sequence or can have mismatches.
  • Each Cas9 enzyme can have a particular PAM sequence that it can recognize in a target DNA.
  • S. pyogenes can recognize in a target nucleic acid a PAM that comprises the sequence 5’-NRG-3’, where R can comprise either A or G, where N can be any nucleotide and N can be immediately 3’ of the target nucleic acid sequence targeted by the spacer sequence.
  • the target nucleic acid sequence can have about 20 nucleotides in length. In some embodiments, the target nucleic acid can have less than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have more than about 20 nucleotides in length.
  • the target nucleic acid can have at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid can have at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence can have 20 bases immediately 5’ of the first nucleotide of the PAM. For example, in a sequence comprising 5′-NNNNNNNNNNNNNNNNNNNRG 3′, the target nucleic acid can be the sequence that coresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence can be the S. pyogenes PAM.
  • the guide RNA can target any sequence of interest via the spacer sequence in the crRNA.
  • the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
  • the spacer sequence of the guide RNA and the target sequence in the target gene can be 100% complementary.
  • the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.
  • the length of the spacer sequence in gRNAs can depend on the CRISPR/Cas9 system and components used for editing any of the target genes (e.g., DNAI1 or CFTR).
  • the target genes e.g., DNAI1 or CFTR.
  • the spacer sequence can have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length.
  • the spacer sequence can have 18-24 nucleotides in length.
  • the targeting sequence can have 19-21 nucleotides in length.
  • the spacer sequence can comprise 20 nucleotides in length.
  • the gRNA can be an sgRNA, which can comprise a 20- nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a less than 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a more than 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a variable length spacer sequence with about 17-30 nucleotides at the 5’ end of the sgRNA sequence.
  • the gRNAs can comprise unmodified ribonucleic acid. In some embodiments, the gRNAs can comprise modified ribonucleic acid.
  • RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that can enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other atributes, as described in the art.
  • non-natural modified nucleobases can be introduced into any of the gRNAs during synthesis or post-synthesis. In some embodiments, modifications can be on internucleoside linkages, purine or pyrimidine bases, or sugar.
  • a modification can be introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme.
  • more than one guide RNAs can be used with a CRISPR/Cas nuclease system.
  • Each guide RNA can contain a diferent targeting sequence, such that the CRISPR/Cas system can cleave more than one target nucleic acid.
  • one or more guide RNAs can have the same or differing properties, such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA can be used, each guide RNA can be encoded on the same or on different vectors.
  • the promoters used to drive expression of the more than one guide RNA can be the same or different.
  • enzymatic or chemical ligation methods can be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, and the like.
  • the CRISPR/Cas nuclease system can contain multiple gRNAs, for example, 2, 3, or 4 gRNAs. Such multiple gRNAs can target diferent sites in a same target gene. Alternatively, the multiple gRNAs can target diferent genes.
  • the guide RNA(s) and the Cas protein can form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex.
  • RNP ribonucleoprotein
  • the guide RNAs can guide the Cas protein to a target sequence(s) on one or more target genes (e.g., DNAI1 and CFTR), where the Cas protein can cleave the target gene at the target site.
  • the CRISPR/Cas complex can be a Cpf1/guide RNA complex.
  • the CRISPR complex can be a Type- I CRISPR/Cas9 complex.
  • the Cas protein can be a Cas9 protein.
  • the CRISPR/Cas9 complex can be a Cas9/guide RNA complex.
  • the indel frequency (editing frequency) of a particular CRISPR/Cas nuclease system, comprising one or more specific gRNAs can be determined using a TIDE analysis, which can be used to identify highly available gRNA molecules for editing a target gene.
  • a highly available gRNA can yield a gene editing frequency of higher than 80%.
  • a gRNA can be considered to be highly efficient if it can yield a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • additional gene editing systems as known in the art can also be used as a payload of the LNPs described herein.
  • the additional gene editing system can comprise zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, or the like.
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which can be a polypeptide domain that can bind DNA in a sequence-specific manner through one or more zinc fingers.
  • ZFBD zinc finger DNA binding domain
  • a zinc finger can be a domain of about 30 amino acids within the zinc finger binding domain whose structure can be stabilized through coordination of a zinc ion.
  • Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • a designed zinc finger domain can be a domain not occuring in nature whose design/composition results principaly from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.
  • a selected zinc finger domain can be a domain not found in nature whose production can result primarily from an empirical process such as phage display, interaction trap or hybrid selection.
  • a ZFN can be a fusion of the FokI nuclease with a zinc finger DNA binding domain.
  • a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
  • a “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins can be secreted by plant pathogens of the genus Xanthomonas during infection.
  • TAL effector DNA binding domain specificity can depend on an effector-variable number of imperfect 34 amino acid repeats, which can comprise polymorphisms at select repeat positions caled repeat variable-diresidues (RVD).
  • a TALEN can be a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.
  • Bxb1 nuclease also known as the Bxb1 integrase, is a site-specific recombinase enzyme derived from the mycobacteriophase Bxb1.
  • the Bxb1 integrase can catalyze site-specific recombination between two specific DNA sequences, refered to as atachment (at) sites.
  • the Bxb1 integrase can recognize a specific 48 base-pair sequence within the atachment sites.
  • the phiC31 nuclease also known as the phiC31 integrase, is derived from the bacteriophage phiC31.
  • the phiC31 nuclease can catalyze site-specific recombination between two specific DNA sequences, refered to as atB (atachment site in bacteriophage) and atP (atachment site in the phage).
  • the phiC31 nuclease can promote integration of a DNA fragment flanked by atB and atP into the genome in cels of target organ.
  • the phiBT1 nuclease can integrate into a diferent atachment site than phiC31.
  • the W ⁇ /SPBc/TP901-1 nuclease also known as bacteriophage P2 Bxb1 Cre nuclease, is a site- specific recombination enzyme derived from the temperate bacteriophage P2.
  • D. Lipids [0373] The present disclosure contemplates LNP compositions comprising an antigen (e.g., a pathogen-associated antigen, or a cancer antigen) or a polynucleotide encoding an antigen as described herein and a mixture of lipids for delivery to a host cel.
  • Exemplary lipids contemplated for the LNP compositions described herein comprise ionizable cationic lipids, selective organ targeting lipids, helper lipids, structural lipids and polyethylene glycol- conjugated lipids (PEG-lipids).
  • the disclosure provides a lipid nanoparticle composition comprising an antigen (e.g., a pathogen-associated antigen, or a cancer antigen) or a polynucleotide encoding an antigen, and a selective organ targeting (SORT) lipid; and an ionizable cationic lipid, a helper lipid, a sterol, and/or a polyethylene glycol-conjugated lipid (PEG-lipid).
  • an antigen e.g., a pathogen-associated antigen, or a cancer antigen
  • SORT selective organ targeting
  • the LNP composition comprises one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., a pathogen-associated antigen). In some embodiments, the LNP composition comprises one or more, two or more, three or more polynucleotides encoding antigens from diferent pathogens. [0376] In some embodiments, the LNP composition comprises one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., a cancer antigen).
  • a cancer antigen e.g., a cancer antigen
  • the LNP composition comprises one or more, two or more, three or more polynucleotides encoding antigens from diferent cancer cel types. [0377] In some embodiments, the LNP composition delivers one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., a pathogen-associated antigen). In some embodiments, the LNP compositions delivers one or more, two or more, three or more polynucleotides encoding antigens from diferent pathogens.
  • the LNP composition delivers one or more, two or more, three or more polynucleotides encoding one or more, two or more, three or more antigens (e.g., a cancer antigen). In some embodiments, the LNP compositions delivers one or more, two or more, three or more polynucleotides encoding antigens from diferent cancer cel types. [0379] In some embodiments, the lipid nanoparticle delivers one or more, two or more three or more antigens (e.g., a pathogen-associated antigen) to a host cel. In some embodiments, the lipid nanoparticle delivers one or more, two or more, three or more antigens from diferent pathogens.
  • the lipid nanoparticle delivers one or more, two or more, three or more antigens from diferent pathogens.
  • the lipid nanoparticle delivers one or more, two or more three or more antigens (e.g., a cancer antigen) to a host cel. In some embodiments, the lipid nanoparticle delivers one or more, two or more, three or more antigens from diferent cancer cel types.
  • the LNP composition comprises mRNA at a lipid:mRNA (weight/weight) ratio between 5:1 and 40:1.
  • the LNP composition comprises mRNA at a lipid:mRNA ratio between 10:1 and 40:1, between 15:1 and 40:1, between 20:1 and 40:1, between 25:1 and 40:1, between 30:1 and 40:1, between 35:1 and 40:1, between 20:1 and 35:1, between 25:1 and 35:1, between 30:1 and 35:1, between 20:1 and 30:1, between 25:1 and 30:1, between 20:1 and 25:1, between 25:1 and 30:1, between 25:1 and 35:1, between 20:1 and 36:1, between 25:1 and 36:1, or between 5:1 and 45:1.
  • the lipid composition comprises an ionizable cationic lipid.
  • the ionizable cationic lipids contains one or more groups which is protonated at physiological pH but may deprotonate and has no charge at a pH above the pKa of the lipid.
  • the ionizable group may contain one or more protonatable amines which are able to form a cationic group at physiological pH.
  • the ionizable cationic lipid compound may also further comprise one or more lipid components such as two or more faty acids with C6-C24 alkyl or alkenyl carbon groups.
  • a lipid nanoparticle composition may include one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH).
  • Ionizable cationic lipids may be selected from the non-limiting group consisting of 3- (didodecylamino)-N1,N1,4-tridodecy1-1-piperazineethanamine (KL10), N1-[2- (didodecylamino)ethyl]N1,N4,N4-tridodecy1-1,4-piperazinediethanamine (KL22), 14,25- ditridecy1-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoley1-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-y1-4-(dimethylamino)butanoate (DLin-
  • an ionizable cationic lipid may also be a lipid including a cyclic amine group.
  • Ionizable cationic lipids can also be the compounds disclosed in International Publication No. WO2017075531, the entire contents of which is incorporated herein by reference.
  • Ionizable cationic lipids can also be the compounds disclosed in International Publication No. WO2015199952, the entire contents of which is incorporated herein by reference.
  • the ionizable cationic lipid may be selected from, but not limited to, an ionizable cationic lipid described in International Publication Nos.
  • an ionizable cationic lipid comprises between 2 and 6 hydrophobic tails, often alkyl or alkenyl such as C6-C24 alkyl or alkenyl groups, but may have at least 1, at least 2, at least 3, at least 4, at least 5, or more than 6 tails.
  • Dendrimers [0386] In some embodiments, the ionizable cationic lipid is a dendrimer.
  • Dendrimers are a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core and are characterized by a core, at least one interior branched layer, and a surface branched layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in England, 641-645, August 1994.)
  • the term “dendrimer” as used herein is intended to include, but is not limited to, a molecular architecture with an interior core, interior layers (or “generations”) of repeating units regularly atached to this initiator core, and an exterior surface of terminal groups atached to the outermost generation.
  • a “dendron” is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a larger dendrimer.
  • the dendrimer structures have radiating repeating groups from a central core which doubles with each repeating unit for each branch.
  • the dendrimers described herein may be described as a smal molecule, medium- sized molecules, lipids, or lipid-like material. These terms may be used to describe compounds described herein which have a dendron like appearance (e.g., molecules which radiate from a single focal point).
  • dendrimers are polymers, dendrimers may be preferable to traditional polymers because they have a controlable structure, a single molecular weight, numerous and controlable surface functionalities, and traditionaly adopt a globular conformation after reaching a specific generation.
  • Dendrimers can be prepared by sequentialy reactions of each repeating unit to produce monodisperse, tree-like and/or generational structure polymeric structures. Individual dendrimers consist of a central core molecule, with a dendritic wedge atached to one or more functional sites on that central core.
  • the dendrimeric surface layer can have a variety of functional groups disposed thereon including anionic, cationic, hydrophilic, or lipophilic groups, according to the assembly monomers used during the preparation.
  • Modifying the functional groups and/or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can be modulated. Some properties which can be varied include, but are not limited to, solubility, toxicity, immunogenicity and bioatachment capability. Dendrimers are often described by their generation or number of repeating units in the branches. A dendrimer consisting of only the core molecule is refered to as Generation 0, while each consecutive repeating unit along al branches is Generation 1, Generation 2, and so on until the terminating or surface group. In some embodiments, half generations are possible resulting from only the first condensation reaction with the amine and not the second condensation reaction with the thiol.
  • Dendrimer synthesis can be of the convergent or divergent type. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process involving ataching one generation to the previous and then changing functional groups for the next stage of reaction. Functional group transformation is necessary to prevent uncontroled polymerization. Such polymerization would lead to a highly branched molecule that is not monodisperse and is otherwise known as a hyperbranched polymer.
  • the dendrimers of G1-G10 generation are specificaly contemplated.
  • the dendrimers comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein.
  • the dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by reducing the spacing units in the branching polymer.
  • dendrimers have two major chemical environments: the environment created by the specific surface groups on the termination generation and the interior of the dendritic structure which due to the higher order structure can be shielded from the bulk media and the surface groups. Because of these diferent chemical environments, dendrimers have found numerous diferent potential uses including in therapeutic applications. [0391] In some embodiments of the lipid composition, the dendrimers are assembled using the diferential reactivity of the acrylate and methacrylate groups with amines and thiols. The dendrimers may include secondary or tertiary amines and thioethers formed by the reaction of an acrylate group with a primary or secondary amine and a methacrylate with a mercapto group.
  • the repeating units of the dendrimers may contain groups which are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diethers, esters, amides, or disulfides groups.
  • the core molecule is a monoamine which alows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple diferent dendritic branches which each may comprise one or more repeating units.
  • the dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom.
  • the terminating group is a lipophilic groups such as a long chain alkyl or alkenyl group. In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine ( ⁇ NH2) or a carboxylic acid ( ⁇ CO2H). In stil other embodiments, the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.
  • the ionizable cationic lipids may contain one or more asymmetricaly-substituted carbon or nitrogen atoms, and may be isolated in opticaly active or racemic form. Thus, al chiral, diastereomeric, racemic form, epimeric form, and al geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specificaly indicated. Ionizable cationic lipids may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained.
  • the chiral centers of the ionizable cationic lipids can have the S or the R configuration.
  • one or more of the ionizable cationic lipids may be present as constitutional isomers.
  • the compounds have the same formula but diferent connectivity to the nitrogen atoms of the core.
  • the constitutional isomers may present the fuly reacted primary amines and then a mixture of reacted secondary amines.
  • ketone groups are known to exist in equilibrium with coresponding enol groups.
  • imine groups exist in equilibrium with enamine groups.
  • the ionizable cationic lipids of the present disclosure may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effectss than, be more easily absorbed than, and/or have a beter pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
  • atoms making up the ionizable cationic lipids are intended to include al isotopic forms of such atoms.
  • Isotopes include those atoms having the same atomic number but diferent mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include 13C and 14C.
  • the ionizable lipid is a dendrimer or dendron.
  • the ionizable cationic lipid comprises an ammonium group which is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH from about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C6-C24 alkyl or alkenyl groups.
  • the ionizable cationic lipid comprises at least two C8-C24 alkyl groups.
  • the ionizable cationic lipid is a dendrimer further defined by the formula: Core-(Repeating Unit)n-Terminating Group (D-I) wherein one or more hydrogen atoms of the core are replaced with a repeating unit and wherein: the core has the formula: wherein: X1 is amino or C1-C12 alkylamino, C1-C12 dialkylamino, C3-C12 heterocycloalkyl, C5-C12 heteroaryl, or a substituted version thereof; R1 is amino, hydroxy, mercapto, C1-C12 alkylamino, or C1-C12 dialkylamino, or a substituted version of either of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has the formula: wherein
  • the terminating group is further defined by the formula: ) wherein: Y4 is C1-C18 alkanediyl; and R10 is hydrogen.
  • A1 and A2 are each independently ⁇ O ⁇ or ⁇ NRa ⁇ .
  • the terminating group is a structure selected from the structures in Table 6.
  • the core is further defined by the formula: ) wherein: X2 is N(R5)y; R5 is hydrogen or C1-C8 alkyl, or substituted C1-C18 alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or C1-C12 alkylamino, C1-C12 dialkylamino, or a substituted version of either of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, 3; provided that the sum of z and y is 3.
  • the core is further defined by the formula: ) wherein: X3 is ⁇ NR6 ⁇ , wherein R6 is hydrogen, C1-C8 alkyl, or substituted C1-C8 alkyl, ⁇ O ⁇ , or C1-C8 alkylaminodiyl, C1-C8 alkoxydiyl, C1-C8 arenediyl, C1-C8 heteroarenediyl, C1-C8 heterocycloalkanediyl, or a substituted version of any of these groups; R3 and R4 are each independently amino, hydroxy, or mercapto, or C1-C12 alkylamino, dialkylamino, or a substituted version of either of these groups; or a group of , wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3; Rc, Rd, and Rf are each independently
  • the terminating group is represented by the formula: , wherein: Y4 is alkanediyl(C ⁇ 18); and R10 is hydrogen.
  • a core of the structure of formula (D-IV) is: , , or a pharmaceuticaly acceptable salt thereof.
  • the core comprises a structural formula set forth in Table 12 and pharmaceuticaly acceptable salts thereof, wherein * indicates a point of atachment of the core to a repeating unit (i.e., where a hydrogen of the core is replaced with a repeating unit).
  • the degradable diacyl is further defined as: .
  • the linker is further defined ), wherein Y1 is C1-C8 alkanediyl or substituted C1-C12 alkanediyl.
  • R6 is H. In some embodiments, in the core of formula (D-IV), R6 is C1-C8 alkyl.
  • R6 is substituted alkyl (e.g., alkyl substituted with -NH2, alkyl substituted with - NHCH3, or alkyl substituted with -NHCH2CH3).
  • one or two hydrogen atoms of the core are replaced with a repeating unit.
  • three or four hydrogen atoms of the core is replaced with a repeating unit.
  • five hydrogen atoms of the core is replaced with a repeating unit.
  • six hydrogen atoms of the core is replaced with a repeating unit.
  • the ionizable lipid is a compound having a structure of Formula D-A: ), wherein: RD1 is a C1-C4 alkyl; z1 and z2 are each independently 1, 2, or 3; and z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. [0411] In some embodiments, in the compound of Formula DA, RD1 is methyl. [0412] In some embodiments, in the compound of Formula DA, z1 and z2 are each 2. [0413] In some embodiments, the core of Formula D-I, D-II, or D-IV has a structure of Table 9 and the terminating group of formula D-VI has a structure of Table 10.
  • the dendrimer is selected from the group consisting of:
  • the ionizable cationic lipid is a dendrimer of the formula . In some embodiments, the ionizable cationic lipid is a dendrimer of the formula: .
  • the ionizable cationic lipid is a dendrimer of a generation (g) having a structural formula: or a pharmaceuticaly acceptable salt thereof, wherein: (a)the core comprises a structural formula (XCore): wherein: Q is independently at each occurence a covalent bond, -O-, -S-, -NR2-, or - CR3aR3b-; R2 is independently at each occurrence R1g or -L2-NR1eR1f; R3a and R3b are each independently at each occurence hydrogen or an optionaly substituted (e.g., C1-C6, such as C1-C3) alkyl; R1a, R1b, R1c, R1d, R1e, R1f, and R1g (if present) are each independently at each occurence a point of connection to a branch, hydrogen, or an optionaly substituted (e.g., C1-C12) alkyl;
  • XCore structural formula
  • Q is independently at each occurence a covalent bond, -O-, -S-, -NR2-, or -CR3aR3b.
  • XCore Q is independently at each occurence a covalent bond.
  • XCore Q is independently at each occurence an -O-.
  • XCore Q is independently at each occurence a -S- .
  • X Q is independently at each occurre 2 2 Core nce a -NR and R is independently at each occurence R1g or -L2-NR1eR1f.
  • XCore Q is independently at each occurence a -CR3aR3b R3a, and R3a and R3b are each independently at each occurrence hydrogen or an optionaly substituted alkyl (e.g., C1-C6, such as C1-C3).
  • R, R , R, R, R, and R are each independently at each occurence a point of connection to a branch, hydrogen, or an optionaly substituted alkyl.
  • R, R , R, R, R, and R are each independently at each occurence a point of connection to a branch, hydrogen.
  • R1a, R1b, R1c, R1d, R1e, R1f, and R1g Core are each independently at each occurence a point of connection to a branch an optionaly substituted alkyl (e.g., C1- C12).
  • L0, L1, and L2 are each independently at each occurence selected from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]- [alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L1 form a heterocycloalkyl (e.g., C4-C6 and containing one or two nitrogen atoms and, optionaly, an additional heteroatom selected from oxygen and sulfur) with one of R1c and R1d.
  • a heterocycloalkyl e.g., C4-C6 and containing one or two nitrogen atoms and, optionaly, an additional heteroatom selected from oxygen and sulfur
  • L0, L1, and L2 are each independently at each occurence can be a covalent bond.
  • X ,0 1 2 Core L, L, and L are each independently at each occurence can be a hydrogen.
  • X 0 1 2 Core, L, L, and L are each independently at each occurence can be an alkylene (e.g., C1-C12, such as C1-C6 or C1-C3).
  • L0, L1, and L2 are each independently at each occurence can be a heteroalkylene (e.g., C-C , suc 0 1 1 12 h as C1-C8 or C1-C6).
  • L, L, and L2 are each independently at each occurrence can be a heteroalkylene (e.g., C2-C8 alkyleneoxide, such as oligo(ethyleneoxide).
  • X 0 1 2 Core, L, L, and L are each independently at each occurrence can be a [alkylene]-[heterocycloalkyl]-[alkylene] [(e.g., C1-C6) alkylene]-[(e.g., C4-C6) heterocycloalkyl]-[(e.g., C1-C6) alkylene].
  • L0, L1, and L2 are each independently at each occurence can be a [alkylene]-(arylene)-[alkylene] [(e.g., C1-C6) alkylene]-(arylene)-[(e.g., C1-C6) alkylene].
  • L0, L1, and L2 are each independently at each occurence can be a [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6) alkylene]-phenylene-[(e.g., C1-C6) alkylene]).
  • X , L0, L1 2 Core , and L are each independently at each occurence can be a heterocycloalkyl (e.g., C4-C6heterocycloalkyl).
  • L0, L1, and L2 are each independently at each occurence can be an arylene (e.g., phenylene).
  • part of L form a heterocycloalkyl with one of R and R1d.
  • part of L form a heterocycloalkyl (e.g., C4-C6 heterocycloalkyl) with one of R1c and R1d and the heterocycloalkyl can contain one or two nitrogen atoms and, optionaly, an additional heteroatom selected from oxygen and sulfur.
  • a heterocycloalkyl e.g., C4-C6 heterocycloalkyl
  • the heterocycloalkyl can contain one or two nitrogen atoms and, optionaly, an additional heteroatom selected from oxygen and sulfur.
  • X 0 1 2 Core, L, L, and L are each independently at each occurence selected from a covalent bond, C1-C6 alkylene (e.g., C1-C3 alkylene), C2-C12 (e.g., C2-C8) alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH2CH2O)1-4-(CH2CH2)-), [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g.
  • C1-C6 alkylene e.g., C1-C3 alkylene
  • C2-C12 e.g., C2-C8 alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH2CH2O)1-4-(CH2CH2)-)
  • X , L0 1 2 Core , L, and L are each independently at each occurrence selected from C1-C6 alkylene (e.g., C1-C3 alkylene), -(C1-C3 alkylene-O)1-4-(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene- (C1-C3 alkylene)-, and -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-.
  • L0, L1, and L2 are each independently at each occurence C1-C6 alkylene (e.g., C1-C3 alkylene). In some embodiments, L0, L1, and L2 are each independently at each occurrence C2- C12 (e.g., C2-C8) alkyleneoxide (e.g., -(C1-C3 alkylene-O)1-4-(C1-C3 alkylene).
  • C2- C12 e.g., C2-C8 alkyleneoxide (e.g., -(C1-C3 alkylene-O)1-4-(C1-C3 alkylene).
  • L0, L1, and L2 are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)- phenylene-(C1-C3 alkylene)-) and [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-).
  • x1 is 0, 1, 2, 3, 4, 5, or 6. In some embodiments of XCore, x1 is 0. In some embodiments of X , x1 is 1. In som 1 Core e embodiments of XCore, x is 2. In some embodiments of X 1 1 Core, x is 3. In some embodiments of XCore, x is 4. In some embodiments of X x1 is 5. 1 Core, In some embodiments of XCore, x is 6. [0422] In some embodiments of XCore, the core comprises a structural formula: ). In some embodiments of XCore, the core comprises a structural formula: .
  • the core comprises a structural formula: ). In some embodiments of XCore, the core comprises a structural formula: . In some embodiments of XCore, the core comprises a structural formula: , . In some embodiments of XCore, the core comprises a structural formula: (e.g., , ). In some embodiments of X , the core comprise 2 Core s a structural formula: , wherein Q’ is -NR- or -CR3aR3b-; q1 and q2 are each independently 1 or 2. In some embodiments of XCore, the core comprises a structural formula: , ).
  • the core comprises a structural formula ., , , , or ), wherein ring A is an optionaly substituted aryl or an optionaly substituted (e.g., C3-C12, such as C3-C5) heteroaryl.
  • the core comprises has a structural formula .
  • the core comprises a structural formula set forth in Table 11 and pharmaceuticaly acceptable salts thereof, wherein * indicates a point of atachment of the core to a branch of the plurality of branches.
  • the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches.
  • each branch of the plurality of branches comprises a structural formula each branch of the plurality of branches comprises a structural formula
  • each branch of the plurality of branches comprises a structural formula .
  • An example formulation of the dendrimers described herein for generations 1-4 is shown in Table 11. The number of diacyl groups, linker groups, and terminating groups can be calculated based on g. Table 11.
  • the diacyl group independently comprises a structural formula , * indicates a point of atachment of the diacyl group at the proximal end thereof, and ** indicates a point of atachment of the diacyl group at the distal end thereof.
  • Y3 is independently at each occurence an optionaly substituted; alkylene, an optionaly substituted alkenylene, or an optionaly substituted arenylene.
  • Y is independently at each occurence an optionaly substituted alkylene (e.g., C1-C12).
  • Y is independently at each occurence an optionaly substituted alkenylene (e.g., C1-C12).
  • Y3 is independently at each occurence an optionaly substituted arenylene (e.g., C1- C12).
  • a and A are each independently at each occurence -O-, -S-, or -NR4-.
  • a and A2 are each independently at each occurrence -O-.
  • a and A are each independently at each occurence -S-. In some embodiments of the diacyl group of XBranch, A1 and A2 are each independently at each occurence -NR4- and R4 is hydrogen or optionaly substituted alkyl (e.g., C1-C6). In some embodiments of the diacyl group of X 1 2 Branch, m and m are each independently at each occurence 1, 2, or 3. In some embodiments of the diacyl group of X 1 2 Branch, m and m are each independently at each occurence 1. In some embodiments of the diacyl group of X 1 Branch, m and m2 are each independently at each occurence 2.
  • m1 and m2 are each independently at each occurence 3.
  • R3c, R3d, R3e, and R3f are each independently at each occurence hydrogen or an optionaly substituted alkyl.
  • R3c, R3d, R3e, and 3f Branch R are each independently at each occurence hydrogen.
  • R3c, R3d, R3e, and R3f Branch are each independently at each occurrence an optionaly substituted (e.g., C1-C8) alkyl.
  • A1 is -O- or -NH-. In some embodiments of the diacyl group, A1 is -O-. In some embodiments of the diacyl group, A2 is -O- or -NH-. In some embodiments of the diacyl group, A2 is -O-. In some embodiments of the diacyl group, Y3 is C1-C12 (e.g., C1-C6, such as C1-C3) alkylene.
  • the diacyl group independently at each occurence comprises a structural formula (e.g., , R3e, and R3f are each independently at each occurrence hydrogen or C1-C3 alkyl.
  • linker group independently comprises a structural formula , ** indicates a point of atachment of the linker to a proximal diacyl group, and *** indicates a point of atachment of the linker to a distal diacyl group.
  • Y1 is independently at each occurence an optionaly substituted alkylene, an optionaly substituted alkenylene, or an optionaly substituted arenylene. In some embodiments of the linker group of XBranch if present, Y1 is independently at each occurence an optionaly substituted alkylene (e.g., C1-C12). In some embodiments of the linker group of XBranch if present, Y1 is independently at each occurence an optionaly substituted alkenylene (e.g., C1-C12).
  • each terminating group is independently selected from optionaly substituted alkylthiol and optionaly substituted alkenylthiol. In some embodiments of the terminating group of XBranch, each terminating group is an optionaly substituted alkylthiol (e.g., C1-C18, such as C4-C18).
  • each terminating group is optionaly substituted alkenylthiol (e.g., C1-C18, such as C4-C18).
  • each terminating group is independently C1-C18 alkenylthiol or C1-C18 alkylthiol, and the alkyl or alkenyl moiety is optionaly substituted with one or more substituents each independently selected from halogen, C6-C12 aryl, C1-C12 alkylamino, C4-C6 N-heterocycloalkyl , -OH, -C(O)OH, ⁇ C(O)N(C1-C3 alkyl) ⁇ (C1-C6 alkylene) ⁇ (C1-C12 alkylamino), ⁇ C(O)N(C1-C3 alkyl) ⁇ (C1-C6 alkylene) ⁇ (C4-C18).
  • each terminating group is independently C1-C18 (e.g., C4-C18) alkenylthiol or C1-C18 (e.g., C4-C18) alkylthiol, wherein the alkyl or alkenyl moiety is optionaly substituted with one or more substituents each independently selected from halogen, C6-C12 aryl (e.g., phenyl), C1-C12 (e.g., C1-C8) alkylamino (e.g., C1-C6 mono-alkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 di- alkylamino (such -heterocycloalkyl (e.g., N-pyrolidinyl ), N-piperidinyl ), N-azepanyl , -OH, -C(O)OH, ⁇ C(O)
  • each terminating group is independently C1-C18 (e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionaly substituted with one substituent -OH.
  • each terminating group is independently C1-C18 (e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionaly substituted with one substituent selected from C1-C12 (e.g., C1-C8) alkylamino (e.g., C1-C6 mono-alkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 di-alkylamino (such - heterocycloalkyl (e.g., N-pyrolidinyl ), N-piperidinyl ), N-azepanyl ).
  • C1-C12 e.g., C1-C8 alkylamino
  • C1-C6 mono-alkylamino such as -NHCH2CH2CH2CH3
  • C1-C8 di-alkylamino such - heterocycloalkyl (e.g., N-pyrolidinyl ), N-piperidinyl
  • each terminating group is independently C1-C18 (e.g., C4-C18) alkenylthiol or C1-C18 (e.g., C4-C18) alkylthiol. In some embodiments of the terminating group of XBranch, each terminating group is independently C1- C18 (e.g., C4-C18) alkylthiol. Table 12.
  • Example core structures [0444] In some embodiments of XCore, the core comprises a structural formula selected from the group consisting of: , , and pharmaceuticaly acceptable salts thereof, wherein * indicates a point of atachment of the core to a branch of the plurality of branches.
  • each terminating group is independently a structure selected from the structures in Table 13.
  • the dendrimers described herein can comprise a terminating group or pharmaceuticaly acceptable salt, or thereof selected in Table 13. Table 13. Example terminating group / peripheries structures
  • the dendrimer of Formula (X) is selected from those set forth in Table 14 and pharmaceuticaly acceptable salts thereof. Table 9.
  • the dendrimer is 2A2-SC14. In some embodiments, the dendrimer is 2A6-SC14. In some embodiments, the dendrimer is 2A9-SC14. In some embodiments, the dendrimer is 3A3-SC10. In some embodiments, the dendrimer is 3A3-SC14. In some embodiments, the dendrimer is 4A5-SC10. In some embodiments, the dendrimer is 3A5-SC14. In some embodiments, the dendrimer is 4A1-SC12. In some embodiments, the dendrimer is 4A3-SC12. In some embodiments, the dendrimer is 5A1-SC12.
  • the dendrimer is 5A1-SC8. In some embodiments, the dendrimer is 5A2-2- SC12. In some embodiments, the dendrimer is 5A3-1-SC12. In some embodiments, the dendrimer is 5A3-1-SC8. In some embodiments, the dendrimer is 5A4-1-SC12. In some embodiments, the dendrimer is 5A4-1-SC8. In some embodiments, the dendrimer is 5A5-SC8. In some embodiments, the dendrimer is 5A5-SC12. In some embodiments, the dendrimer is 5A2-4-SC12. In some embodiments, the dendrimer is 5A2-4-SC10.
  • the dendrimer is 5A3-2-SC8. In some embodiments, the dendrimer is 5A3-2-SC12. In some embodiments, the dendrimer is 5A4-2-SC8. In some embodiments, the dendrimer is 5A4-2- SC12. In some embodiments, the dendrimer is 6A4-SC8. In some embodiments, the dendrimer is 6A4-SC12. In some embodiments, the dendrimer is 2A2-g2-SC12. In some embodiments, the dendrimer is 2A2-g2-SC8. In some embodiments, the dendrimer is 2A11-g2-SC12. In some embodiments, the dendrimer is 2A11-g2-SC8.
  • the dendrimer is 3A3- g2-SC12. In some embodiments, the dendrimer is 3A3-g2-SC8. In some embodiments, the dendrimer is 3A5-g2-SC12. In some embodiments, the dendrimer is 2A11-g3-SC12. In some embodiments, the dendrimer is 2A11-g3-SC8. In some embodiments, the dendrimer is 1A2- g4-SC12. In some embodiments, the dendrimer is 4A1-g2-SC12. In some embodiments, the dendrimer is 1A2-g4-SC8. In some embodiments, the dendrimer is 4A1-g2-SC8.
  • the dendrimer is 4A3-g2-SC12. In some embodiments, the dendrimer is 4A3- g2-SC8. In some embodiments, the dendrimer is 1A2-g3-SC12. In some embodiments, the dendrimer is 1A2-g3-SC8. In some embodiments, the dendrimer is 2A2-g3-SC12. In some embodiments, the dendrimer is 2A2-g3-SC8. In some embodiments, the dendrimer is 5A2-4- SC8. In some embodiments, the dendrimer is 5A5-SC8. In some embodiments, the dendrimer is 5A2-6-SC8. In some embodiments, the dendrimer is 5A2-1-SC8.
  • the dendrimer is 5A2-2-SC8. In some embodiments, the dendrimer is 4A1-SC5. In some embodiments, the dendrimer is 4A1-SC8. In some embodiments, the dendrimer is 4A3-SC6. In some embodiments, the dendrimer is 4A3-SC7. In some embodiments, the dendrimer is 4A3-SC8. In some embodiments, the dendrimer is 5A4-2-SC5. In some embodiments, the dendrimer is 5A4-2-SC6. In some embodiments, the dendrimer is 5A2-4-SC8. In some embodiments, the dendrimer is 3A5-g2-SC8.
  • the dendrimer is 5A2- SC8.
  • Other Ionizable cationic lipids [0448]
  • a is 1. In some embodiments of the ionizable cationic lipid of formula (D-I’), b is 2. In some embodiments of the ionizable cationic lipid of formula (D-I’), m is 1. In some embodiments of the ionizable cationic lipid of formula (D-I’), n is 1. In some embodiments of the ionizable cationic lipid of formula (D-I’), R1, R2, R3, R4, R5, and R6 are each independently H or -CH 7 2CH(OH)R.
  • R1, R2, R3, R4, R5, and R6 are each independently H or . In some embodiments of the ionizable cationic lipid of formula (D-I’), R1, R2, R3, R4, R5, and R6 are each independently H or . In some embodiments of the ionizable cationic lipid of formula (D-I’), R7 is C3-C18 alkyl (e.g., C6-C12 alkyl).
  • the ionizable cationic lipid of formula (D-I’) is 13,16,20-tris(2- hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: .
  • the ionizable cationic lipid of formula (D-I’) is (11R,25R)- 13,16,20-tris(R)-2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: .
  • Additional ionizable cationic lipids that can be used in the compositions and methods described herein include those ionizable cationic lipids as described in International Patent Publication WO2010144740, WO2013149140, WO2016118725, WO2016118724, WO2013063468, WO2016205691, WO2015184256, WO2016004202, WO2015199952, WO2017004143, WO2017075531, WO2017117528, WO2017049245, WO2017173054 and WO2015095340, the entire contents of which is incorporated herein by reference for al purposes.
  • Examples of those ionizable cationic lipids include but are not limited to those as shown in Table 15. Table 15.
  • the ionizable cationic lipid is present in the composition at a molar percentage from about 10% to about 25%.
  • the ionizable cationic lipid is present in the composition at a molar percentage about 5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.
  • the ionizable cationic lipid is present in the composition at a molar percentage from about 5% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%.
  • the ionizable lipid is present at a molar percentage of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%. In some embodiments of the lipid composition, the ionizable lipid is present at a molar percentage of at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, or at most about 30%.
  • SORT selective organ targeting
  • the lipid (e.g., nanoparticle) composition is preferentialy delivered to a target organ.
  • the additional lipid comprises a permanently positively charged moiety (i.e., is a permanently cationic lipid).
  • the permanently positively charged moiety may be positively charged at a physiological pH such that the additional lipid (e.g., SORT lipid) comprises a positive charge upon delivery of a polynucleotide to a cel.
  • the positively charged moiety is quaternary amine or quaternary ammonium ion.
  • the additional lipid (e.g., SORT lipid) comprises, or is otherwise complexed to or interacting with, a counterion.
  • the additional lipid is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group).
  • the permanently cationic lipid may contain a group which has a positive charge regardless of the pH.
  • One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammonium group.
  • the permanently cationic lipid may comprise a structural formula: (S-I), wherein: Y, Y, or Y are each indepen + 1 2 3 dently X1C(O)R1 or X2NR3R4R5; provided at least one of Y, Y, and Y + 1 2 3 is X2NR3R4R5; R1 is C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, C1-C24 substituted alkenyl; X1 is O or NRa, wherein Ra is hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl; X2 is C1-C6 alkanediyl or C1-C6 substituted alkanediyl; R3, R4, and R5 are each independently C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, C1-C24 substituted al
  • the permanently cationic additional lipid e.g., SORT lipid
  • the permanently cationic lipids is 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn- glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC), 1,2- dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), Dimethyldioct
  • the SORT (additional) lipid is an ionizable cationic lipid (e.g., comprising one or more hydrophobic components and an ionizable group, e.g., a tertiary amino group).
  • the ionizable positively charged moiety may be positively charged at a physiological pH.
  • One ionizable group that may be used in the ionizable cationic lipid is a tertiary ammine group.
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) has a structural formula , wherein: R1 and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; and R3 and R3′ are each independently C1-C6 alkyl or substituted C1-C6 alkyl. [0463] In some embodiments of formula (S-I’a) R1 and R2 are each independently C8-C24 alkenyl (e.g., hexadecane, heptadecene, or octadecene).
  • R1 and R2 are each independently C8-C24 alkenyl (e.g., hexadecane, heptadecene, or octadecene).
  • R3 and R3′ are each independently C1-C6 alkyl (e.g., methyl or ethyl).
  • R1 and R2 are each independently C8-C24 alkenyl, (e.g., hexadecane, heptadecene, or octadecene) and R3 and R3′ are each independently C1-C6 alkyl (e.g., methyl or ethyl).
  • the ionizable cationic lipids is 1,2-distearoyl-3- dimethylammonium-propane (18:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dioleoyl-3- dimethylammonium-propane (18:1 DAP, DODAP), or 1,2-dioleyloxy-3- dimethylaminopropane (DODMA).
  • the additional ionizable cationic lipid or permanently cationic lipid comprises a head group of a particular structure.
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) comprises a headgroup having a structural formula: , wherein L is a linker; Z+ is positively charged moiety and X- is a counterion.
  • the linker is a biodegradable linker.
  • the biodegradable linker may be degradable under physiological pH and temperature.
  • the biodegradable linker may be degraded by proteins or enzymes from a subject.
  • the positively charged moiety is a quaternary ammonium ion or quaternary amine.
  • the SORT (additional ionizable cationic lipid or permanently cationic) lipid has a structural formula: , wherein R1 and R2 are each independently an optionaly substituted C6-C24 alkyl, or an optionaly substituted C6-C24 alkenyl.
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) has a structural formula: .
  • the additional lipid (e.g., additional lipid (e.g., SORT lipid) comprises a Linker (L).
  • L is , wherein: p and q are each independently 1, 2, or 3; and R4 is an optionaly substituted C1-C6 alkyl
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) has a structural formula: ), wherein: R1 and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R3, R3′, and R3′ are each independently C1-C6 alkyl or substituted C1-C6 alkyl; R4 is C1-C6 alkyl or substituted C1-C6 alkyl; and X ⁇ is a monovalent anion.
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) is a phosphatidylcholine (e.g., 14:0 EPC).
  • the phosphatidylcholine compound is further defined ), wherein: R1 and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R3, R3′, and R3′ are each independently C1-C6 alkyl or substituted C1-C6 alkyl; and X ⁇ is a monovalent anion.
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) is a phosphocholine lipid. In some embodiments, the additional lipid (e.g., additional lipid (e.g., SORT lipid) is an ethylphosphocholine.
  • the ethylphosphocholine may be, by way of example, without being limited to, 1,2-dimyristoleoyl-sn-glycero-3- ethylphosphocholine (14:1 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dipalmitoyl-sn-glycero-3- ethylphosphocholine (16:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), 1-palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine (16:0-18:0 EPC).
  • the lipid has a structural formula: ), wherein: R1 and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R3, R3′, and R3′ are each independently C1-C6 alkyl or substituted C1-C6 alkyl; X ⁇ is a monovalent anion.
  • an additional lipid e.g., additional lipid (e.g., SORT lipid) of the structural formula of the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) has a structural formula: (S-I’), wherein: R4 and R4′ are each independently alkyl(C6-C24), alkenyl(C6-C24), or a substituted version of either group; R4′ is alkyl(C ⁇ 24), alkenyl(C ⁇ 24), or a substituted version of either group; R4′′ is alkyl(C1-C8), alkenyl(C2-C8), or a substituted version of either group; and X2 is a monovalent anion.
  • R4 and R4′ are each independently alkyl(C6-C24), alkenyl(C6-C24), or a substituted version of either group
  • R4′ is alkyl(C ⁇ 24), alkenyl(C ⁇ 24), or a substituted version of either group
  • R4′′ is alkyl(C1-C8), alkenyl(C2-C8)
  • an additional lipid e.g., additional lipid (e.g., SORT lipid) of the structural formula of the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB).
  • the additional lipid e.g., additional lipid (e.g., SORT lipid) is: 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA).
  • the additional lipid is selected from the lipids set forth in Table 16A and Table 16B.
  • Example additional lipid e.g., SORT lipids
  • Example additional lipid e.g., SORT lipids
  • the permanently cationic lipid is selected from 1,2-dilauroyl-sn- glycero-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycero- 3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), Dimethyldio
  • the ethylphosphocholine is 1,2-dipalmitoyl-sn-glycero-3– ethylphosphocholine (16:0 EPC) or 2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC).
  • the SORT lipid is 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn- glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC), 1,2- dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), Dimethyldioctadec
  • the SORT lipid selected from the group shown in Table 16A and Table 16B. In some embodiments, the SORT lipid is present in the composition at a molar percentage from about 5% to about 65%. In some embodiments, the SORT lipid is present in the composition at a molar percentage from about 5% to about 35%. [0481] In some embodiments of the lipid composition, the SORT lipid is present in the composition at a molar percentage from about 5% to about 50%.
  • the SORT lipid is present in the composition at a molar percentage about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.
  • the SORT lipid is present in the composition at a molar percentage from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 10%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 20% to about 25%.
  • the SORT lipid is present at a molar percentage of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or at least about 55%.
  • the ionizable lipid is present at a molar percentage of at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, or at most about 25%.
  • Helper lipids are phospholipids.
  • Phospholipids are any lipids that comprise a phosphate group.
  • the lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly) unsaturated lipids.
  • Phospholipids may assemble into one or more lipid bilayers.
  • phospholipids may include a phospholipid moiety and one or more faty acid moieties.
  • a phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a faty acid moiety may be selected 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.
  • Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or celular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids useful or potentialy useful in the compositions and methods may be selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn- glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero
  • the phospholipid may contain one or two long chain (e.g., C6- C24) alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionaly, a smal organic molecule.
  • the smal organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine.
  • the phospholipid is a phosphatidylcholine.
  • the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.
  • helperionic lipids are used, where zwiterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.
  • the helper lipid is 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).
  • DOPE 1,2-distearoyl-sn- glycero-3-phosphocholine
  • the helper lipid is present in the composition at a molar percentage from about 5% to about 35%.
  • the helper lipid is present in the composition at a molar percentage from about 7.5% to about 30%. [0491] In some embodiments of the lipid composition, the helper lipid is present in the composition at a molar percentage about 5%, about 7.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.
  • the helper lipid is present in the composition at a molar percentage from about 5% to about 50%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%.
  • the helper lipid is present at a molar percentage of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%.
  • the ionizable lipid is present at a molar percentage of at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, or at most about 30%.
  • Structural lipids [0494]
  • the lipid nanoparticle may include one or more structural lipids. Structural lipids can be steroids or steroid derivatives. In some embodiments of the lipid composition, the lipid composition further comprises a steroid or steroid derivative.
  • the steroid or steroid derivative comprises any steroid or steroid derivative.
  • the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.
  • the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula: .
  • a steroid derivative comprises the ring structure above with one or more non- alkyl substitutions.
  • the steroid or steroid derivative is a sterol wherein the formula is further defined as: n some embodiments, the steroid or steroid derivative is a cholestane or cholestane derivative.
  • the ring structure is further defined by the formula: s described above, a cholestane derivative includes one or more non-alkyl substitution of the above ring system.
  • the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative.
  • the cholestane or cholestane derivative is both a cholesterol and a sterol or a derivative thereof.
  • Sterols useful in the compositions and methods described herein may be selected from: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.
  • the sterol is present in the composition at a molar percentage from about 20% to about 50%.
  • the sterol is cholesterol.
  • the sterol is sitosterol.
  • the cholesterol is present in the composition at a molar percentage about 10%, about 15%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 50%, about 55%, or about 60%.
  • the sterol is present in the composition at a molar percentage from about 10% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 20% to about 25%, from about 25% to about 50%, from about 25% to about 40%, from about 25% to about 30%, from about 30% to about 50%, from about 30% to about 40%, from about 30% to about 35%, from about 35% to about 50%, from about 35% to about 45%, from about 35% to about 40%, from about 40% to about 50%, from about 40% to about 45%, or from about 45% to about 50%.
  • the sterol is present at a molar percentage of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or at least about 50%.
  • the ionizable lipid is present at a molar percentage of at most about 60%, at most about 15%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, or at most about 20%.
  • Polyethylene glycol-conjugated lipids PEG-lipids
  • the lipid compositions of the disclosure may include lipids conjugated to polymers, such as lipids conjugated to polyethylene glycol (“PEG-lipid”).
  • 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-lipids can be PEG-lipids described in Int’l Pat. Pub. No. WO 2012/099755, the entire contents of which is incorporated herein by reference. 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.
  • a “PEG-OH lipid” is a PEG-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-PEG-lipid comprises an — OH group at the terminus of the PEG chain.
  • the lipid composition further comprises a polymer conjugated lipid.
  • the polymer conjugated lipid is a PEG-lipid.
  • the PEG-lipid is a diglyceride which also comprises a PEG chain atached to the glycerol group.
  • the PEG-lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 faty acid group atached to a linker group with a PEG chain.
  • Some non-limiting examples of a PEG-lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified 1,2-diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols.
  • PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-sn-glycerol is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000.
  • the molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000.
  • Some non-limiting examples of lipids that may be used are taught by U.S. Patent 5,820,873, WO 2010/141069, or U.S. Patent 8,450,298, the entire contents of these are incorporated herein by reference.
  • the PEG-lipid has a structural formula: , wherein: R12 and R13 are each independently alkyl(C ⁇ 24), alkenyl(C ⁇ 24), or a substituted version of either of these groups; Re is hydrogen, alkyl(C ⁇ 8), or substituted alkyl(C ⁇ 8); and x is 1-250. In some embodiments, Re is alkyl(C ⁇ 8) such as methyl. R12 and R13 are each independently alkyl(C ⁇ 4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250.
  • the PEG-lipid is 1,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol.
  • the PEG-lipid has a structural formula: , wherein: n1 is an integer between 1 and 100 and n2 and n3 are each independently selected from an integer between 1 and 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein.
  • n1 is from about 30 to about 50. In some embodiments, n2 is from 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is from 5 to 23. In some embodiments, n3 is 11 to about 17. [0506] In some embodiments of the lipid composition, the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%. [0507] In some embodiments of the lipid composition, the PEG-lipid is present in the composition at a molar percentage about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.
  • the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, from about 1% to about 5%, from about 1% to about 4.5%, from about 1% to about 4%, from about 1% to about 3.5%, from about 1% to about 3%, from about 1% to about 2%, from about 2% to about 5%, from about 2% to about 4.5%, from about 2% to about 4%, from about 2% to about 3.5%, from about 2% to about 3%, from about 3% to about 5%, from about 3% to about 4.5%, from about 3% to about 4%, from about 3% to about 3.5%, from about 4% to about 3% to about 3.5%, from about 4% to about 3% to about 3.5%, from about 4% to about 3% to about 3.5%, from about 4% to about 3% to about
  • the PEG-lipid is present at a molar percentage of at least about 0.5%, at least about 1%, at least about 2%, at least about 2.5%, at least about 3%, or at least about 3.5%.
  • the ionizable lipid is present at a molar percentage of at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, or at most about 5%.
  • the LNP composition comprises DOPE at a molar percentage of about 8 to about 23 %; cholesterol at a molar percentage of about 20 to about 50%; DMG-PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and 16:0 EPC at a molar percentage of about 30 to about 40%.
  • the LNP composition comprises mRNA at a lipid:mRNA (weight/weight) ratio between 5:1 and 40:1.
  • the LNP composition comprises mRNA at a lipid:mRNA ratio between 10:1 and 40:1, between 15:1 and 40:1, between 20:1 and 40:1, between 25:1 and 40:1, between 30:1 and 40:1, between 35:1 and 40:1, between 20:1 and 35:1, between 25:1 and 35:1, between 30:1 and 35:1, between 20:1 and 30:1, between 25:1 and 30:1, between 20:1 and 25:1, between 25:1 and 30:1, between 25:1 and 35:1, between 20:1 and 36:1, between 25:1 and 36:1, or between 5:1 and 45:1.
  • the composition comprises 4A3-SC7 at a molar percentage of about 5 to about 30%, SORT lipid at a molar percentage of about 5 to about 30%, DOPE at a molar percentage of about 8 to about 23%, cholesterol at a molar percentage of about 15 to about 46%, and/or DMG-PEG at a molar percentage of about 0.5 to about 10%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 13 to about 20%, SORT at a molar percentage of about 10 to about 30%, DOPE at a molar percentage of about 13 to about 20%, cholesterol at a molar percentage of about 30 to about 40%, and/or DMG-PEG at a molar percentage of about 3 to about 5%, wherein the SORT lipid is an ethylphosphocholine.
  • the composition comprises 4A3-SC7 at a molar percentage of about 13 to about 20%, SORT at a molar percentage of about 5 to about 30%, DOPE at a molar percentage of about 13 to about 20%, cholesterol at a molar percentage of about 30 to about 40%, and/or DMG-PEG at a molar percentage of about 3 to about 5%, wherein the SORT lipid is an anionic lipid.
  • the composition comprises 4A3-SC7 at a molar percentage of about 19%, 14:0 EPC at a molar percentage of about 20%, DOPE at a molar percentage of about 19%, cholesterol at a molar percentage of about 38%, and/or DMG-PEG at a molar percentage of about 4%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 17%, 16:0 EPC at a molar percentage of about 30%, DOPE at a molar percentage of about 17%, cholesterol at a molar percentage of about 33%, and/or DMG-PEG at a molar percentage of about 3%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 17%, 14:0 EPC at a molar percentage of about 30%, DOPE at a molar percentage of about 17%, cholesterol at a molar percentage of about 33%, and/or DMG-PEG at a molar percentage of about 3%.
  • the composition comprises 4A3-SC7 at a molar percentage of about 23%, 18:1 PA at a molar percentage of about 5%, DOPE at a molar percentage of about 23%, cholesterol at a molar percentage of about 45%, and/or DMG-PEG at a molar percentage of about 5%.
  • the composition is capable of delivering mRNA to a cel in the central nervous system (CNS) of a subject in an amount effete to increase expression and/or function of a gene encoded by the mRNA.
  • the cel is an endothelial cel.
  • the cel is a pericyte.
  • the cel is a smooth muscle cel (SMA).
  • the LNP composition comprises DOPE at a molar percentage of about 8 to about 23%; cholesterol at a molar percentage of about 20 to about 50%; DMG-PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and DODAP at a molar percentage of about 10 to about 30%.
  • the LNP composition comprises DOPE at a molar percentage of about 8 to about 23%; cholesterol at a molar percentage of about 20 to about 50%; DMG-PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and 18:1 PA at a molar percentage of about 5 to about 20%.
  • the LNP composition comprises DOPE at a molar percentage of about 8 to about 23%; cholesterol at a molar percentage of about 20 to about 50%; DMG-PEG at a molar percentage of about 0.5 to about 10%; ionizable cationic lipid at a molar percentage of about 10 to about 25%; and DOTAP at a molar percentage of about 30 to about 50%.
  • the LNP composition comprises DOPE at a molar percentage of about 15%; cholesterol at a molar percentage of about 30%; DMG-PEG at a molar percentage of about 3%; ionizable cationic lipid at a molar percentage of about 15%; and 16:0 EPC at a molar percentage of about 30%.
  • the LNP composition comprises DOPE at a molar percentage of about 20%; cholesterol at a molar percentage of about 40%; DMG-PEG at a molar percentage of about 3%; ionizable cationic lipid at a molar percentage of about 15%; and DODAP at a molar percentage of about 15%.
  • the LNP composition comprises DOPE at a molar percentage of about 20%; cholesterol at a molar percentage of about 40%; DMG-PEG at a molar percentage of about 5%; ionizable cationic lipid at a molar percentage of about 20%; and 18:1 PA at a molar percentage of about 5%.
  • the LNP composition comprises DOPE at a molar percentage of about 10%; cholesterol at a molar percentage of about 20%; DMG-PEG at a molar percentage of about 3%; ionizable cationic lipid at a molar percentage of about 10%; and DOTAP at a molar percentage of about 50%.
  • the LNP composition comprises mRNA at a lipid:mRNA (weight/weight) ratio between 5:1 and 40:1. In some embodiments, the LNP composition comprises mRNA at a lipid:mRNA ratio between 10:1 and 40:1, between 15:1 and 40:1, between 20:1 and 40:1, between 25:1 and 40:1, between 30:1 and 40:1, between 35:1 and 40:1, between 20:1 and 35:1, between 25:1 and 35:1, between 30:1 and 35:1, between 20:1 and 30:1, between 25:1 and 30:1, between 20:1 and 25:1, between 25:1 and 30:1, between 25:1 and 35:1, between 20:1 and 36:1, between 25:1 and 36:1, or between 5:1 and 45:1.
  • compositions for the LNP compositions described herein. Such pharmaceutical compositions can be used for generating an immune response against an infectious disease, or against cancer cels and/or tumors in a subject.
  • the pharmaceutical compositions of the disclosure may include a pharmaceuticaly acceptable carier. A thorough discussion of such cariers is available in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rd ed., 2021), the entire contents of which is incorporated herein by reference.
  • the pharmaceutical composition comprises Tris bufer, optionaly at a pH from 6-9.
  • the pharmaceutical composition comprises sucrose, optionaly at 5-15%.
  • the pharmaceutical composition comprises citrate buffer, optionaly at a pH 4-6.
  • the pharmaceutical composition comprises 15 mM Tris bufer, optionaly at a pH from 6-9, and/or 5-15% sucrose. In some embodiments, the composition comprises 10 mM citrate bufer, optionaly at a pH from 4-6.
  • the pharmaceutical compositions include one or more of a poloxamer (e.g., Poloxamer 188) polyethylene glycol (“PEG”), sucrose, and a bufer, wherein the bufer comprises a citrate bufer, an acetate bufer, or a Tris buffer.
  • the pharmaceutical composition comprises a citrate bufer. For example, the citrate bufer is at a pH from 4 to 8.
  • the bufer is an acetate bufer and has a pH from 4 to 8.
  • the pharmaceutical composition comprises a Tris buffer, and the Tris bufer has a pH from 4 to 8.
  • the pharmaceutical composition comprises sucrose.
  • the sucrose is at a concentration from 1% to 15% w/v, 5% to 15% w/v, 1% to 10% w/v, or 5% to 10% w/v.
  • the pharmaceutical composition can also include excipients and/or additives.
  • surfactants examples include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof.
  • EDTA ethylenediaminetetraacetic acid
  • preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate.
  • Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, salts or esters thereof.
  • pharmaceuticaly acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, filers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, faty acid esters, hydroxymethycelluloseose, polyvinyl pyrolidine, and colors, and the like.
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, bufers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, bufers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, weting agents, emulsifiers, salts for influencing osmotic pressure, bufers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • tonicity agents may be added to provide the desired ionic strength.
  • the disclosure provides a vaccine comprising the LNP compositions or the pharmaceutical compositions described herein.
  • the vaccine is a influenza virus vaccine and comprises a polynucleotide encoding a influenza HA protein, NA protein, NP (nucleoprotein) protein, M1 (matrix protein 1) protein, M2 (matrix protein 2) protein, non-structural protein 1 (NS1), non- structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2), or an antigenic fragment thereof.
  • a influenza virus vaccine comprises a polynucleotide encoding a influenza HA protein, NA protein, NP (nucleoprotein) protein, M1 (matrix protein 1) protein, M2 (matrix protein 2) protein, non-structural protein 1 (NS1), non- structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic
  • anthe influenza virus vaccine of the present disclosure comprises a polynucleotide encoding one or more, two or more, three or more influenza virus proteins.
  • the vaccine is a SARS-CoV-2 vaccine and comprises a polynucleotide encoding a SARS-CoV-2 S protein, or an antigenic fragment thereof.
  • the vaccine is a SARS-CoV-2 vaccine and comprises a polynucleotide encoding S protein, smal envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein, or an antigenic fragment thereof.
  • the vaccine is an RSV vaccine and comprises a polynucleotide encoding one or more, two or more, three or more RSV proteins in Table 1, or an antigenic fragment thereof.
  • the vaccine is a PIV3 vaccine and comprises a polynucleotide encoding one or more, two or more, three or more PIV3 proteins, or an antigenic fragment thereof.
  • the vaccine is a hMPV vaccine and comprises one or more, two or more, three or more hMPV proteins, or an antigenic fragment thereof.
  • the vaccine is a MERS-CoV vaccine and comprises a polynucleotide encoding one or more, two or more, three or more MERS-CoV proteins, or an antigenic fragment thereof.
  • the vaccine is a MeV vaccine and comprises a polynucleotide encoding one or more, two or more, three or more MeV proteins, or an antigenic fragment thereof.
  • the vaccine is a lung cancer vaccine and comprises a polynucleotide encoding 5T4 protein, surviving protein, NY ESO-1 protein, MAGE-C1 protein, or MAGE-C2 protein, or an antigenic fragment thereof.
  • another lung cacner vaccine of the present disclosure comprises a polynucleotide encoding one or more, two or more, three or more lung cancer proteins.
  • the vaccine is a lung cancer vaccine and comprises a polynucleotide encoding one or more, two or more, three or more lung cancer proteins in Table 6, or an antigenic fragment thereof.
  • the vaccine is a prostate cancer vaccine and comprises a polynucleotide encoding PSA protein, PSMA protein, PSCA protein, STEAP protein, PAP protein, or MUC1 protein, or an antigenic fragment thereof.
  • another prostate cacner vaccine of the present disclosure comprises a polynucleotide encoding one or more, two or more, three or more prostate cancer proteins.
  • the vaccine is a prostate cancer vaccine and comprises a polynucleotide encoding one or more, two or more, three or more prostate cancer proteins in Table 6, or an antigenic fragment thereof.
  • the vaccine is neoantigen vaccine and comprises a polynucleotide encoding ACAD5 protein, AGAP6 protein, AKT1 protein, ANAPC1 protein, ANKRC36C protein, BLC11A protein, BRAF protein, EGFR protein, ERBB3 protein, KRAS protein, MAGE-C1 protein, NRAS protein, PIK3CA protein, RAC1 protein, or ROS1 protein, or an antigenic fragment thereof.
  • another neoantigen vaccine of the present disclosure comprises a polynucleotide encoding one or more, two or more, three or more tumor-speccific antigens or an antigenic fragment thereof.
  • the vaccine is a neoantigen vaccine and comprises a polynucleotide encoding one or more, two or more, three or more neoantigens in Table 7, or an antigenic fragment thereof.
  • the vaccine comprises or encodes an antigenic fragment from one or more of the folowing genes: 4-IBB, 5T4, ACAD5, AGAP6, AGS-16, AGS-5, AKT1, ANAPC1, Angiopoietin 2, ANKRC36C, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BLC11A, BRAF, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CD 19, CD137, CD2, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, c-MET, Cripto, CTLA4, ED-B,
  • the vaccine comprises an adjuvant.
  • adjuvants Adjuvants or immune potentiators may also be administered with or in combination with lipid nanoparticle composition. Advantages of adjuvants include the enhancement of the immunogenicity of antigens (e.g., pathogen-associated antigens, or cancer antigens), modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccinees. These may be co-administered by any route, e.g., intramuscularly, subcutaneous, IV, or intradermal injections.
  • route e.g., intramuscularly, subcutaneous, IV, or intradermal injections.
  • Adjuvants may include, but are not limited to, a natural or a synthetic adjuvant. Adjuvants may be organic or inorganic. [0555] Adjuvants may be selected from any of the classes (1) mineral salts, e.g., aluminum hydroxide and aluminum or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidised detergent stabilized oil-in-water emulsion, purified saponin, oil-in-water emulsion, stabilized water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes (unilamelar liposomal vehicles incorporating influenza hemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and/or (6) inert vehicles, such as
  • Adjuvants for nucleic acid vaccine have been disclosed in, for example, Kobiyama, et al., Vaccines, 2013, 1(3), 278-292, the entire contents of which is incorporated herein by reference. Even though the present disclosure is not limited to nucleic acid vaccines, any of the adjuvants disclosed by Kobiyama et al., may be used in the vaccines as described herein. [0557] Other adjuvants which may be utilized and include any of those listed on the web-based vaccine adjuvant database, on the World Wide Web at violinet dot org/vaxjo/ and as described in for example Sayers, et al., J.
  • Specific adjuvants may include cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM(s)TM, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium-derived P40 Vaccine Adjuvant, MPLTM
  • the adjuvant comprises squalene.
  • G. Methods of Administration [0560] In another aspect, the disclosure provides methods of administration for the LNP composition, the pharmaceutical composition, or the vaccine described herein. [0561] In some embodiments, the administering to the subject is done by intravenous (I.V.) delivery. In some embodiments, the administering to the subject is done by intrathecal (I.T.) delivery. In some embodiments the administering to the subject is done by subcutaneous injection (S.C.) delivery. In some embodiments the administering to the subject is done by intramuscular (I.M.) delivery. In some embodiments, the administering to the subject is done by intradermal (I.D.) delivery.
  • I.V. intravenous
  • the administering to the subject is done by intrathecal (I.T.) delivery.
  • the administering to the subject is done by subcutaneous injection (S.C.) delivery.
  • the administering to the subject is done by intramuscular (I.M.) delivery.
  • the administering to the subject is done by intranasal delivery. In some embodiments, the administering is done via nebulization. In some embodiments, the administering is done via injection. [0562] In some embodiments, the administration may be given pre-exposure to an infective agent. In some embodiments, the administration may be given post-exposure to an infective agent. In some embodiments, the administration may be given pre-diagnosis. In some embodiments, the administration may be given post-diagnosis. [0563] In some embodiments, the administration may be given before a subject has been diagnosed with a cancer. In some embodiments, the administration may be given after a subject has been diagnosed with a cancer.
  • the administration may be given before pre-diagnosis and given post-diagnosis.
  • the administration is single administration.
  • the administration is multiple administration.
  • the multiple administrations occur three times a day, twice a day, once a day, every other day, every third day, weekly, biweekly, every three weeks, every four weeks, or monthly.
  • the disclosure provides a method of using the LNP composition, the pharmaceutical composition, or the vaccine described herein.
  • the LNP composition, the pharmaceutical composition, or the vaccine can be used to prevent, treat, or ameliorate an infectious disease.
  • the disclosure provides a method for treating and/or preventing a CNS disease in a subject in need thereof, wherein the method comprises administering the composition described herein to the subject by intrathecal injection.
  • the disclosure provides a method of delivering a lipid nanoparticle composition the method comprising administering the LNP composition to a subject in need thereof, wherein the LNP composition comprises an antigen (e.g., a pathogen-associated antigen) or a polynucleotide encoding an antigen, a helper lipid, a sterol, a PEG-lipid, an ionizable cationic lipid, and a selective organ targeting (SORT) lipid.
  • an antigen e.g., a pathogen-associated antigen
  • SORT selective organ targeting
  • the disclosure provides a method of preventing a disease in a subject in need thereof, wherein the method comprises administering the LNP composition, the pharmaceutical composition, and/or vaccine described herein.
  • the disclosure provides a method of immunizing a subject in need thereof to an infectious disease, the method comprising administering the LNP composition, the pharmaceutical composition, and/or vaccine described herein.
  • the disclosure provides a method of immunizing a subject in need thereof against infection by influenza virus, wherein the method comprises administering the vaccine described herein.
  • the disclosure provides a method of immunizing a subject in need thereof against infection by coronavirus, wherein the method comprises administering the vaccine described herein.
  • the disclosure provides a method of immunizing a subject in need thereof against infection by paramyxovirus, wherein the method comprises administering the vaccine described herein.
  • the LNP composition delivery to a cel results in increased level of expression of influenza virus proteins, such as HA, NA, NP, M1, M2, NS1, NS2, NEP, PA, PB1, PB1-F2 and PB2, or an antigenic fragment thereof.
  • the LNP composition delivery to a cel results in increased level of expression of SARS-CoV proteins, such as Spike (S) protein, smal envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein.
  • the LNP composition delivery to a cel results in increased level of expression of respiratory syncytial virus proteins, such as F protein, G protein, SH protein, L protein, P protein, N protein, M2, protein, M protein, NS2 protein and NS2 protein.
  • the LNP composition delivery results in increased level of expression of hMPV proteins, such as F protein, G protein, SH protein, P protein, N protein, and M protein.
  • the LNP composition delivery results in increased level of expression of PIV proteins, such as F protein, HN protein, L protein, N protein, and M protein.
  • the LNP composition delivery results in increased level of expression of MERS- CoV proteins, such as S protein, S1 subunit protein, S2 subunit protein, E protein, N protein, and M protein.
  • the LNP composition delivery results in increased level of expression of Measles virus proteins, such as F protein, HA protein, P protein, V protein, and C protein.
  • the LNP composition, the pharmaceutical composition, or the vaccine can be used to prevent a cancer; to treat a cancer or to reduce the severity of a cancer; and/or to inhibit the growth, proliferation, progression, and/or metastasis of cancer cels.
  • the disclosure provides a method of delivering a lipid nanoparticle composition the method comprising administering the LNP composition to a subject in need thereof, wherein the LNP composition comprises a cancer antigen or a polynucleotide encoding a cancer antigen, a helper lipid, a sterol, a PEG-lipid, an ionizable cationic lipid, and a selective organ targeting (SORT) lipid.
  • the disclosure provides a method of preventing a disease in a subject in need thereof, wherein the method comprises administering the LNP composition, the pharmaceutical composition, and/or vaccine described herein.
  • an LNP composition, pharmaceutical composition, and/or vaccine described herein comprises or encodes an antigenic fragment from one or more of the folowing genes: 4-IBB, 5T4, ACAD5, AGAP6, AGS-16, AGS-5, AKT1, ANAPC1, Angiopoietin 2, ANKRC36C, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BLC11A, BRAF, BT-062, BTLA, CAIX, Carcinoembryonic antigen, CD 19, CD137, CD2, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, c-MET,
  • use of an LNP composition, pharmaceutical composition, and/or vaccine described herein prevents a cancer; treats a cancer or reduces the severity of a cancer; and/or inhibits the growth, proliferation, or metastasis of cancer cels.
  • a herein-disclosed method prevents a cancer; treats a cancer or reduces the severity of a cancer; and/or inhibits the growth, proliferation, or metastasis of cancer cels.
  • the cancer is one or more of acute myeloid leukemia (LAML), adrenocortical carcinoma (ACC), bladder cancer, bladder urothelial carcinoma (BLCA), brain cancer, breast cancer, breast invasive carcinoma (BRCA), Burkit lymphoma, cervical squamous cel carcinoma and endocervical adenocarcinoma (CESC), Chronic lymphocytic Leukaemia (CLL), colon adenocarcinoma (COAD), colon cancer, colorectal cancer (CRC), Difuse large B-cel lymphoma (DLBCL), Ewing's sarcoma, glioblastoma multiforme (GBM), glioma, head and neck carcinomas, head and neck squamous cel carcinoma (HNSC), hepatoma, human carcinomas (including colorectal, gastric, renal, and ovarian cancers), kidney chromophobe (KICH), kidney renal clear cel carcinoma (KIRC), kidney renal papa, a,
  • the method results in immune response to the subject, optionaly wherein the immune response is greater than the immune response after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in antibody response to the subject, optionaly wherein the antibody response is greater than the antibody response after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in T cel proliferation in the subject, optionaly wherein the T cel proliferation is greater than the T cel proliferation after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the method results in T cel activation in the subject, optionaly wherein the T cel activation is greater than the T cel activation after administration of a Composition 2H (50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG).
  • the T cel is T helper cel.
  • the T cel is cytotoxic T cel.
  • the disclosure provides a kit comprising an LNP composition, a pharmaceutical composition, or a vaccine described herein and instructions for use thereof.
  • the composition comprises a payload wherein the payload is a messenger RNA (mRNA) and wherein the method results in delivery of the payload to the CNS in an amount effective to increase expression and/or function of a gene encoded by the mRNA.
  • mRNA messenger RNA
  • the method results in expression of a polypeptide in a CNS of the subject.
  • the method results in expression of the polypeptide in the CNS of the subject between 1 and 72 hours after administration of the composition to the subject.
  • the CNS disease is acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, atention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold- Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosaminuria
  • ADHD tention deficit hyperactivity disorder
  • the disclosure provides a method of delivering a payload to a cel in a CNS of a subject, wherein the method comprises administering to the subject, by intrathecal injection, the composition described herein.
  • the disclosure provides use of the composition described herein for treatment of a CNS disease.
  • the disclosure provides use of the composition described herein for treatment of a CNS disease by intrathecal administration.
  • the disclosure provides the composition described herein or the pharmaceutical composition described herein for use in the treatment of a CNS disease in a subject in need thereof.
  • the CNS disease is acid lipase disease, acid maltase deficiency, acid storage disease, acquired epileptiform aphasia, acute disseminated encephalomyelitis, atention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoiditis, Arnold- Chiari malformation, aromatic L-amino acid decarboxylase deficiency (AADC deficiency), aspartylglucosamin
  • ADHD tention deficit hyperactivity disorder
  • Adie's pupil A
  • lipid nanoparticles For the formation of a lipid nanoparticle (LNP) composition, mRNA was dissolved in 1x PBS or citrate bufer (10mM, pH 4.0), and mixed rapidly into ethanol containing ionizable lipids, DOPE, cholesterol, DMG-PEG, and 16:0 EPC, fixing the weight ratio of 40:1 (total lipid:mRNA) and volume ratio of 3:1 (lipid:mRNA).
  • LNP lipid nanoparticle
  • LNP characterization The diferent LNP compositions were characterized by size, polydispersity index (PDI), and zeta-potential as assessed by Dynamic Light Scatering (DLS, Malvern, 173° Scatering angle). DLS measures the scatering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (at or around the mean particle diameter) in a population, with a perfectly uniform population having a PDI of zero. The encapsulation eficacy (EE %) was tested using RiboGreen RNA Assay (Zhao et al., 2016).
  • IVIS image Subjects were intravenously or intrathecaly injected with mRNA- containing LNP compositions at given dose. At the given time point, subjects were injected intraperitoneal (IP) with D-Luciferin and incubated for 5 min. Luciferase expression of whole body and ex vivo images were imaged by IVIS Lumina system (Perkin Elmer®). Images were processed using Living Image analysis software (Perkin Elmer®). [0592] Readouts: antibody response (H1N1 antibody), T proliferation (T helper, cytotoxic T), T activation (T helper, cytotoxic T), cytokine response. [0593] Western blot analysis: A549 cels were transfected with H1N1 mRNA.
  • Cel lysates were cleared by centrifugation at 21,000 g for 10 min at 4°C. Supernatants were then colected and quantified by BCA assay (Pierce Technology, NJ, USA). Samples were denatured at 65°C for 20 min. Samples were then analyzed by running on an automated capilary-based JESS protein simple system (Bio-Techne, CO, USA) with the 12–230 kDa separation module.100 ng of each sample was adjusted in 0.1X sample bufer and 1X fluorescent master mix (EZ standard pack, Bio-Techne, CO, USA) to a total final volume of 3 ⁇ l.
  • the rabbit polyclonal anti Influenza H1N1 HA (Cat# PA581656, Thermo Fisher, CA, USA) was used at 20 ⁇ g/mL, diluted in milk free antibody diluent (Bio-techne, CO, USA) and 5 ⁇ l was added per wel.
  • the secondary anti-rabbit-HRP antibody and enhanced chemiluminescence reagents were used according to the kit’s instructions. The samples were run folowing the machine’s RePlex program.
  • Fragment analyzer Evaluation of the in vitro generated transcript on a Fragment Analyzer indicated that transcripts maintained good integrity. Briefly, 2 ⁇ L of 200 ng/ ⁇ L samples were analyzed on a Fragment Analyzer.
  • ELISA assay to determine anti-HA mouse IgG/IgM 384-wel high binding plates (Greiner cat # 781061) were coated with 1 ⁇ g/ml H1N1 antigen (Sino Biological cat # 11055- V08B) in carbonate-bicarbonate bufer (Sigma Aldrich cat # C3041) O/N at 4°C.
  • ELISA assay to determine anti-RSV 384-wel high binding plates (Greiner cat # 781061) were coated with 0.5 ⁇ g/ml RSV antigen (Sino Biological cat # 40832) in carbonate- bicarbonate bufer (Sigma Aldrich cat # C3041) overnight (O/N) at 4°C. Plates were then blocked with Superblock (Thermo Fisher cat # 37535) for 2 hrs at RT, then washed 3 times with TBS (Thermo Fisher cat # 28372) containing 0.05% Tween (Thermo Fisher cat # 85113). An antibody standard curve was generated from RSV monoclonal antibody 9C5 (Thermo Fisher cat # MA1-20009).
  • Mouse splenocyte isolation Mouse spleens were harvested and put in the ice-cold PBS under sterile condition. In the biosafety cabinet, spleens were mechanicaly disrupted into single cels in 4ml of Red Blood Cel lysis bufer (Cat# 00433357, Invitrogen).
  • T cel proliferation assay T cel proliferation in immunized mice were assessed using a SA3800 Spectral Flow Cytometer (Sony Biotechnology).
  • 1x 107 mouse splenocytes were labeled with 5 ⁇ M CelTraceTM CFSE (Cat# C34554, Thermo Fisher) for 30 min at 37 °C, then, a total of 5x 106 splenocytes were plated in a 24 wel plate and stimulated either with a negative control (culture medium) or 5 ⁇ g/ml of HA antigen (Cat# 11055-V08B, Sino Biological) for 7 days.
  • T cel activation assay T cel activation in immunized mice were assessed using a SA3800 Spectral Flow Cytometer (Sony Biotechnology). Briefly, after single splenocyte isolation, a total of 5x 106 splenocytes were plated in a 24 wel plate and stimulated either with control (culture medium) or 5 ⁇ g/ml of HA antigen (Cat#, Sino Biological) in a total volume of 1ml.
  • cels were colected and centrifuged at 500g for 10 min at 4°C.
  • the supernatants were colected and stored at -80°C for cytokine release assay, while the pelets (cels) were washed with PBS and stained with PE anti-mouse CD3 (Cat#100206, Biolegend), FITC anti-mouse CD4 (Cat#100510, Biolegend), BV421 anti- mouse CD8a (Cat#100737, Biolegend), BV605 anti-mouse CD69 (Cat#104530, Biolegend) and AF700 anti-mouse CD25 (Cat# 102024, Biolegend) for 30 min at 4°C.7-AAD (Biolegend, Cat: 420404) were used as cel viability dye to stain the dead cels.20,000 live cels were recorded.
  • Cytokine release assay Cytokine release from supernatant was detected by MSD (Meso Scale Discovery, MD, USA).29 cytokines (including IFN- ⁇ , IL-10, IL-12p70, IL-1 ⁇ , IL-2, IL-4, IL-5, IL-6, KC/GRO, TNF- ⁇ , IL-16, IL-17A, IL-17C, IL-21, IL-22, IL-23, IL- 17E/IL-25, IL-15, IL-17F, IL-27P28/IL-30, IL-31, IL-33, IL-17A/F, IL-9, IP-10, MCP-1, MIP- 1 ⁇ , MIP-2, and MIP-3 ⁇ ) from Th1, Th2, Th17 and T memory cels were detected by U-PLEX Biomarker Group 1 (mouse) (Cat# K15355K-2, MSD).
  • the assay was operated according to the manufacturer’s guidelines. Briefly, 200 ⁇ l of biotinylated antibodies were first coupled to 300 ⁇ l of specific U-PLEX linkers and then incubated for 30 min at RT.200 ⁇ l of stop solution was added and incubated for 30 min at RT. Next, 600 ⁇ l of each antibody-linker-stop solution was combined to form the U-PLEX multiplex coating solutions.50 ⁇ l of the U-PLEX multiplex coating solutions were added to each wel of the plate and shaken 1h at RT with 700 rpm to alow the linkers to self-assemble onto unique spots on the U-PLEX plate.
  • Control groups received respective formulation bufers. Blood samples were colected on Day 14, 21, 28, and 35 and processed to serum to monitor immune responses. Two animals in each formulation group were sacrificed and spleen and lymph nodes were colected for T cel activation and proliferation analysis on day 14 and 21. An exemplary schematic of the mouse study is shown in FIG.1.
  • H1N1 mRNA construct design This example describes the design, production, and expression of H1N1 mRNA constructs.
  • H1N1 ORF open reading frame
  • the mRNA was produced in vitro according to the method described in Chen et al., Scientific Reports (2022) 12, 13017 and included pseudouridine bases.
  • Sequence-optimized H1N1 ORF was further modified with Cap1 vaccinia capping system (m7GpppGm), Kozak sequence (GCCACCAUGx), and a template-encoded polyA tail.
  • the estimated mRNA size, including the polyA tail was 2,190 nucleotides.
  • LNPs comprising the H1N1 HA mRNA were produced as described in Example 1. The resulting LNPs were analyzed by Fragment Analyzer to determine the content of mRNA in the lipid nanoparticles. The results show a high content of ful-length mRNA (predicted size of 2,190 nucleotides) (FIG.2A, left panel).
  • FPGA Fragment Analyzer
  • Example 4 Formulation Characteristics
  • Composition 2B Composition V, Composition K, Composition X, and Composition 2H were used in this study.
  • LNP compositions used in the experiments are shown in FIG.2B.
  • Composition 2B comprised: 16.67 % 4A3-SC7, 16.67 % DOPE, 33.3 % cholesterol, 3.33 % DMG-PEG, and 30 % 16:0 EPC.
  • the lipid:mRNA ratio was 30:1.
  • Composition V comprised: 11.9 % 4A3-SC7, 11.9 % DOPE, 23.81 % cholesterol, 2.38 % DMG-PEG, and 50 % DOTAP.
  • the lipid:mRNA ratio was 40:1.
  • Composition K comprised: 22.62 % 4A3-SC7, 22.62 % DOPE, 45.24 % cholesterol, 4.52 % DMG-PEG, and 5 % 18:1 PA.
  • the lipid:mRNA ratio was 40:1.
  • Composition X comprised: 14.8 % 4A3-SC7, 22.2 % DOPE, 44.4 % cholesterol, 3 % DMG-PEG, and 15.6 % DODAP.
  • Composition 2H comprised: 50 % SM-102, 10 % DSPC, 38.5 % cholesterol, and 1.5 % DMG-PEG. The lipid:mRNA ratio was 20.6:1.
  • Al LNPs were loaded with H1N1 HA mRNA.
  • LNPs were characterized as described in Example 1. The results show, that the LNP size of Composition 2B, Composition V, Composition K, and Composition X were smaler than the LNP size of Composition 2H.
  • composition 2B, Composition V, and Composition K showed higher encapsulation eficiencies (EE%) after freeze-thaw cycle at day 21, when compared to Composition 2H (shown in FIG.2B).
  • Example 5 anti-H1N1-HA antibody titer analysis
  • This example describes the determination of anti-H1N1-HA antibody titer in mice injected with H1N1 HA mRNA LNPs.
  • ⁇ -H1N1 HA IgG/IgM antibody levels were determined by enzyme-linked immunoassay (ELISA) as described in Example 1 on days 14, 21, 28, and 35.
  • FIG.3A The results show that mice injected with Composition V had no antibody response due to toxicity.
  • Antibody responses for Composition 2B, Composition K, Composition X, and Composition 2H peaked at day 28. Peak response for Composition 2H is shown as a horizontal line in each graph. Composition 2B, Composition K, and Composition X showed higher antibody response at various doses compared to Composition 2H. [0619]
  • the results show that the antibody response was mRNA dose-dependent (FIG.3B). Peak antibody responses from Composition 2B, Composition K, and Composition X were statisticaly significant.
  • T cel proliferation analysis This example describes the evaluation of T cel proliferation in mice treated with H1N1 HA mRNA LNPs. [0621] Briefly, T cels were prepared from mouse bleeds on day 14 and day 28. Proliferation of cels was analyzed as described in Example 1. [0622] T cel proliferation was observed at day 14 (FIG.4A to FIG.4C) and day 28 (FIG. 5A to FIG.5C). FIG.4A and FIG.5A shows a cel proliferation analysis. FIG.4B and FIG. 5B shows proliferation analysis of T helper cels.
  • FIG.4C and FIG.5C shows proliferation analysis of cytotoxic T cels.
  • Mice treated with Composition 2H showed increased proliferation of both T helper cel and cytotoxic T cels at day 14.
  • Mice treated with Composition 2B showed peak T cel proliferation at day 28.
  • Mice treated with Composition X showed increased proliferation of both T helper cel and cytotoxic T cels at day 28. These results indicated that Composition 2B and Composition X have higher cytotoxic T cel induction compared to other Compositions at day 28.
  • Example 7 T cel activation analysis [0623] This example describes the evaluation of T cel activation in mice treated with H1N1 HA mRNA LNPs. [0624] Briefly, T cels were prepared from mouse bleeds on day 14 and day 28.
  • T cel activation of T cels was analyzed as described in Example 1. [0625] T cel activation was observed at day 14 (FIG.6A to FIG.6C) and day 28 (FIG.7A to FIG.7C). Mice treated with Composition 2H showed higher T cel activation at day 14. At day 28, mice treated with either Composition 2B or Composition X showed higher T cel activation compared to Composition 2H. Peak T cel response was observed at 0.03 - 10 ⁇ g doses.
  • Example 8. Cytokine expression analysis [0626] This example describes cytokine expression analysis of T cels from mice treated with H1N1 HA mRNA LNPs. [0627] Briefly, T cels were prepared from mouse bleeds on day 14 and day 28.
  • a panel of cytokines (including IFN ⁇ , IL-10, IL-12p70, IL-1 ⁇ , IL-2, IL-4, IL-5, IL-6, KC/GRO, TNF- ⁇ , IL-16, IL-17A, IL-17C, IL-21, IL-22, IL-23, IL-17E/IL-25, IL-15, IL-17F, IL-27P28/IL-30, IL-31, IL-33, IL-17A/F, Il-9, OP-10, MCP-1, MIP-1 ⁇ , MIP-2, and MIP-3 ⁇ ) was probed as described in Example 1 and ploted as heatmaps.
  • cytokines including IFN ⁇ , IL-10, IL-12p70, IL-1 ⁇ , IL-2, IL-4, IL-5, IL-6, KC/GRO, TNF- ⁇ , IL-16, IL-17A, IL-17C, IL-21, IL-22, IL-23, IL-17E/
  • T cel subtypes expressing the respective cytokines are shown (Th1/2 cels, Tmem/Th2, Th2, and Innate).
  • Cytokine heatmaps for 1, 3, or 10 ⁇ g dose are shown in FIG.8A (day 14) and FIG.8B (day 28). Cytokine response in Composition 2B-treated mice were higher at day 14 and lower at day 28.
  • Composition X induced similar or higher cytokine response compared to the other Compositions. Th1/2 and innate immune responses were similar in magnitude at al tested concentrations.
  • T memory (Tmem) cel-related cytokine (IL-22) was upregulated in Composition K- treated mice and in Composition X-treated mice at 10 ⁇ g dose at day 28.
  • FIG.8C and FIG.8D Cytokine heatmap analysis at micro-dosing (0.03 ⁇ g and 0.1 ⁇ g) are shown in FIG.8C and FIG.8D.
  • Micro dosing Composition 2B induced higher cytokine responses compared to other Compositions at day 14 indicating a potential early innate and T cel response.
  • Example 9 Anti-H1N1-HA antibody titers
  • This example describes the determination of anti-H1N1-HA antibody titer in mice injected with H1N1 HA mRNA LNPs.
  • ⁇ -H1N1 HA IgG/M antibody levels were determined by enzyme-linked immunoassay (ELISA) as described in Example 1 on days 7, 14, 21, 28, and 35.
  • Composition X (pH6.5)-treated mice showed lower antibody response and cytokine response compared to Composition X (pH4)-treated mice.
  • Composition 2B-treated mice showed robust response in Th1/2 cytokine responses, with robust of IL-22 response.
  • Lymph node cytokine heatmaps at day 7 and day 28 are shown in FIG.11C and FIG. 11B, respectively. The analysis shows low cytokine response in Composition 2B-treated mice compared to spleen cytokine heatmap.
  • Example 10 Anti-RSV antibody titers
  • ⁇ -RSV antibody levels were determined by enzyme-linked immunoassay (ELISA) (FIG.12A). Cytokine heatmaps for Composition 2B / RSV-injected mice were analyzed in spleen (FIG.12B) and lymph node (FIG.12C) showing higher cytokine response at day 28 compared to day 7.
  • FIG.12A Cytokine heatmaps for Composition 2B / RSV-injected mice were analyzed in spleen (FIG.12B) and lymph node (FIG.12C) showing higher cytokine response at day 28 compared to day 7.
  • Example 11 Cel specific gene expression profile on Day 1
  • This example describes cel specific gene expression profile using scRNAseq to support beter tolerability of lipid nanoparticle formulations on Day 1.
  • Lymph node cels from vaccine-injected mice showed significantly elevated transcription level of interferon-induced genes, indicating a robust and effective anti- viral immune response.
  • Gene expression levels in immune cels suggest that immune responses can be modulated through diferent formulations and pH conditions. The modulation aligns with observed cytokine responses at diferent time points, potentialy enhancing overal tolerability of Composition 2B and Composition X/pH 6.5 compared to Composition 2H.
  • Example 12 Viral chalenge study
  • This example describes a viral chalenge study to determine eficacy of novel lipid nanoparticle-encapsulated mRNA vaccines, delivered by intramuscular injection, for protection against infection with seasonal H1N1 influenza infection.
  • Study design for virus chalenge at day 28 post initial vaccine dose is shown in Table 19. Table 19. Study design for virus chalenge – Day 28
  • influenza virus H1N1, A/Puerto Rico/8/34 (PR8) in sterile saline at a dose of 60 PFU (plaque forming unit) was administered to each animal by intranasal instilation under isoflurane anesthesia. Al animals were weighted weekly during the study beginning on Day 0 and daily after chalenge on Day 84.
  • FIG.18A shows conditional survival rate of mice dosed with either Composition V, Composition 2B, Composition X, or Composition 2H.
  • FIG.18B shows comparison results of conditional survival rate of mice dosed with either Composition V, Composition 2B, or Composition X to Composition 2H.
  • Composition V was used as a negative control.
  • Mice dosed with either Composition 2B, Composition X showed higher conditional survival rates (FIGs.18B and 18C). These results are consistant with body weight change shown in FIGs.19A to 19C.
  • Example 13 Formulation Characteristics for CNS related Assays [0647]This example describes the characteristics of LNP compositions Composition W, B, G, A, and I used in this study. [0648]Briefly, LNP compositions used in the experiments are shown in FIG.1. [0649]Composition W comprised: 50% D-Lin-MC3-DMA, 10% DSPC, 38.5 % cholesterol, and 1.5% DMG-PEG.
  • Composition B comprised: 19.05% 4A3-SC7, 19.05% DOPE, 38.09% cholesterol, 3.81% DMG-PEG, and 20% DODAP.
  • Composition G comprised: 14.29% 5A2-SC8, 14.29% DOPE, 28.56% cholesterol, 2.86% DMG-PEG, and 40% 14:0 TAP.
  • Composition A comprised: 19.05% 4A3-SC7, 19.05% DOPE, 38.09% cholesterol, 3.81% DMG-PEG, and 20% 14:0 EPC.
  • Composition I comprised: 22.62% 5A2-SC8, 22.62% DOPE, 45.24% cholesterol, 4.52% DMG-PEG, and 5% 18:1 PA.
  • mice were intrathecaly administered with Fluc-0.13.5a mRNA- containing lipid nanoparticle Composition W, B, G, A, I.
  • D-Luciferin was injected intraperitoneal (IP) into each mouse and the mice were imaged by an IVIS Lumina system (Perkin Elmer®).
  • IVIS Lumina system Perkin Elmer®
  • Several organs were explanted for further imaging. Brains were rinsed in ice-cold PBS and its images were taken at dorsal and ventral sides at 10 seconds exposure.
  • MAV Meninges and associated vasculature
  • FIG.21A-21E Brain images of LNP-treated mice showed the strongest signal at dorsal cerebelum and at the center of the ventral brain with MAV, while litle or no signal was observed after the MAV had been removed.
  • Luciferase signal in Composition A-treated mice showed that higher expression of luciferase on cerebelum.
  • Quantitative results in FIG.21F showed that CNS luciferase signal is SORT lipid dependent with Composition A showing higher signal in both whole brain and coronal sections compared to other Compositions.
  • Whole body in vivo imaging (FIG.22A) showed that Composition A showed the highest signal in brain compared to other Compositions.
  • FIG.22C Bioluminescence images of organs (FIG.22C) showed expression of luciferase in each organ (liver, spleen, lung, and bone).
  • Composition A can be delivered to the CNS via intrathecal administration in vivo.
  • Further studies were conducted to evaluate brain distribution, parenchyma penetration, and colocalization of Cre induced tdTomato expression with key cel types. Briefly, Ai14 tdTomato transgenic mice were intrathecaly administered with Cre mRNA-containing Composition A. Mice were transcardialy perfused with PBS folowed by 4% paraformaldehyde (PFA) at 48 hour post-injection.
  • PFA paraformaldehyde
  • Ai14 tdTomato mice express robust tdTomato fluorescence folowing Cre-mediated recombination.
  • Brains were harvested, imaged by an IVIS Lumina system (Perkin Elmer®), and soaked in 10, 20, 30% sucrose. Then, sections were taken at defined Bregma coordinates throughout the brain. Immunofluorescent staining confirmed the result of bioluminescence images that most LNP accumulates in the meninges and ventricles (FIG.23). Immunofluorescent also strongly marked the endothelial cels throughout the parenchyma and signal was slightly brighter in ventral/inferior portion of the brain sections (FIGs.24A and 24B).
  • the results shown in FIGs.26 and 27 further show no of colocalization with astrocytes in olfactory bulbs (FIG.26) and neurons in cortex/thalamus/hypothalamus (FIG. 27).
  • composition 2I and Composition 2B This example describes delivery and distribution of Composition 2I and Composition 2B in endothelial cels and after intrathecal delivery in vivo.
  • LNP compositions used in the experiments are shown in FIG.28 and Table 21.
  • Composition 2F comprised: 23.81% 4A3-SC7, 23.81% DOPE, 47.62% cholesterol, and 4.76% DMG-PEG. N/P ratio is 8.9. Lipid:mRNA ratio was 30.
  • Composition 2I comprised: 16.67% 4A3-SC7, 16.67% DOPE, 33.33% cholesterol, 3.33% DMG-PEG, and 30% 14:0 EPC. N/P ratio is 10.3.
  • Lipid:mRNA ratio was 30.
  • Composition 2B comprised: 16.67% 4A3-SC7, 16.67% DOPE, 33.33% cholesterol, 3.33% DMG-PEG, and 30% 16:0 EPC. N/P ratio is 10.1. Lipid:mRNA ratio was 30.
  • Al LNPs were loaded with Cre mRNA.
  • LNPs were prepared and characterized as described in Example 15. The results show that the LNP sizes of Compositions L and Composition 2B are smaler than Composition 2F, while encapsulation eficiencies (EE%) are higher. (shown in FIG.28).
  • FIG.29C further shows widely distributed colocalization with tdTomato and endothelial cels (CD31) throughout the brain in Composition 2I and Composition 2B-treated mice.
  • Composition 2F-treated mice showed limited, sparsely distributed colocalization with tdTomato and endothelial cels throughout the brain.
  • Composition 2F-treated mice showed less tdTomato signal throughout the brain. This suggests that Composition 2I or Composition 2B can deliver Cre mRNA into the CNS.
  • PDGRF ⁇ pericyte marker
  • FIG.30C further shows widely distributed colocalization with tdTomato and pericyte cels throughout the brain in Composition 2I and Composition 2B-treated mice.
  • Composition 2F-treated mice showed limited, sparsely distributed colocalization with tdTomato and pericyte cels throughout the brain.
  • Composition 2F-treated mice showed less tdTomato signal throughout the brain. This suggests that Composition 2I or Composition 2B can deliver Cre mRNA into the CNS.
  • FIG.31B More colocalization with tdTomato and SMA were observed in Composition 2I-treated mice compared to Composition 2B-treated mice (FIG.31A).
  • FIG.31C further shows colocalization with tdTomato and smooth muscle cels (SMA) throughout the brain.
  • Composition 2F-treated mice showed litle colocalization with tdTomato and SMA.
  • composition 2B-treated mice showed sparse colocalization with tdTomato and SMA.
  • Composition 2I-treated mice showed broad colocalization with tdTomato and SMA, suggesting Composition 2I-mediated delivery to smooth muscle cels is slightly favored.
  • the results show that Composition 2I and M can deliver mRNA cargo to the CNS in vivo.
  • Example 16 [0671]This example describes delivery and distribution of Composition K in the central nervous system (CNS) after intrathecal delivery in vivo.
  • CNS central nervous system
  • IVIS images showed expression of luciferase in brains.

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Abstract

L'invention concerne des nanoparticules lipidiques (LNP) ainsi que des compositions pharmaceutiques et des vaccins les comprenant. L'invention concerne également des méthodes d'administration d'une composition de LNP, des compositions pharmaceutiques et des vaccins décrits dans la description pour prévenir une maladie infectieuse et/ou pour immuniser un sujet ou pour administrer une charge utile à une cellule dans le système nerveux central (SNC) d'un sujet. L'invention concerne également des méthodes de traitement et/ou de prévention d'une maladie du SNC.
PCT/US2024/057850 2023-12-01 2024-11-27 Compositions de nanoparticules lipidiques et leurs utilisations Pending WO2025117816A1 (fr)

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CN110505877A (zh) * 2017-02-01 2019-11-26 摩登纳特斯有限公司 Rna癌症疫苗
WO2020051220A1 (fr) * 2018-09-04 2020-03-12 The Board of the Regents of the University of Texas System Compositions et procédés pour la délivrance spécifique d'organe d'acides nucléiques
US20220125899A1 (en) * 2018-11-07 2022-04-28 Modernatx, Inc. Rna cancer vaccines
US20220409708A1 (en) * 2019-11-01 2022-12-29 Korea Advanced Institute Of Science And Technology Small lipid nanoparticles, and cancer vaccine including same
US20230226217A1 (en) * 2012-03-26 2023-07-20 TRON-Translationale Onkologie an der Universitatsmedizin der Johannes Gutenberg-Universitat Mainz ad Rna formulation for immunotherapy
US20230338411A1 (en) * 2021-03-22 2023-10-26 Recode Therapeutics, Inc. Compositions and methods for targeted delivery to cells

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Publication number Priority date Publication date Assignee Title
US20230226217A1 (en) * 2012-03-26 2023-07-20 TRON-Translationale Onkologie an der Universitatsmedizin der Johannes Gutenberg-Universitat Mainz ad Rna formulation for immunotherapy
CN110505877A (zh) * 2017-02-01 2019-11-26 摩登纳特斯有限公司 Rna癌症疫苗
WO2020051220A1 (fr) * 2018-09-04 2020-03-12 The Board of the Regents of the University of Texas System Compositions et procédés pour la délivrance spécifique d'organe d'acides nucléiques
US20220125899A1 (en) * 2018-11-07 2022-04-28 Modernatx, Inc. Rna cancer vaccines
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