WO2024131810A1 - Lipid nanoparticles comprising sterol-modified phospholipids - Google Patents

Lipid nanoparticles comprising sterol-modified phospholipids Download PDF

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WO2024131810A1
WO2024131810A1 PCT/CN2023/140052 CN2023140052W WO2024131810A1 WO 2024131810 A1 WO2024131810 A1 WO 2024131810A1 CN 2023140052 W CN2023140052 W CN 2023140052W WO 2024131810 A1 WO2024131810 A1 WO 2024131810A1
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lnp
lipid
independently
mol
integer
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PCT/CN2023/140052
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French (fr)
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Dandan LING
Jianxiu XUE
Jerry C. ZHANG
Bo YING
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Suzhou Abogen Biosciences Co., Ltd.
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Publication of WO2024131810A1 publication Critical patent/WO2024131810A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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

Definitions

  • the invention relates to lipid nanoparticles comprising phospholipids that contain a sterol moiety.
  • LNPs Lipid nanoparticles
  • mRNA vaccines One important application of nucleic acid-loaded LNPs are mRNA vaccines.
  • the present application provides phospholipids containing a sterol moiety that can be used to construct lipid nanoparticles.
  • the lipid nanoparticles are useful for delivering nucleic acids, e.g. mRNA, to one or more cells.
  • the application provides a method for expressing protein in a cell by delivering nucleic acids to the cell via the lipid nanoparticle comprising a phospholipid that contains a sterol moiety.
  • lipid nanoparticles comprising
  • the phospholipid has a structure selected from:
  • the phospholipid has the structure:
  • the phospholipid has the structure:
  • the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20 ⁇ 1 to 2 ⁇ 1. In some embodiments, the molar ratio of ionizable lipid to phospholipid is from 15 ⁇ 1 to 5 ⁇ 1. In some embodiments, the i onizable lipid comprises from 40 to 80 mol%of a total amount of lipids in the LNP. In some embodiments, the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP. In some embodiments, the ionizable lipid is a cationic lipid.
  • the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
  • the polymer conjugated lipid comprises from 0.5 to 5 mol%of the total amount of lipids in the LNP. In some embodiments, the polymer conjugated lipid comprises from 1 to 2 mol%of the total amount of lipids in the LNP. In some embodiments, the polymer conjugated lipid comprises 1.5 mol%of the total amount of lipids in the LNP. In some embodiments, the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid of from 1 ⁇ 2 to 1 ⁇ 20. In some embodiments, the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid of from 1 ⁇ 3 to 1 ⁇ 18.
  • the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 5 to 1 ⁇ 10.
  • the polymer conjugated lipid is a PEGylated lipid.
  • the polymer conjugated lipid is a PEGylated lipid with the structure:
  • R 12 and R 13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w is an integer ranging from 30 to 60.
  • the polymer conjugated lipid is a PEGylated lipid with the structure:
  • w is an integer ranging from 30 to 60.
  • w is an integer ranging from 45 to 55.
  • w is about 49.
  • the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
  • the LNP further comprises a lipid stabilizer.
  • the LNP has a molar ratio of the lipid stabilizer to the phospholipid of from 10 ⁇ 1 to 1 ⁇ 4. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 5 ⁇ 1 to 1 ⁇ 3. In some embodiments, the LNP has a molar ratio of the lipid stabilizer to the phospholipid of from 10 ⁇ 1 to 1 ⁇ 2. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 5 ⁇ 1 to 1 ⁇ 1. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 4 ⁇ 1 to 3 ⁇ 1.
  • the lipid stabilizer comprises from 5 to 50 mol%of the total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
  • the phospholipid comprises from 1 to 30 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP.
  • the LNP has a size of from 20 nm to 300 nm, as determined using dynamic light scattering. In some embodiments, the LNP has a size of from 50 nm to 150 nm, as determined using dynamic light scattering. In some embodiments, the size is from 60 nm to 140 nm. In some embodiments, the size is from 80 nm to 100 nm. In some embodiments, the size is from 85 nm to 95 nm.
  • the LNP encapsulates mRNA.
  • LNPs comprising a phospholipid, wherein the phospholipid has a structure:
  • the phospholipid has the structure:
  • the phospholipid has the structure:
  • the LNP further comprises an ionizable lipid.
  • the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20 ⁇ 1 to 2 ⁇ 1. In some embodiments, the molar ratio of ionizable lipid to phospholipid is from 15 ⁇ 1 to 5 ⁇ 1. In some embodiments, the i onizable lipid comprises from 40 to 80 mol%of a total amount of lipids in the LNP. In some embodiments, the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP.
  • the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5. In some embodiments, the ionizable lipid is a cationic lipid.
  • the LNP further comprises a polymer conjugated lipid.
  • the polymer conjugated lipid comprises from 1 to 2 mol%of a total amount of lipids in the LNP. In some embodiments, the polymer conjugated lipid comprises 1.5%of the total amount of lipids in the LNP.
  • the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 5 to 1 ⁇ 10. In some embodiments, the polymer conjugated lipid is a PEGylated lipid.
  • the polymer conjugated lipid is a PEGylated lipid with the structure:
  • R 12 and R 13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w is an integer ranging from 30 to 60.
  • the polymer conjugated lipid is a PEGylated lipid with the structure:
  • w is an integer ranging from 30 to 60.
  • w is an integer ranging from 45 to 55. In some embodiments, w is about 49.
  • the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
  • the LNP further comprises a lipid stabilizer.
  • the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10 ⁇ 1 to 1 ⁇ 4. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 5 ⁇ 1 to 1 ⁇ 3. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 4 ⁇ 1 to 3 ⁇ 1. In some embodiments, the lipid stabilizer comprises from 5 to 50 mol%of a total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
  • the phospholipid comprises from 1 to 30 mol%of a total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP. In some embodiments, the LNP has a size of from 20 nm to 300 nm, as determined using dynamic light scattering.
  • the LNP has a size of from 50 nm to 150 nm, as determined using dynamic light scattering. In some embodiments, the size is from 60 nm to 140 nm. In some embodiments, the size is from 80 nm to 100 nm. In some embodiments, the size is from 85 nm to 95 nm.
  • the LNP encapsulates mRNA.
  • compositions of the lipid nanoparticles (LNPs) described herein are also provided herein.
  • LNPs lipid nanoparticles
  • at least 80%of the LNPs encapsulate mRNA.
  • at least 85%of the LNPs encapsulate mRNA.
  • the cell is a mammalian cell.
  • the LNP or composition thereof is administered systemically.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • FIG. 1 shows expression levels of hEPO in LNP formulations containing PChemsPC or OChemsPC with a 60 ⁇ 10 ⁇ 28.5 ⁇ 1.5 molar ratio of compound 01-1/OChemsPC or PChemsPC/Chol/DMG-PEG in Example 15.
  • FIG. 2 shows expression levels of hEPO in LNP formulations containing DSPC or PChemsPC at various molar percentages in Example 16.
  • FIG. 3 shows expression levels of hEPO in LNP formulations containing PChemsPC at various molar ratios of PChemsPC/Chol in Example 17.
  • FIG. 4 shows the luminescence levels measured from harvest liver tissues in Example 19.
  • Fig. 5 shows the percentage of luminescence intensity in different tissues in Example 19.
  • FIG. 6 shows hEPO expression fold change of LNPs with or without a steroid containing phospholipid in Example 20.
  • FIG. 7 shows serum cytokines levels boosted by compound 01-1 LNP in Example 21.
  • FIG. 8 shows serum cytokines levels boosted by Lipid 5 LNP in Example 21.
  • FIG. 9 shows serum cytokines levels boosted by SM-102 LNP in Example 21.
  • FIG. 10 shows serum cytokines levels boosted by ALC-0315 LNP in Example 21.
  • FIG. 11 shows serum cytokines levels boosted by Compound 03-135 LNP in Example 21.
  • FIG. 12 shows serum cytokines levels boosted by Compound 01-1 saRNA-LNP in Example 22.
  • FIG. 13 shows serum cytokines levels boosted by Compound 03-135 saRNA-LNP in Example 22.
  • FIG. 14 shows CD3-CD19 antibody levels of LNPs with or without a steroid containing phospholipid in Example 23.
  • LNPs comprising a phospholipid containing a sterol moiety.
  • the LNPs can be loaded with mRNA, such as in mRNA vaccine technology.
  • Sterol-modified phospholipids stabilize bilayers but do not exchange between membranes as freely as cholesterol.
  • the mRNA-loaded LNPs demonstrate an ability to increase protein expression in target cells compared to mRNA-loaded LNPs with more conventional phospholipids. Increasing protein expression helps increase the effectiveness and efficiency of mRNA-based treatments and therapies.
  • pharmaceutically acceptable or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
  • a pharmaceutically acceptable carrier refers to a pharmaceutically acceptable substrate, composition or vehicle used in the process of drug delivery, which may have one or more ingredients including, but not limited to, excipient (s) , binder (s) , diluent (s) , solvent (s) , filler (s) , and/or stabilizer (s) .
  • lipid refers to a group of compounds including, without limitation, fats, sterols, waxes, fat-soluble vitamins, monoglycerides, diglycerides, sphingolipids, and phospholipids.
  • phospholipids, ionizable lipids, polymer conjugated lipids, and lipid stabilizers are considered lipids.
  • the term “ionizable lipid” refers to a lipid that has a non-zero net electric charge at physiological pH.
  • the term is inclusive with respect to cationic lipids, including lipids that have a partial positive charge at physiological pH.
  • the term is also inclusive with respect to mixtures of ionizable lipids, which can contain two or more ionizable lipids.
  • ionizable lipid it is likewise contemplated with a “cationic lipid, ” as though all embodiments were specifically and individually listed with both ionizable and cationic lipids.
  • polymer conjugated lipid refers to a lipid comprising a polymer moiety.
  • the term is inclusive with respect to PEGylated lipids, including PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, and PEGylated dialkylglycerols.
  • the term is also inclusive with respect to mixture of polymer conjugated lipids, which may contain two or more polymer conjugated lipids.
  • lipid stabilizer refers to a component of the lipid nanoparticle that thought to help stabilize the LNP structure. Without being bound by theory, it is believed that the lipid stabilizer component of LNPs helps favor the liquid-ordered phase of the lipid membrane in LNPs. See, for example, section 3.3.1 of Albertsen, H.C.; et al., “The role of lipid components in lipid nanoparticles for vaccines and gene therapy. ” Adv Drug Deliv Rev. 2022 Sep; 188: 114416. Compounds that can serve as lipid stabilizers include sterols, corticosteroids, vitamins, and other compounds comprising a steroid core.
  • alkyl refers to a chain of carbon atoms wherein all bonds between the carbon atoms in the alkyl group are single bonds.
  • the term is inclusive with respect to straight and branched chains (e.g., the term includes both n-propyl and isopropyl groups) .
  • C x -C y alkyl refers to an alkyl with at least x carbon atoms and no more than y carbon atoms in the alkyl chain.
  • C 1 -C 3 alkyl includes, without limitation, methyl, ethyl, n-propyl, and isopropyl.
  • alkylene refers to an alkyl chain that connects in at least two locations to other chemical groups.
  • C x -C y alkylene refers to an alkylene with at least x carbon atoms and no more than y carbon atoms in the alkylene chain.
  • C 1 -C 3 alkylene includes, without limitation, methylene, ethylene, n-propylene, and iso-propylene.
  • alkenyl refers to a chain of carbon atoms with at least one double bond between two carbon atoms in the chain.
  • the term is inclusive with respect to straight and branched chains (e.g., the term includes both 1 -propenyl and iso-propenyl groups) .
  • C x -C y alkenyl refers to an alkenyl with at least x carbon atoms and no more than y carbon atoms in the alkenyl chain.
  • C 2 -C 4 alkenyl includes, without limitation, vinyl and 1-propenyl.
  • alkenylene refers to an alkenyl chain that connects in at least two locations to other chemical groups.
  • C x -C y alkenylene refers to an alkenylene with at least x carbon atoms and no more than y carbon atoms.
  • alkynyl refers to a chain of carbon atoms with at least one triple bond between two carbon atoms in the chain.
  • the term is inclusive with respect to straight and branched chains (e.g., the term includes both 1-propynyl and iso-propynyl groups) .
  • C x -C y alkynyl refers to an alkynyl with at least x carbon atoms and no more than y carbon atoms in the alkynyl chain.
  • cycloalkyl refers to a cyclic group of carbon atoms wherein all the bonds between the carbon atoms are single bonds.
  • C x -C y cycloalkyl refers to a cycloalkyl with at least x carbon atoms and no more than y carbon atoms.
  • C 6 -C 10 cycloalkyl includes, without limitation, cyclohexyl and cyclo-octyl.
  • cycloalkylene has the same meaning as cycloalkyl, except that the cycloalkylene connects to at least two other chemical groups.
  • cycloalkenyl refers to a cyclic group of carbon atoms where at least one bond between two carbon atoms in the cycloalkenyl group is a double bond.
  • C x -C y cycloalkenyl refers to a cycloalkenyl with at least x carbon atoms and no more than y carbon atoms.
  • C x -C y aryl refers to an aryl with at least x carbon atoms and no more than y carbon atoms.
  • C 6 -C 10 aryl includes, without limitation, phenyl and naphthyl.
  • arylene has the same meaning as aryl, except that the arylene connects to at least two other chemical groups.
  • heterocycloalkyl refers to a cyclic group of atoms wherein all the bonds between the atoms in the ring are single bonds.
  • C x -C y heterocycloalkyl refers to a heterocycloalkyl with at least x atoms and no more than y atoms.
  • C 5 -C 6 heterocycloalkyl includes, without limitation, pyrrolidinyl and 1, 4-dioxanyl.
  • heterocycloalkylene has the same meaning as heterocycloalkyl, except that the heterocycloalkylene connects to at least two other chemical groups.
  • x-to y-membered heteroaryl refers to a cyclic group of atoms with at least x atoms and no more than y atoms.
  • 5-or 6-membered heteroaryl includes, without limitation, pyridinyl and furanyl.
  • the term “carbocycle” refers to a cycloalkyl or an aryl group. Likewise, the term “heterocycle” refers to a heterocycloalkyl or a heteroaryl group.
  • Possible atoms that make up the ring in heterocycloalkyl and heteroaryl groups, as well as derivatives thereof, include, without limitation, carbon, nitrogen, oxygen, and sulfur.
  • the term “optionally substituted” means the indicated group may be substituted or unsubstituted.
  • substituted refers to another chemical moiety that decorates the indicated group by replacement of one H atom.
  • ethanol is an example of ethane substituted with OH.
  • a group that is optionally substituted is optionally substituted by chloro, fluoro, bromo, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 7 cycloalkyl, C 6 -C 10 aryl, or 5-or 6-membered heteroaryl.
  • the terms “individual, ” “subject, ” and “patient” are used interchangeably herein to describe a mammal, including humans.
  • the individual is in need of treatment, for example, the individual may have been diagnosed with, or is suspected of having, a cancer.
  • references to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se.
  • description referring to "about X” includes description of "X” .
  • the term “about” a value or parameter means a range within 20%, in either direction, of the value or parameter recited.
  • the mole percentages of lipids and lipid stabilizers in lipid nanoparticles are calculated based on the total mole number of components in the lipid nanoparticles.
  • LNPs Lipid Nanoparticles
  • the lipid nanoparticles (LNPs) herein comprise a phospholipid containing a sterol moiety and optionally one or more of an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer.
  • the LNPs comprise a phospholipid containing a sterol moiety, an ionizable lipid, and a polymer conjugated lipid.
  • the LNPs comprise a phospholipid containing a sterol moiety, a polymer conjugated lipid, and a lipid stabilizer.
  • the LNPs comprise a phospholipid containing a sterol moiety, an ionizable lipid, and a lipid stabilizer.
  • the LNPs comprise a phospholipid containing a sterol moiety, an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer.
  • the phospholipid comprises from 1 to 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 2 to 25 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 3 to 20 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 10, 15, 20, 25, or 30 mol%of the total lipids in the LNP.
  • the phospholipid comprises from 10 to 15, 20, 25, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 15 to 20, 25, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 20 to 25 or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 25 to 30 mol%of the total lipids in the LNP.
  • the ionizable lipid comprises from 40 to 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 40 to 50, 60, 70, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 45 to 50, 60, 70, 75, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 50 to 60, 70, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 60 to 65, 70 or 80 mol%of the total lipids in the LNP.
  • the ionizable lipid comprises from 70 to 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipids comprise about 40, 50, 60, 70, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipids comprise about 50, 60, or 70 mol%of the total lipids in the LNP.
  • the LNP has a molar ratio of the ionizable lipid to the phospholipid of from 20 ⁇ 1 to 2 ⁇ 1. In some embodiments, the molar ratio is from 20 ⁇ 1 to 15 ⁇ 1, 10 ⁇ 1, 5 ⁇ 1, or 2 ⁇ 1. In some embodiments, the molar ratio is from 18 ⁇ 1 to 2.5 ⁇ 1. In some embodiments, the molar ratio is from 16 ⁇ 1 to 4 ⁇ 1. In some embodiments, the molar ratio is from 15 ⁇ 1 to 10 ⁇ 1, 5 ⁇ 1, or 2 ⁇ 1. In some embodiments, the molar ratio is from 10 ⁇ 1 to 5 ⁇ 1 or 2 ⁇ 1. In some embodiments, the ratio is from 5 ⁇ 1 to 2 ⁇ 1. In some embodiments, the ratio is from 15 ⁇ 1 to 5 ⁇ 1.
  • the polymer conjugated lipid has a molar ratio of 0.5 to 5 mol%of the total lipids in the LNP. In some embodiments, the polymer conjugated lipid has a molar ratio of 1 to 2 mol%of the total lipids in the LNP. In some embodiments, the polymer conjugated lipid has a molar ratio of 1.5 mol%of the total lipids in the LNP.
  • the molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 2 to 1 ⁇ 20. In some embodiments, the molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 3 to 1 ⁇ 18. In some embodiments, the molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 5 to 1 ⁇ 10.
  • the lipid stabilizer comprises from 5 to 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 5 to 10, 20, 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 8 to 20, 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 10 to 20, 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 20 to 30, 40, or 50 mol%of the total lipids in the LNP.
  • the lipid stabilizer comprises from 30 to 40 or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 40 to 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises about 5, 10, 20, 30, 40, or 50 mol%of the total lipids in the LNP.
  • any of the molar percentages or molar ratios described above for the sterol-containing phospholipid, the ionizable lipid, the polymer conjugated lipid, and the lipid stabilizer may be combined with each other in any embodiment describing the lipid composition of the LNPs described herein, such that every combination is contemplated as though each and every combination were specifically and individually disclosed.
  • the phospholipid comprises from 1 to 30 mol%, the ionizable lipid comprises from 40 to 80 mol%, the polymer conjugated lipid comprises from 1 to 2 mol%, and the lipid stabilizer comprises from 5 to 50 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 25 mol%, the ionizable lipid comprises from 45 to 75 mol%, the polymer conjugated lipid comprises from 1 to 2 mol%, and the lipid stabilizer comprises from 20 to 40%of the total lipids in the LNP.
  • the phospholipid comprises from 5 to 15 mol%, the ionizable lipid comprises from 40 to 60 mol%, the polymer conjugated lipid comprises from 1 to 2 mol%, and the lipid stabilizer comprises from 20 to 40 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises about 10 mol%, the ionizable lipid comprises about 50 mol%, the polymer conjugated lipid comprises about 38.5 mol%, and the lipid stabilizer comprises about 1.5 mol%of the total lipids in the LNP.
  • the LNP has a size from 20 to 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 40 to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 50 to 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 60 to 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm.
  • the size is from 70 to 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 80 to 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 90 to 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 100 to 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 110 to 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 120 to 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 120 to 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 120 to 130, 140, 150, 200, 250, or 300 nm.
  • the size is from 130 to 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 140 to 150, 200, 250, or 300 nm. In some embodiments, the size is from 150 to 200, 250, or 300 nm. In some embodiments, the size is from 200 to 300 nm. In some embodiments, the size is from 60 to 150 nm. In some embodiments, the size is from 65 to 90 nm. In some embodiments, the size is from 70 to 80 nm. In some embodiments, the size is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 nm. In some embodiments, the size is about 85 or about 90 nm. In some embodiments, the size is about 85 nm. In some embodiments, the size is about 90 nm. In some embodiments, the size is about 90 nm. In some embodiments, the size is about 90 nm. In some embodiments, the size is about 90 nm.
  • the LNPs herein comprise a phospholipid containing a sterol moiety.
  • the phospholipid containing a sterol moiety is any phospholipid that incorporates a sterol moiety into the lipid structure.
  • the sterol moiety is incorporated in place of one or more alkyl chains on the phospholipid.
  • the sterol moiety is incorporated into one alkyl chain of the phospholipid.
  • the sterol moiety is connected through the O atom of the sterol (e.g. converting the sterol moiety into the -O-atom of an ester connection to the remainder of the phospholipid) .
  • the sterol moiety is cholesterol.
  • the sterol moiety is a cholesterol moiety connected through O atom of the sterol (e.g. by converting the sterol O atom of cholesterol into the -O-atom of an ester connection to the remainder of the phospholipid) .
  • the phospholipid has a structure of selected from:
  • the LNPs comprise an ionizable lipid.
  • the ionizable lipid is a cationic lipid.
  • the cationic lipid is a cationic lipid described in International Patent Publication No. WO 2021/204175, the entirety of which is incorporated herein by reference.
  • the cationic lipid is a compound of Formula (01-I) :
  • G 1 and G 2 are each independently a bond, C 2 -C 12 alkylene, or C 2 -C 12 alkenylene, wherein one or more -CH 2 -in the alkylene or alkenylene is optionally replaced by -O-;
  • R 1 and R 2 are each independently C 6 -C 32 alkyl or C 6 -C 32 alkenyl
  • R a , R b , R d , and R e are each independently H, C 1 -C 24 alkyl, or C 2 -C 24 alkenyl;
  • R c and R f are each independently C 1 -C 32 alkyl or C 2 -C 32 alkenyl
  • G 3 is C 2 -C 24 alkylene, C 2 -C 24 alkenylene, C 3 -C 8 cycloalkylene, or C 3 -C 8 cycloalkenylene;
  • R 3 is -N (R 4 ) R 5 ;
  • R 4 is C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, 4-to 8-membered heterocyclyl, or C 6 -C 10 aryl; or R 4 , G 3 or part of G 3 , together with the nitrogen to which they are attached form a cyclic moiety;
  • R 5 is C 1 -C 12 alkyl or C 3 -C 8 cycloalkyl; or R 4 , R 5 , together with the nitrogen to which they are attached form a cyclic moiety;
  • x 0, 1, or 2;
  • alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
  • the cationic lipid is a compound of Formula (01-II) :
  • G 1 and G 2 are each independently a bond, C 2 -C 12 alkylene, or C 2 -C 12 alkenylene, wherein one or more -CH 2 -in the alkylene or alkenylene is optionally replaced by -O-;
  • R 1 and R 2 are each independently C 6 -C 32 alkyl or C 6 -C 32 alkenyl
  • R a , R b , R d , and R e are each independently H, C 1 -C 24 alkyl, or C 2 -C 24 alkenyl;
  • R c and R f are each independently C 1 -C 32 alkyl or C 2 -C 32 alkenyl
  • G 4 is a bond, C 1 -C 23 alkylene, C 2 -C 23 alkenylene, C 3 -C 8 cycloalkylene, or C 3 -C 8 cycloalkenylene;
  • R 3 is -N (R 4 ) R 5 ;
  • R 4 is C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, 4-to 8-membered heterocyclyl, or C 6 -C 10 aryl; or R 4 , G 3 or part of G 3 , together with the nitrogen to which they are attached form a cyclic moiety;
  • R 5 is C 1 -C 12 alkyl or C 3 -C 8 cycloalkyl; or R 4 and R 5 , together with the nitrogen to which they are attached form a cyclic moiety;
  • x 0, 1, or 2;
  • alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
  • the cationic lipid is a compound of Formula (01-I-B) , (01-I-B’) , (01-I-B”) , (01-I-C) , (01-I-D) , or (01-I-E) :
  • G 1 and G 2 are each independently C 3 -C 7 alkylene. In some embodiments, G 1 and G 2 are each independently C 5 alkylene. In some embodiments, G 3 is C 2 -C 4 alkylene. In some embodiments, G 3 is C 2 alkylene. In some embodiments, G 3 is C 4 alkylene.
  • R 3 has one of the following structures:
  • R 1 , R 2 , R c , and R f are each independently branched C 6 -C 32 alkyl or branched C 6 -C 32 alkenyl. In some embodiments, R 1 , R 2 , R c , and R f are each independently branched C 6 -C 24 alkyl or branched C 6 -C 24 alkenyl. In some embodiments, R 1 , R 2 , R c , and R f are each independently -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl.
  • R 1 , R 2 , R c , and R f are each independently -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 1 alkylene, and R 8 and R 9 are independently C 4 -C 8 alkyl.
  • the cationic lipid is a compound in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
  • the cationic lipid is a cationic lipid described in International Patent Application No. PCT/CN2022/072694, the entirety of which is incorporated herein by reference. In some embodiments, the cationic lipid is a compound of Formula (02-I) :
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R a , R b , R d , and R e are each independently H, C 1 -C 24 alkyl, or C 2 -C 24 alkenyl;
  • R c and R f are each independently C 1 -C 24 alkyl or C 2 -C 24 alkenyl
  • G 3 is C 2 -C 12 alkylene or C 2 -C 12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by a C 3 -C 8 cycloalkylene or C 3 -C 8 cycloalkenylene;
  • R 3 is -N (R 4 ) R 5 , -OR 6 , or-SR 6 ;
  • R 4 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • R 5 is H, C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • R 6 is hydrogen, C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, or C 6 -C 10 aryl;
  • x 0, 1, or 2;
  • each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.
  • the cationic lipid is a compound of Formula (02-II) :
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R a , R b , R d , and R e are each independently H, C 1 -C 24 alkyl, or C 2 -C 24 alkenyl;
  • R c and R f are each independently C 1 -C 24 alkyl or C 2 -C 24 alkenyl
  • G 3 is C 2 -C 12 alkylene or C 2 -C 12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by a C 3 -C 8 cycloalkylene or C 3 -C 8 cycloalkenylene;
  • R 3 is -N (R 4 ) R 5 , -OR 6 , or-SR 6 ;
  • R 4 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • R 5 is H, C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • R 6 is hydrogen, C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, or C 6 -C 10 aryl;
  • x 0, 1, or 2;
  • each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.
  • the compound is a compound of Formula (02-V-A) , (02-V-B) , (02-V-C) , (02-V-D) , (02-V-E) , (02-V-F) :
  • z is an integer from 2 to 12
  • x0 is an integer from 1 to 11;
  • y0 is an integer from 1 to 11;
  • x1 is an integer from 0 to 9;
  • y1 is an integer from 0 to 9;
  • x2 is an integer from 2 to 9;
  • x3 is an integer from 1 to 5;
  • x4 is an integer from 0 to 3;
  • y2 is an integer from 2 to 9;
  • y3 is an integer from 1 to 5;
  • y4 is an integer from 0 to 3;
  • z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 and y0 are independently 2 to 6. In some embodiments, x0 and y0 are independently 4 or 5. In some embodiments, x1 and y1 are independently 2 to 6. In some embodiments, x1 and y1 are independently 4 or 5. In some embodiments, x2 and y2 are independently an integer from 2 to 8. In some embodiments, x2 and y2 are independently 3, 5, or 7. In some embodiments, x3 and y3 are both 1. In some embodiments, x4 and y4 are independently 0 or 1.
  • R 1 and R 2 are independently straight C 6 -C 10 alkyl, or -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl or C 2 -C 10 alkenyl.
  • the compound is a compound of formula (02-VI-A) , (02-VI-B) , (02-VI-C) , (02-VI-D) , (02-VI-E) , or (02-VI-F) :
  • z is an integer from 2 to 12;
  • y is an integer from 2 to 12;
  • x0 is an integer from 1 to 11;
  • x1 is an integer from 0 to 9;
  • x2 is an integer from 2 to 5;
  • x3 is an integer from 1 to 5;
  • x4 is an integer from 0 to 3;
  • z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 is 4 or 5. In some embodiments, x1 is 4 or 5. In some embodiments, x2 is an integer from 2 to 5. In some embodiments, x2 is 3 or 5. In some embodiments, x3 is 0 or 1. In some embodiments, y is an integer from 2 to 6. In some embodiments, y is 5.
  • R 1 is straight C 6 -C 10 alkyl or -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl or C 2 -C 10 alkenyl.
  • R 2 and R f are each independently straight C 6 -C 18 alkyl, C 6 -C 18 alkenyl, or -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl or C 2 -C 10 alkenyl.
  • R d and R e are each independently H.
  • the compound is a compound in Table 2, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
  • the cationic lipid described herein is a cationic lipid described in International Patent Publication No. WO 2022/152109, the entirety of which is incorporated herein by reference.
  • the cationic lipid is a compound of Formula (03-I) :
  • G 1 and G 2 are each independently a bond, C 2 -C 12 alkylene, or C 2 -C 12 alkenylene, wherein one or more -CH 2 -in G 1 and G 2 is optionally replaced by -O-;
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R a , R b , R d , and R e are each independently H, C 1 -C 24 alkyl, or C 2 -C 24 alkenyl;
  • R c and R f are each independently C 1 -C 24 alkyl or C 2 -C 24 alkenyl
  • G 3 is C 2 -C 12 alkylene or C 2 -C 12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene, C 3 -C 8 cycloalkynylene, 4-to 8-membered heterocyclylene, C 6 -C 10 arylene, or 5-to 10-membered heteroarylene;
  • R 3 is hydrogen, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 3 -C 8 cycloalkynyl, 4-to 8-membered heterocyclyl, C 6 -C 10 aryl, or 5-to 10-membered heteroaryl; or R 3 and G 1 , or part of G 1 , together with the nitrogen to which they are attached form a cyclic moiety; or R 3 and G 3 or part ofG 3 , together with the nitrogen to which they are attached form a cyclic moiety;
  • R 4 is C 1 -C 12 alkyl or C 3 -C 8 cycloalkyl
  • x 0, 1, or 2;
  • n I or 2;
  • n 1 or 2;
  • alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
  • the cationic lipid is a compound of Formula (03-II-A) :
  • the cationic lipid is a compound of Formula (03-II-B) :
  • the cationic lipid is a compound of Formula (03-II-C) :
  • the cationic lipid is a compound of Formula (03-II-D) :
  • G 1 and G 2 are each independently C 2 -C 12 alkylene. In some embodiments, G 1 and G 2 are each independently C 5 alkylene. In some embodiments, G 3 is C 2 -C 6 alkylene.
  • R 3 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, or C 3 -C 8 cycloalkyl. In some embodiments, R 3 is C 3 -C 8 cycloalkyl. In some embodiments, R 3 is unsubstituted. In some embodiments, R 4 is substituted C 1 -C 12 alkyl. In some embodiments, R 4 is-CH 2 CH 2 OH.
  • R 1 , R 2 , R c , and R f are each independently straight C 6 -C 18 alkyl, straight C 6 -C 18 alkenyl, or -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl or C 2 -C 10 alkenyl.
  • R 1 , R 2 , R c , and R f are each independently straight C 7 -C 15 alkyl, straight C 7 -C 15 alkenyl, or-R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 0 -C 1 alkylene, and R 8 and R 9 are independently C 4 -C 8 alkyl or C 6 -C 10 alkenyl.
  • R a , R b , R d , and R e are each independently H.
  • the cationic lipid is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
  • the cationic lipid is a cationic lipid described in International Patent Application No. PCT/CN2022/094227, the entirety of which is incorporated herein by reference.
  • the cationic lipid is a compound of Formula (04-I) :
  • G 1 and G 2 are each independently a bond, C 2 -C 12 alkylene, or C 2 -C 12 alkenylene;
  • R 1 and R 2 are each independently C 5 -C 32 alkyl or C 5 -C 32 alkenyl
  • R a , R b , R d , and R e are each independently H, C 1 -C 24 alkyl, or C 2 -C 24 alkenyl;
  • R c and R f are each independently C 1 -C 32 alkyl or C 2 -C 32 alkenyl
  • R 0 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • G 3 is C 2 -C 12 alkylene or C 2 -C 12 alkenylene
  • R 4 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • R 5 is C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • x 0, 1, or 2;
  • s is 0 or 1;
  • each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene, is independently optionally substituted.
  • the cationic lipid is a compound of Formula (04-III) :
  • R 1 and R 2 are each independently C 5 -C 32 alkyl or C 5 -C 32 alkenyl
  • R 0 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • G 3 is C 2 -C 12 alkylene or C 2 -C 12 alkenylene
  • G4 is C 2 -C 12 alkylene or C 2 -C 12 alkenylene
  • R 3 is -N (R 4 ) R 5 or -OR 6 ;
  • R 4 is C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl;
  • R 5 is C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, C 6 -C 10 aryl, or 4-to 8-membered heterocycloalkyl; or R 4 and R 5 , together with the nitrogen to which they are attached form a cyclic moiety;
  • R 6 is hydrogen, C 1 -C 12 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkenyl, or C 6 -C 10 aryl; and wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently optionally substituted.
  • the cationic lipid is a compound of Formula (04-IV) :
  • G 3 is C 2 -C 4 alkylene. In some embodiments, G4 is C 2 -C 4 alkylene.
  • R 0 is C 1 -C 6 alkyl.
  • R 3 is -OH.
  • R 3 is -N (R 4 ) R 5 .
  • R 4 is C 3 -C 8 cycloalkyl.
  • R 4 is unsubstituted.
  • R 5 is -CH 2 CH 2 OH.
  • R 1 and R 2 are each independently branched C 6 -C 24 alkyl or branched C 6 -C 24 alkenyl.
  • R 1 and R 2 are each independently -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 1 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl or C 2 -C 10 alkenyl.
  • R 1 is straight C 6 -C 24 alkyl and R 2 is branched C 6 -C 24 alkyl.
  • R 1 is straight C 6 -C 24 alkyl and R 2 is -R 7 -CH (R 8 ) (R 9 ) , wherein R 7 is C 1 -C 5 alkylene, and R 8 and R 9 are independently C 2 -C 10 alkyl.
  • the cationic lipid is a compound in Table 4, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
  • the cationic lipid contained in the particles or compositions provided herein is a cationic lipid described in U.S. Patent Nos. US 10442756B2, US9868691B2, or US9868692B2, all of which are incorporated herein by reference.
  • the cationic lipid is a compound Formula (05-I) :
  • 1 is selected from 1, 2, 3, 4, and 5;
  • n is selected from 5, 6, 7, 8, and 9;
  • M 1 is a bond or M′
  • M and M′ are independently selected from -C (O) O-, -OC (O) -, -C (O) N (R′) -, -P (O) (OR′) O-, -S-S-, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are both C 1 -C14 alkyl, or C 2 -C14 alkenyl
  • R 8 is selected from the group consisting of C 3 -C 6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1 -C 6 alkyl, -OR, -S (O) 2 R, -S (O) 2 N (R) 2 , C 2 -C 6 alkenyl, C 3 -C 6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1 -C 3 alkyl, C 2 -C 3 alkenyl, and H;
  • R′ is a linear alkyl
  • the cationic lipid is SM102 or Lipid 5:
  • the cationic lipid is a cationic lipid described in U.S. Patent No. US 10166298B2, the entire teachings of which are incorporated herein by reference.
  • the cationic lipid is a compound of Formula (06-I) :
  • G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene;
  • R a is H or C 1 -C 12 alkyl
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 4 is C 1 -C 12 alkyl
  • R 5 is H or C 1 -C 6 alkyl
  • the cationic lipid is a compound of Table 5, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
  • the cationic lipids of the present disclosure are the same as those disclosed in International Application Publication No. WO 2010/144740, the entire teachings of which are incorporated herein by reference.
  • the cationic lipid is a compound represented by Formula (07-I) , also named as compound 07-I:
  • the ionizable lipid used in the LNPs according to the present invention is selected from
  • y and z are each independently an integer from 4 to 6,
  • s is an integer from 2 to 4,
  • t is an integer from 1 to 3
  • R 1 and R 2 are each independently C 12 -C 22 alkyl
  • R 4 is C 3 -C 8 cycloalkyl
  • R 6 is hydrogen or hydroxyl
  • 1 is selected from 1, 2, 3, 4, and 5;
  • n is selected from 5, 6, 7, 8, and 9;
  • M 1 is -C (O) O-;
  • R 4 is - (CH 2 ) n OH, and n is selected from 1, 2, 3, 4, or 5;
  • M is -OC (O) -
  • R 2 and R 3 are both C 6-10 alkyl
  • G 1 and G 2 are each independently unsubstituted C 4 -C 8 alkylene
  • G 3 is C 3 -C 8 alkylene
  • R 1 and R 2 are each independently C 12 -C 22 alkyl
  • R 3 is H or OH
  • R 1 and R 2 are each independently C 6 -C 24 alky
  • R 3 is-OR 6 ;
  • R 6 is hydrogen
  • z is an integer from 2 to 12;
  • x1 is an integer from 0 to 9;
  • y1 is an integer from 0 to 9;
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 3 is -OR 6 ;
  • R 6 is hydrogen
  • z is an integer from 2 to 12;
  • y is an integer from 2 to 12;
  • x1 is an integer from 2 to 5;
  • R 1 and R 2 are each independently C 6 -C 24 alkyl
  • R 3 is -OR 6 ;
  • R 6 is hydrogen
  • z is an integer from 2 to 12;
  • x2 is an integer from 2 to 9;
  • x4 is an integer from 0 to 3;
  • y2 is an integer from 2 to 9;
  • y4 is an integer from 0 to 3
  • G 1 and G 2 are each independently C 3 -C 8 alkylene
  • R 1 is independently C 6 -C 24 alkyl
  • R 2 is independently C 6 -C 24 alkyl
  • G 3 is C 2 -C 12 alkylene
  • R 3 is C 3 -C 8 cycloalkyl
  • R 4 is C 1 -C 4 hydroxylalkyl
  • n 1 or 2;
  • n 1 or 2
  • the ionizable lipid used in the LNPs according to the present invention is selected from the following compounds:
  • the LNP comprises a polymer conjugated lipid.
  • Polymers that can be incorporated into polymer conjugated lipids include polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • a polymer may include poly (caprolactone) (PCL) , ethylene vinyl acetate polymer (EVA) , poly (lactic acid) (PLA) , poly (L-lactic acid) (PLLA) , poly (glycolic acid) (PGA) , poly (lactic acid-co-glycolic acid) (PLGA) , poly (L-lactic acid-co-glycolic acid) (PLLGA) , poly (D, L-lactide) (PDLA) , poly (L-lactide) (PLLA) , poly (D, L-lactide-co-caprolactone) , poly (D, L-lactide-co-caprolactone-co-glycolide) , poly (D, L-lactide-co-PEO-co-D, L-lactide) , poly (D, L-lactide-co-PPO-co-D, L-lactide) , polyalkyl cyanoacralate, poly (
  • the polymer conjugated lipid is a PEGylated lipid (PEG lipids) .
  • PEG lipids PEGylated lipids
  • a polymer conjugated lipid component in an LNP can improve colloidal stability and/or reduce protein absorption of the nanoparticles.
  • Exemplary PEGylated lipids that can be used in connection with the present disclosure include but are not limited to PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • the PEG-conjugated lipid may be a PEG-modifiedphosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or a PEG-modified dialkylglycerol.
  • the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, Ceramide-PEG2000, or ChoI-PEG2000.
  • the PEGylated lipid is a PEGylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a pegylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2’, 3’-di (tetradecanoyloxy) propyl-1-O- ( ⁇ -methoxy (polyethoxy) ethyl) butanedioate (PEG-S-DMG) , a pegylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as ⁇ -methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecanoxy) prop
  • the PEGylated lipid is present in a concentration ranging from 1.0 to 2.5 molar percent. In some embodiments, the polymer conjugated lipid is present in a concentration of about 1.7 molar percent. In some embodiments, the polymer conjugated lipid is present in a concentration of about 1.5 molar percent.
  • the molar ratio of the ionizable lipid to the polymer conjugated lipid ranges from about 20 ⁇ 1 to about 100 ⁇ 1. In some embodiments, the molar ratio of the ionizable lipid to polymer conjugated lipid ranges from about 25 ⁇ 1 to about 80 ⁇ 1 . In some embodiments, the molar ratio of the ionizable lipid to polymer conjugated lipid ranges from about 30 ⁇ 1 to about 60 ⁇ 1. In some embodiments, the molar ratio of the ionizable lipid to polymer conjugated lipid ranges from about 30 ⁇ 1 to about 50 ⁇ 1.
  • the PEGylated lipid has the following Formula:
  • R 12 and R 13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w has a mean value ranging from 30 to 60.
  • R 12 and R 13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • the average w ranges from 42 to 55, for example, the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some embodiments, the average w is about 49.
  • the PEGylated lipid has the following Formula:
  • the LNPs comprise a lipid stabilizer.
  • the lipid stabilizer comprises a sterol.
  • the lipid stabilizer comprises a corticosteroid.
  • the lipid stabilizer comprises two or more components.
  • the lipid stabilizer comprises a corticosteroid and a sterol.
  • the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, and brassicasterol.
  • the corticosteroid is selected from the group consisting ofprednisolone, dexamethasone, prednisone, and hydrocortisone.
  • the lipid stabilizer comprises tomatidine, tomatine, ursolic acid, or alpha-tocopherol.
  • the lipid stabilizer comprises one or more compounds selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, prednisolone, dexamethasone, prednisone, hydrocortisone, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.
  • the lipid stabilizer is cholesterol.
  • the LNPs disclosed herein further comprise a therapeutic payload.
  • the payload can be any substance or compound that has a therapeutic or prophylactic effect.
  • the therapeutic payload is a small molecule, a cytotoxin, a radioactive ion, a chemotherapeutic compound, a vaccine, or a compound that elicits an immune response.
  • the LNPs disclosed herein comprise a nucleic acid.
  • the nucleic acid is a DNA.
  • the DNA is catalytic DNA, plasmid DNA, aptamer, or complementary DNA (cDNA) .
  • the nucleic acid is an RNA.
  • the RNA is a messenger RNA (mRNA) , antisense oligonucleotide, microRNA (miRNA) , miRNA inhibitor (e.g., antagomir or antimir) , messenger-RNA-interfering complementary RNA (micRNA) , multivalent RNA, dicer substrate RNA (dsRNA) , small hairpin RNA (shRNA) , antisense RNA, transfer RNA (tRNA) , asymmetrical interfering RNA (aiRNA) , a ribozyme, an aptamer, or a vector.
  • the RNA is an mRNA hybrid.
  • the nucleic acid is an mRNA.
  • the mRNA encodes a protein.
  • the protein is an antibody.
  • the antibody is a bispecific antibody.
  • the LNPs comprise an RNAi agent or RNAi-inducing agent.
  • the weight ratio of the ionizable lipid to the therapeutic payload is from 5 ⁇ 1 to 20 ⁇ 1.
  • compositions comprising Lipid Nanoparticles (LNPs)
  • the present disclosure is inclusive with respect to compositions comprising the LNPs described herein.
  • the composition comprises a plurality of LNPs.
  • the plurality of LNPs have a polydispersity index (PDI) of 0.001 to 0.2.
  • the plurality of LNPs have a polydispersity index (PDI) of 0.001 to 0.1.
  • the LNPs have a PDI of 0.005 to 0.05.
  • some of the LNPs of the LNP composition comprise mRNA.
  • the mRNA encapsulation efficiency (EE%-i.e., the percentage of individual LNPs in the composition that encapsulate the mRNA) of the LNP composition is from 70%to 100%.
  • the EE%of the LNP composition is from 80 to 95%.
  • the EE%of the LNP composition is from 85 to 95%.
  • the EE%of the LNP composition is from 90 to 95%.
  • the EE% is 80%or greater.
  • the EE% is 85%or greater.
  • the LNP composition is able to increase protein expression compared to comparable LNP compositions that do not comprise a steroid-containing phospholipid.
  • any of the molar percentages or molar ratios described above for the sterol-containing phospholipid, the ionizable lipid, the polymer conjugated lipid, and the lipid stabilizer may be combined with each other in any embodiment describing the composition of LNPs described herein, such that every combination is contemplated as though each and every combination were specifically and individually disclosed.
  • the LNPs here can be made according to the methods that are well known in the art.
  • the method comprises solubilizing the lipid components (e.g., a phospholipid, an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer) in a solvent.
  • the method comprises the steps:
  • lipid components e.g., a phospholipid, an ionizable lipid, a polymer conjugated lipid, and optionally a lipid stabilizer
  • the solvent in step (a) is a polar solvent. In some embodiments, the solvent in step (a) is an alcohol solvent. In some embodiments, the solvent in step (a) is methanol, ethanol, n-propanol, or isopropanol. In some embodiments, the solvent in step (a) is ethanol.
  • the solvent in step (b) is an aqueous solvent. In some embodiments, the solvent in step (b) is an aqueous buffer. In some embodiments, the solvent in step (b) is a citrate buffer. In some embodiments, the citrate buffer has a citrate concentration of 5 to 100 mM.In some embodiments, the citrate buffer has a citrate concentration of 10 to 50 mM. In some embodiments, the aqueous solvent of step (b) has a pH of 2-6. In some embodiments, the aqueous solvent of step (b) has a pH of 3-5.
  • the mixing of step (c) occurs with a weight ratio of lipid ⁇ mRNA of 10 ⁇ 1 to 30 ⁇ 1. In some embodiments, the mixing of step (c) occurs at a volume ratio of lipid ⁇ mRNA of 1 ⁇ 1 to 1 ⁇ 5. In some embodiments, the volume ratio is 1 ⁇ 2 to 1 ⁇ 4. In some embodiments, the volume ratio is about 1 ⁇ 3. In some embodiments, the mixing of step (c) is performed with a microfluidic apparatus. In some embodiments, the microfluidic apparatus has a flow rate of 9 to 30 mL/min.
  • the mRNA mixture has an mRNA concentration from 1 to 3, 5, 7, 10, 12, 15, 20, 30, 40 or 50 mM. In some embodiments, the concentration is from 3 to 5, 7, 10, 12, 15, 20, or 30mM. In some embodiments, the concentration is from 5 to 7, 10, 12, 15, 20, or 30 mM. In some embodiments, the concentration is from 7 to 10, 12, 15, 20, or 30 mM. In some embodiments, the concentration is from 10 to 12, 15, 20, or 30 mM. In some embodiments, the concentration is from 12 to 15, 20, or 30 mM. In some embodiments, the concentration is from 15 to 20 or 30 mM. In some embodiments, the concentration is from 20 to 30 mM. In some embodiments, the concentration is about 5, 7, 10, 12, 15, or 20 mM.
  • the concentration is about 5, 7, 10, 12, or 15 mM. In some embodiments, the concentration is about 10 mM. In some embodiments, the concentration is about 12 mM. In some embodiments, the concentration is about 15 mM. In some embodiments, the concentration is about 20 mM.
  • the method further comprises a step (d) :
  • the sterile filter is a 0.2 ⁇ m sterile filter.
  • the present disclosure is inclusive with respect to potentially useful methods that make use of the LNPs described herein.
  • a method for expressing protein in a cell comprising introducing an LNP or composition thereof, as described above, to a cell.
  • the cell is a mammalian cell.
  • the LNP or composition thereof is administered systemically to a mammal.
  • the mammal is a human.
  • the LNP composition is able to increase protein expression compared to comparable LNP compositions that do not comprise a steroid-containing phospholipid.
  • Kits comprising a phospholipid containing a sterol moiety
  • kits comprising a phospholipid containing a sterol moiety and packaging for said phospholipid.
  • the kit further comprises an ionizable lipid.
  • the kit further comprises a polymer conjugated lipid.
  • the kit further comprises a lipid stabilizer.
  • the kit further comprises an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer.
  • the ionizable lipid is a cationic lipid.
  • the polymer conjugated lipid is a PEGylated lipid.
  • the lipid stabilizer is cholesterol.
  • the kit further comprises a cationic lipid, a PEGylated lipid, and cholesterol.
  • any embodiment of the compounds provided herein, as set forth above, and any specific substituent and/or variable in the compound provided herein, as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of the compounds to form embodiments not specifically set forth above.
  • substituents and/or variables may be listed for any particular group or variable, it is understood that each individual substituent and/or variable may be deleted from the particular embodiment and/or claim and that the remaining list of substituents and/or variables will be considered to be within the scope of embodiments provided herein.
  • Embodiment 1 A lipid nanoparticle (LNP) comprising
  • Embodiment 2 The LNP of embodiment 1, wherein the phospholipid has a structure selected from:
  • Embodiment 3 The LNP of embodiment 1 or 2, wherein the phospholipid has the structure:
  • Embodiment 4 The LNP of embodiment 1 or 2, wherein the phospholipid has the structure:
  • Embodiment 5 The LNP of any one of embodiments 1 to 4, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20 ⁇ 1 to 2 ⁇ 1.
  • Embodiment 6 The LNP of embodiment 5, wherein the molar ratio of the ionizable lipid to the phospholipid is from 15 ⁇ 1 to 5 ⁇ 1.
  • Embodiment 7 The LNP of any one of embodiments 1 to 4, wherein the ionizable lipid comprises from 40 to 80 mol%of a total amount of lipids in the LNP.
  • Embodiment 8 The LNP of embodiment 7, wherein the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP.
  • Embodiment 9 The LNP of any one of embodiments 1 to 8, wherein the ionizable lipid is a cationic lipid.
  • Embodiment 10 The LNP of any one of embodiments 1 to 8, wherein the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
  • Embodiment 11 The LNP of any one of embodiments 1 to 10, wherein the polymer conjugated lipid comprises from 1 to 2%of a total amount of lipids in the LNP.
  • Embodiment 12 The LNP of embodiment 11, wherein the polymer conjugated lipid comprises 1.5%of the total amount of lipids in the LNP.
  • Embodiment 13 The LNP of any one of embodiments 1 to 12, wherein the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 5 to 1 ⁇ 10.
  • Embodiment 14 The LNP of any one of embodiments 1 to 13, wherein the polymer conjugated lipid is a PEGylated lipid.
  • Embodiment 15 The LNP of any one of embodiments 1 to 13, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
  • R 12 and R 13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w is an integer ranging from 30 to 60.
  • Embodiment 16 The LNP of any one of embodiments 1 to 15, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
  • w is an integer ranging from 30 to 60.
  • Embodiment 17 The LNP of embodiment 15 or 16, wherein w is an integer ranging from 45 to 55.
  • Embodiment 18 The LNP of embodiment 15 or 16, wherein w is about 49.
  • Embodiment 19 The LNP of any one of embodiments 1 to 14, wherein the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
  • Embodiment 20 The LNP of any one of embodiments 1 to 19, further comprising a lipid stabilizer.
  • Embodiment 21 The LNP of embodiment 20, wherein the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10 ⁇ 1 to 1 ⁇ 4.
  • Embodiment 22 The LNP of embodiment 21, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 5 ⁇ 1 to 1 ⁇ 1.
  • Embodiment 23 The LNP of embodiment 21, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 4 ⁇ 1 to 3 ⁇ 1.
  • Embodiment 24 The LNP of any one of embodiments 20 to 23, wherein the lipid stabilizer comprises from 5 to 50 mol%of a total amount of lipids in the LNP.
  • Embodiment 25 The LNP of embodiment 24, wherein the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP.
  • Embodiment 26 The LNP of embodiment 24, wherein the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
  • Embodiment 27 The LNP of any one of embodiments 1 to 26, wherein the phospholipid comprises from 1 to 30 mol%of a total amount of lipids in the LNP.
  • Embodiment 28 The LNP of embodiment 27, wherein the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP.
  • Embodiment 29 The LNP of embodiment 27, wherein the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP.
  • Embodiment 30 The LNP of embodiment 27, wherein the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP.
  • Embodiment 31 The LNP of embodiment 27, wherein the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP.
  • Embodiment 32 The LNP of any one of embodiments 1 to 31, wherein the LNP has a size from 50 nm to 150 nm, as determined using dynamic light scattering.
  • Embodiment 33 The LNP of embodiment 32, wherein the size is from 60 nm to 140 nm.
  • Embodiment 34 The LNP of embodiment 32, wherein the size is from 80 nm to 100 nm.
  • Embodiment 35 The LNP of embodiment 32, wherein the size is from 85 nm to 95 nm.
  • Embodiment 36 The LNP of any one of embodiments 1 to 35, wherein the LNP encapsulates mRNA.
  • Embodiment 37 A composition comprising lipid nanoparticles (LNPs) , wherein each LNP is an LNP of any one of embodiments 1 to 36.
  • LNPs lipid nanoparticles
  • Embodiment 38 The composition of embodiment 37, wherein at least 80%of the LNPs encapsulate mRNA.
  • Embodiment 39 The composition of embodiment 37, wherein at least 85%of the LNPs encapsulate mRNA.
  • Embodiment 40 A method for expressing protein in a cell, comprising introducing the LNP of embodiment 36 or the composition of embodiment 38 or 39, to the cell.
  • Embodiment 41 The method of embodiment 40, wherein the cell is a mammalian cell.
  • Embodiment 42 A method for delivering a protein to a subject, comprising administering the LNP of embodiment 36 or the composition of embodiment 38 or 39 to the individual, wherein the mRNA encodes the protein.
  • Embodiment 43 The method of embodiment 42, wherein the LNP or the composition is administered systemically.
  • Embodiment 44 The method of embodiment 42, wherein the subject is a mammal.
  • Embodiment 45 The method of embodiment 42, wherein the subject is a human.
  • Embodiment 46 A lipid nanoparticle (LNP) comprising a phospholipid, wherein the phospholipid has a structure selected from:
  • Embodiment 47 The LNP of embodiment 46, wherein the phospholipid has the structure:
  • Embodiment 48 The LNP of embodiment 46, wherein the phospholipid has the structure:
  • Embodiment 49 The LNP of any one of embodiments 46-48, further comprising an ionizable lipid.
  • Embodiment 50 The LNP of embodiment 49, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20 ⁇ 1 to 2 ⁇ 1.
  • Embodiment 51 The LNP of embodiment 50, wherein the molar ratio of ionizable lipid to phospholipid is from 15 ⁇ 1 to 5 ⁇ 1.
  • Embodiment 52 The LNP of embodiment 49, wherein the ionizable lipid comprises from 40 to 80 mol%of a total amount oflipids in the LNP.
  • Embodiment 53 The LNP of embodiment 52, wherein the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP.
  • Embodiment 54 The LNP of any one of embodiments 49 to 53, wherein the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
  • Embodiment 55 The LNP of any one of embodiments 49 to 54, wherein the ionizable lipid is a cationic lipid.
  • Embodiment 56 The LNP of any one of embodiments 46 to 55, further comprising a polymer conjugated lipid.
  • Embodiment 57 The LNP of embodiment 56, wherein the polymer conjugated lipid comprises from 1 to 2%of a total amount oflipids in the LNP.
  • Embodiment 58 The LNP of embodiment 57, wherein the polymer conjugated lipid comprises 1.5%of the total amount of lipids in the LNP.
  • Embodiment 59 The LNP of any one of embodiments 46 to 58, wherein the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1 ⁇ 5 to 1 ⁇ 10.
  • Embodiment 60 The LNP of any one of embodiments 56 to 59, wherein the polymer conjugated lipid is a PEGylated lipid.
  • Embodiment 61 The LNP of any one of embodiments 56 to 60, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
  • R 12 and R 13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds;
  • w is an integer ranging from 30 to 60.
  • Embodiment 62 The LNP of any one of embodiments 56 to 61, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
  • w is an integer ranging from 30 to 60.
  • Embodiment 63 The LNP of embodiment 61 or 62, wherein w is an integer ranging from 45 to 55.
  • Embodiment 64 The LNP of embodiment 61 or 62, wherein w is about 49.
  • Embodiment 65 The LNP of any one of embodiments 56 to 60, wherein the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
  • Embodiment 66 The LNP of any one of embodiments 46 to 65, further comprising a lipid stabilizer.
  • Embodiment 67 The LNP of embodiment 66, wherein the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10 ⁇ 1 to 1 ⁇ 4.
  • Embodiment 68 The LNP of embodiment 67, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 5 ⁇ 1 to 1 ⁇ 1.
  • Embodiment 69 The LNP of embodiment 67, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 4 ⁇ 1 to 3 ⁇ 1.
  • Embodiment 70 The LNP of any one of embodiments 66 to 69, wherein the lipid stabilizer comprises from 5 to 50 mol%of a total amount of lipids in the LNP.
  • Embodiment 71 The LNP of embodiment 70, wherein the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP.
  • Embodiment 72 The LNP of embodiment 70, wherein the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
  • Embodiment 73 The LNP of any one of embodiments 46 to 72, wherein the phospholipid comprises from 1 to 30 mol%of a total amount of lipids in the LNP.
  • Embodiment 74 The LNP of embodiment 73, wherein the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP.
  • Embodiment 75 The LNP of embodiment 73, wherein the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP.
  • Embodiment 76 The LNP of embodiment 73, wherein the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP.
  • Embodiment 77 The LNP of embodiment 73, wherein the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP.
  • Embodiment 78 The LNP of any one of embodiments 46 to 77, wherein the LNP has a size from 50 nm to 150 nm, as determined using dynamic light scattering.
  • Embodiment 79 The LNP of embodiment 78, wherein the size is from 60 nm to 140 nm.
  • Embodiment 80 The LNP of embodiment 78, wherein the size is from 80 nm to 100 nm.
  • Embodiment 81 The LNP of embodiment 78, wherein the size is from 85 nm to 95 nm.
  • Embodiment 82 The LNP of any one of embodiments 46 to 81, wherein the LNP encapsulates mRNA.
  • Embodiment 83 A composition comprising lipid nanoparticles (LNPs) , wherein each LNP is an LNP of any one of embodiments 46 to 82.
  • LNPs lipid nanoparticles
  • Embodiment 84 The composition of embodiment 83, wherein at least 80%of the LNPs encapsulate mRNA.
  • Embodiment 85 The composition of embodiment 83, wherein at least 85%of the LNPs encapsulate mRNA.
  • Embodiment 86 A method for expressing protein in a cell, comprising introducing the LNP of embodiment 82 or the composition of embodiment 84 or 85, to the cell.
  • Embodiment 87 The method of embodiment 86, wherein the cell is a mammalian cell.
  • Embodiment 88 A method for delivering a protein to a subject, comprising administering the LNP of embodiment 82 or the composition of embodiment 84 or 85 to the individual, wherein the mRNA encodes the protein.
  • Embodiment 89 The method of embodiment 88, wherein the LNP or the composition is administered systemically.
  • Embodiment 90 The method of embodiment 88, wherein the subject is a mammal.
  • Embodiment 91 The method of embodiment 88, wherein the subject is a human.
  • HPLC purification is carried out on a Waters 2767 equipped with a diode array detector (DAD) on an Inertsil Pre-C8 OBD column, generally with water containing 0.1%trifluoroacetic acid (TFA) as solvent A and acetonitrile as solvent B.
  • DAD diode array detector
  • TFA trifluoroacetic acid
  • LCMS analysis is conducted on a Shimadzu (LC-MS2020) System. Chromatography is performed on a SunFire C18, generally with water containing 0.1%formic acid as solvent A and acetonitrile containing 0.1%formic acid as solvent B.
  • OChemsPC refers to the following compound:
  • PChemsPC refers to the following compound:
  • DChemsPC refers to the following compound:
  • DSPC distearoylphosphatidylcholine.
  • compound 01-1 refers to compound 01-1 in Table 1.
  • Cho is an abbreviation for cholesterol.
  • DMG-PEG refers to 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.
  • ALC-0315 refers to compound 06-1, MC3 refers to compound 07-I.
  • Compound 02-1 was prepared according to the scheme below.
  • Compound 02-3 was prepared in analogous fashion as Compound 02-1, using corresponding starting material.
  • Compound 02-2 was prepared according to the scheme below.
  • Compound 02-4 was prepared according to the scheme below.
  • Compound 02-9 was prepared according to the scheme below.
  • Compound 02-14 was prepared in analogous fashion as Compound 02-9, using corresponding starting material.
  • Compound 02-10 was prepared according to the scheme below.
  • Compound 02-11 was prepared in analogous fashion as Compound 02-10, using corresponding starting material.
  • Compound 02-12 was prepared according to the scheme below.
  • Compound 02-20 was prepared according to the scheme below.
  • Compound 04-1 was prepared according to the scheme below.
  • Compound 04-2 was prepared according to the scheme below.
  • Compound 04-7 was prepared according to the scheme below.
  • Compound 04-8 was prepared according to the scheme below.
  • Compound 04-65 was prepared according to the scheme below.
  • Compound 04-68 was prepared according to the scheme below.
  • Example 15 Initial screening of LNPs comprising a steroid containing phospholipid
  • the specified amounts of the lipid components were solubilized in ethanol at the specified molar ratios (see Table 6) .
  • the LNPs were prepared at a total lipid to mRNA weight ratio of approximately 10: 1 to 30: 1 by mixing the ethanolic lipid solution with the aqueous mRNA solution at a volume ratio of 1: 3 using a microfluidic apparatus, total flow rate ranging from 9-30 mL/min. Ethanol was thereby removed and replaced by Dulbecco′sphosphate-buffered saline (DPBS) using dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 ⁇ m sterile filter.
  • DPBS Dulbecco′sphosphate-buffered saline
  • Lipid nanoparticle size were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) using a 173° backscatter detection mode.
  • the encapsulation efficiency of lipid nanoparticles was determined using a Quant-it Ribogreen RNA quantification assay kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.
  • lipid nanoparticle formulations were diluted 20-fold in PBS and transferred 1 mL in measurement cuvette.
  • the LNP encapsulation efficiency (EE%) was determined using a Quant-it RiboGreen RNA assay kit, LNP formulations were diluted to 0.5 ⁇ g/mL in Tris-EDTA and 0.1%Triton respectively.
  • ribogreen reagent were diluted 200-fold with Tris-EDTA buffer and mix at the same volume as diluted LNP formulation.
  • Fluorescence intensity was measured at room temperature in a Molecular Devices Spectramax iD3 spectrometer using excitation and emission wavelengths of 488 nm and 525 nm. EE% was calculated based on the ratio of encapsulated to total RNA fluorescence intensity.
  • Lipid nanoparticles encapsulating human erythropoietin (hEPO) mRNA were prepared as described above, and systemically administered to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at 0.5mg/kg dose by tail vein injection. Mice were euthanized by CO 2 overdoses at 6 hours post administration, and blood samples were taken for hEPO measurement. Particularly, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 °C, snap-frozen and stored at -80 °C for analysis. The serum hEPO level was measured using an ELISA assay carried out using a commercial kit (DEP00, R&D systems) according to manufacturer's instructions. The hEPO expression levels ( ⁇ g/ml) measured from the tested group are plotted in FIG. 1 and summarized in Table 6.
  • Example 16 Expression levels of loaded LNPs comprising a steroid containing phospholipid
  • LNPs containing 45-60 mol%compound 01-1 resulted in the highest protein expression level.
  • PChemsPC LNPs 60 -75 mol%compound 01-1 resulted in the highest protein expression level.
  • Example 17 Screen of %PChemsPC for optimal protein expression
  • PChemsPC LNPs tested 5-10 mol%PChemsPC had the highest protein expression level.
  • the optimal amount of PChemsPC varied with the amount of compound 01-1.
  • the mol%of PChemsPC exceeded 15%, protein expression appeared to decrease.
  • Example 18 Screen of LNP compositions with a steroid containing phospholipid for optimal protein expression
  • Example 19 Tissue-specific Expression of Nucleic Acid Molecules Delivered in LNP formulations.
  • LNP formulations listed in Table 10 containing mRNA encoding luciferase were prepared as described in Example 15.
  • mice Each formulation was systematically administered to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at a 0.25 mg/kg dose by tail vein injection. After 6 hours, the mice were subcutaneously administered with XenoLight D-luciferin (potassium salt) , a substrate ofluciferase that catalyze the production of luminescence. The mice were subsequently euthanized by CO 2 overdoses 15 min thereafter. Mice tissues were harvested and placed in a luminescence imaging scanner to measure the expression level of luciferase in each tissue. The luminescence levels measured from harvest liver tissues were plotted in FIG. 4, showing the mean value and standard deviation (SD) of at least five repeated tested animals for each group.
  • SD standard deviation
  • LNPs composed of a steroid containing phospholipid e.g. PChemsPC, OChemsPC, DChemsPC
  • PChemsPC steroid containing phospholipid
  • DSPC LNP leads to 96%liver distribution with around 3%spleen distribution, while steroid containing phospholipid LNP shows 98 -99%liver distribution, with 0.4%spleen distribution. It can be seen that steroid-modified phospholipid LNP reveals better liver-tropism.
  • Example 20 Characterization of sterol-modified phospholipid LNP with different ionizable lipids
  • LNP formulations containing PChemsPC were prepared with different ionizable lipids.
  • the LNP formulations were composed of a ionizable lipid at a molar ratio of 65%, a PChemsPC lipid at a molar ratio of 10%, a cholesterol-based lipid at a molar ratio of 23.5%and a PEGylated lipid at a molar ratio of 1.5%.
  • Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15.
  • the effect of replacement of DSPC with PChemsPC on in-vivo protein expression level was also studied.
  • the LNP formulations were composed of an ionizable lipid at a molar ratio of 50%-65%, a phospholipid at a molar ratio of 10%, a cholesterol-based lipid at a molar ratio of 23.5%-38.5%and a PEGylated lipid at a molar ratio of 1.5%.
  • LNP characterizations were listed in Table 12.
  • Example 21 Characterization of in vivo serum cytokines post injection of a steroid containing phospholipid LNP
  • LNP injection will lead to significant increase of pro-inflammatory cytokines, such as inteleukin-6 (IL-6) , tumor necrosis factor-alpha (TNF- ⁇ ) , interferon-gamma (INF- ⁇ ) , interferon-alpha (IFN- ⁇ ) , which can cause innate immune response and leads to undesirable side effects.
  • IL-6 inteleukin-6
  • TNF- ⁇ tumor necrosis factor-alpha
  • IFN- ⁇ interferon-gamma
  • IFN- ⁇ interferon-alpha
  • Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15, and systemically administered to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at 0.5mg/kg dose by tail vein injection. Mice were euthanized by CO 2 overdoses at 6 hours post administration, and blood samples were taken for cytokines measurement. Particularly, sera were separated from total blood by centrifugation at 5000g for 10 minutes at 4 °C, snap-frozen and stored at -80 °C for analysis.
  • hEPO human erythropoietin
  • DSPC LNPs were composed of ionizable lipid with a molar ratio of 50%, DSPC with a molar ratio of 10%, cholesterol with a molar ratio of 38.5%and a molar ratio of 1.5%for PEGylated lipid.
  • PChemsPC LNPs were composed of ionizable lipid with a molar ratio of 65%, PChemsPC with a molar ratio of 10%, a molar ratio of 23.5%and 1.5%for cholesterol and PEGylated lipids respectively.
  • ionizable lipids e.g. compound 01-1, lipid 5, SM-102, ALC-0315, Compound 03-135) were tested in this study.
  • Example 22 Characterization of in vivo serum cytokines after administration of a steroid containing phospholipid LNP with self-amplifying mRNA (saRNA)
  • lipid nanoparticles containing human erythropoietin (hEPO) self-amplifying mRNA were prepared as described in Example 15. After tail vein injection, mice were euthanized by CO 2 overdoses at 6 hours post administration, and blood samples were taken for cytokines measurement. Cytokine levels were measured and plotted in FIG. 12 and FIG. 13.
  • PChemsPC LNPs lead to significantly lower IL-6, IFN- ⁇ , TNF- ⁇ levels.
  • Example 23 Characterization of sterol-modified phospholipid LNP with CD3-CD19 mRNA
  • DSPC LNPs were composed of compound 01-1 with a molar ratio of 50%, DSPC with a molar ratio of 10%, cholesterol with a molar ratio of 38.5%and a molar ratio of 1.5%for PEGylated lipid.
  • PChemsPC LNPs were composed of compound 01-1 with a molar ratio of 65%, PChemsPC with a molar ratio of 10%, a molar ratio of 23.5%and 1.5%for cholesterol and PEGylated lipids respectively.
  • Lipid nanoparticles encapsulating CD19-CD3 mRNA were prepared as described in Example 15, and systemically administered to 6-8 week old female Balb/c mice (Xipuer-Bikai, Shanghai) at 0.3mg/kg dose by tail vein injection. Mice were euthanized by CO 2 overdoses at 6 hours post administration, and blood samples were taken for antibody measurement. Particularly, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 °C, snap-frozen and stored at -80 °C for analysis. The serum antibody level was shown in FIG. 14.
  • PChemsPC LNP can significantly improve CD3-CD19 antibody expression level post administration.

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Abstract

Provided is LNPs comprising phospholipids containing a sterol moiety. LNPs comprising such phospholipids have potential applications in mRNA vaccine technology. Provided are compositions comprising the LNPs and methods for using the LNPs or the compositions described above.

Description

LIPID NANOPARTICLES COMPRISING STEROL-MODIFIED PHOSPHOLIPIDS FIELD
The invention relates to lipid nanoparticles comprising phospholipids that contain a sterol moiety.
BACKGROUND
Lipid nanoparticles (or LNPs) are known vehicles for delivering biologically active agents to cells. LNPs have recently become particularly important in delivering nucleic acids to cells, due to issues with in vivo stability and cellular permeability of nucleic acids alone. One important application of nucleic acid-loaded LNPs are mRNA vaccines.
BRIEF SUMMARY
The present application provides phospholipids containing a sterol moiety that can be used to construct lipid nanoparticles. In one aspect, the lipid nanoparticles are useful for delivering nucleic acids, e.g. mRNA, to one or more cells. In one aspect, the application provides a method for expressing protein in a cell by delivering nucleic acids to the cell via the lipid nanoparticle comprising a phospholipid that contains a sterol moiety.
Also provided herein are lipid nanoparticles (LNPs) comprising
a phospholipid containing a sterol moiety;
an ionizable lipid; and
a polymer conjugated lipid.
In some embodiments, the phospholipid has a structure selected from:

In some embodiments, the phospholipid has the structure:
In some embodiments, the phospholipid has the structure:
In some embodiments, the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20∶1 to 2∶1. In some embodiments, the molar ratio of ionizable lipid to phospholipid is from 15∶1 to 5∶1. In some embodiments, the i onizable lipid comprises from 40 to 80 mol%of a total amount of lipids in the LNP. In some embodiments, the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP. In some embodiments, the ionizable lipid is a  cationic lipid. In some embodiments, the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
In some embodiments, the polymer conjugated lipid comprises from 0.5 to 5 mol%of the total amount of lipids in the LNP. In some embodiments, the polymer conjugated lipid comprises from 1 to 2 mol%of the total amount of lipids in the LNP. In some embodiments, the polymer conjugated lipid comprises 1.5 mol%of the total amount of lipids in the LNP. In some embodiments, the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid of from 1∶2 to 1∶20. In some embodiments, the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid of from 1∶3 to 1∶18. In some embodiments, the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1∶5 to 1∶10. In some embodiments, the polymer conjugated lipid is a PEGylated lipid. In some embodiments, the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
w is an integer ranging from 30 to 60.
In some embodiments, the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
w is an integer ranging from 30 to 60.
In some embodiments of either structure above, w is an integer ranging from 45 to 55.
In some embodiments, w is about 49.
In some embodiments, the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
In some embodiments, the LNP further comprises a lipid stabilizer.
In some embodiments, the LNP has a molar ratio of the lipid stabilizer to the phospholipid of from 10∶1 to 1∶4. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 5∶1 to 1∶3. In some embodiments, the LNP has a molar ratio of the lipid stabilizer to the phospholipid of from 10∶1 to 1∶2. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 5∶1 to 1∶1. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 4∶1 to 3∶1.
In some embodiments, the lipid stabilizer comprises from 5 to 50 mol%of the total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
In some embodiments, the phospholipid comprises from 1 to 30 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP.
In some embodiments, the LNP has a size of from 20 nm to 300 nm, as determined using dynamic light scattering. In some embodiments, the LNP has a size of from 50 nm to 150 nm, as determined using dynamic light scattering. In some embodiments, the size is from 60 nm to 140 nm. In some embodiments, the size is from 80 nm to 100 nm. In some embodiments, the size is from 85 nm to 95 nm.
In some embodiments, the LNP encapsulates mRNA.
Also provided herein are LNPs comprising a phospholipid, wherein the phospholipid has a structure:

In some embodiments, the phospholipid has the structure:
In some embodiments, the phospholipid has the structure:
In some embodiments, the LNP further comprises an ionizable lipid.
In some embodiments, the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20∶1 to 2∶1. In some embodiments, the molar ratio of ionizable lipid to phospholipid is from 15∶1 to 5∶1. In some embodiments, the i onizable lipid comprises from 40 to 80 mol%of a total  amount of lipids in the LNP. In some embodiments, the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP. In some embodiments, the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5. In some embodiments, the ionizable lipid is a cationic lipid.
In some embodiments, the LNP further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid comprises from 1 to 2 mol%of a total amount of lipids in the LNP. In some embodiments, the polymer conjugated lipid comprises 1.5%of the total amount of lipids in the LNP. In some embodiments, the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1∶5 to 1∶10. In some embodiments, the polymer conjugated lipid is a PEGylated lipid.
In some embodiments, the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
w is an integer ranging from 30 to 60.
In some embodiments, the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
w is an integer ranging from 30 to 60.
In some embodiments of either structure above, w is an integer ranging from 45 to 55. In some embodiments, w is about 49.
In some embodiments, the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
In some embodiments, the LNP further comprises a lipid stabilizer. In some embodiments, the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10∶1 to 1∶4. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 5∶1 to 1∶3. In some embodiments, the molar ratio of the lipid stabilizer to the phospholipid is from 4∶1 to 3∶1. In some embodiments, the lipid stabilizer comprises from 5 to 50 mol%of a total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
In some embodiments, the phospholipid comprises from 1 to 30 mol%of a total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP. In some embodiments, the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP. In some embodiments, the LNP has a size of from 20 nm to 300 nm, as determined using dynamic light scattering. In some embodiments, the LNP has a size of from 50 nm to 150 nm, as determined using dynamic light scattering. In some embodiments, the size is from 60 nm to 140 nm. In some embodiments, the size is from 80 nm to 100 nm. In some embodiments, the size is from 85 nm to 95 nm.
In some embodiments, the LNP encapsulates mRNA.
Also provided herein are compositions of the lipid nanoparticles (LNPs) described herein. In some embodiments, at least 80%of the LNPs encapsulate mRNA. In some embodiments, at least 85%of the LNPs encapsulate mRNA.
Also provided herein are methods for expressing protein in a cell, comprising introducing an LNP, as described herein, to the cell. In some embodiments, the cell is a mammalian cell.
Also provided herein are methods for delivering a protein to a subject, comprising administering an LNP or composition thereof, as described herein, to the individual, wherein the mRNA encodes the protein. In some embodiments, the LNP or composition thereof is administered systemically. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.
FIG. 1 shows expression levels of hEPO in LNP formulations containing PChemsPC or OChemsPC with a 60∶10∶28.5∶1.5 molar ratio of compound 01-1/OChemsPC or PChemsPC/Chol/DMG-PEG in Example 15.
FIG. 2 shows expression levels of hEPO in LNP formulations containing DSPC or PChemsPC at various molar percentages in Example 16.
FIG. 3 shows expression levels of hEPO in LNP formulations containing PChemsPC at various molar ratios of PChemsPC/Chol in Example 17.
FIG. 4 shows the luminescence levels measured from harvest liver tissues in Example 19.
Fig. 5 shows the percentage of luminescence intensity in different tissues in Example 19.
FIG. 6 shows hEPO expression fold change of LNPs with or without a steroid containing phospholipid in Example 20.
FIG. 7 shows serum cytokines levels boosted by compound 01-1 LNP in Example 21.
FIG. 8 shows serum cytokines levels boosted by Lipid 5 LNP in Example 21.
FIG. 9 shows serum cytokines levels boosted by SM-102 LNP in Example 21.
FIG. 10 shows serum cytokines levels boosted by ALC-0315 LNP in Example 21.
FIG. 11 shows serum cytokines levels boosted by Compound 03-135 LNP in Example 21.
FIG. 12 shows serum cytokines levels boosted by Compound 01-1 saRNA-LNP in Example 22.
FIG. 13 shows serum cytokines levels boosted by Compound 03-135 saRNA-LNP in Example 22.
FIG. 14 shows CD3-CD19 antibody levels of LNPs with or without a steroid containing phospholipid in Example 23.
DETAILED DESCRIPTION
Provided herein are LNPs comprising a phospholipid containing a sterol moiety. The LNPs can be loaded with mRNA, such as in mRNA vaccine technology. Sterol-modified phospholipids stabilize bilayers but do not exchange between membranes as freely as cholesterol. The mRNA-loaded LNPs demonstrate an ability to increase protein expression in target cells compared to  mRNA-loaded LNPs with more conventional phospholipids. Increasing protein expression helps increase the effectiveness and efficiency of mRNA-based treatments and therapies.
I. Definitions
As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
As used herein, “a pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable substrate, composition or vehicle used in the process of drug delivery, which may have one or more ingredients including, but not limited to, excipient (s) , binder (s) , diluent (s) , solvent (s) , filler (s) , and/or stabilizer (s) .
As used herein, the term “lipid” refers to a group of compounds including, without limitation, fats, sterols, waxes, fat-soluble vitamins, monoglycerides, diglycerides, sphingolipids, and phospholipids. In the context of the present disclosure, phospholipids, ionizable lipids, polymer conjugated lipids, and lipid stabilizers are considered lipids.
As used herein, the term “ionizable lipid” refers to a lipid that has a non-zero net electric charge at physiological pH. The term is inclusive with respect to cationic lipids, including lipids that have a partial positive charge at physiological pH. The term is also inclusive with respect to mixtures of ionizable lipids, which can contain two or more ionizable lipids. In every case where an embodiment is contemplated with the term “ionizable lipid, ” it is likewise contemplated with a “cationic lipid, ” as though all embodiments were specifically and individually listed with both ionizable and cationic lipids.
As used herein, the term “polymer conjugated lipid” refers to a lipid comprising a polymer moiety. The term is inclusive with respect to PEGylated lipids, including PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, and PEGylated dialkylglycerols. The term is also inclusive with respect to mixture of polymer conjugated lipids, which may contain two or more  polymer conjugated lipids. In every case where an embodiment is contemplated with the term “polymer conjugated lipid, ” it is likewise contemplated with a “PEGylated lipid, ” as though all embodiments were specifically and individually listed with both polymer conjugated and PEGylated lipids.
As used herein, the term “lipid stabilizer” refers to a component of the lipid nanoparticle that thought to help stabilize the LNP structure. Without being bound by theory, it is believed that the lipid stabilizer component of LNPs helps favor the liquid-ordered phase of the lipid membrane in LNPs. See, for example, section 3.3.1 of Albertsen, H.C.; et al., “The role of lipid components in lipid nanoparticles for vaccines and gene therapy. ” Adv Drug Deliv Rev. 2022 Sep; 188: 114416. Compounds that can serve as lipid stabilizers include sterols, corticosteroids, vitamins, and other compounds comprising a steroid core.
As used herein, the term “alkyl” refers to a chain of carbon atoms wherein all bonds between the carbon atoms in the alkyl group are single bonds. The term is inclusive with respect to straight and branched chains (e.g., the term includes both n-propyl and isopropyl groups) .
As used herein, the term “Cx-Cy alkyl” refers to an alkyl with at least x carbon atoms and no more than y carbon atoms in the alkyl chain. For example, the term “C1-C3 alkyl” includes, without limitation, methyl, ethyl, n-propyl, and isopropyl.
As used herein, the term “alkylene” refers to an alkyl chain that connects in at least two locations to other chemical groups. “Cx-Cy alkylene” refers to an alkylene with at least x carbon atoms and no more than y carbon atoms in the alkylene chain. For example, the term “C1-C3 alkylene” includes, without limitation, methylene, ethylene, n-propylene, and iso-propylene.
As used herein, the term “alkenyl” refers to a chain of carbon atoms with at least one double bond between two carbon atoms in the chain. The term is inclusive with respect to straight and branched chains (e.g., the term includes both 1 -propenyl and iso-propenyl groups) .
As used herein, the term “Cx-Cy alkenyl” refers to an alkenyl with at least x carbon atoms and no more than y carbon atoms in the alkenyl chain. For example, the term “C2-C4 alkenyl” includes, without limitation, vinyl and 1-propenyl.
As used herein, the term “alkenylene” refers to an alkenyl chain that connects in at least two locations to other chemical groups. “Cx-Cy alkenylene” refers to an alkenylene with at least x carbon atoms and no more than y carbon atoms.
As used herein, the term “alkynyl” refers to a chain of carbon atoms with at least one triple bond between two carbon atoms in the chain. The term is inclusive with respect to straight and branched chains (e.g., the term includes both 1-propynyl and iso-propynyl groups) .
As used herein, the term “Cx-Cy alkynyl” refers to an alkynyl with at least x carbon atoms and no more than y carbon atoms in the alkynyl chain.
As used herein, the term “cycloalkyl” refers to a cyclic group of carbon atoms wherein all the bonds between the carbon atoms are single bonds. The term “Cx-Cy cycloalkyl” refers to a cycloalkyl with at least x carbon atoms and no more than y carbon atoms. For example, the term “C6-C10 cycloalkyl” includes, without limitation, cyclohexyl and cyclo-octyl. The term “cycloalkylene” has the same meaning as cycloalkyl, except that the cycloalkylene connects to at least two other chemical groups.
As used herein, the term “cycloalkenyl” refers to a cyclic group of carbon atoms where at least one bond between two carbon atoms in the cycloalkenyl group is a double bond. The term “Cx-Cy cycloalkenyl” refers to a cycloalkenyl with at least x carbon atoms and no more than y carbon atoms.
The term “Cx-Cy aryl” refers to an aryl with at least x carbon atoms and no more than y carbon atoms. For example, the term “C6-C10 aryl” includes, without limitation, phenyl and naphthyl. The term “arylene” has the same meaning as aryl, except that the arylene connects to at least two other chemical groups.
As used herein, the term “heterocycloalkyl” refers to a cyclic group of atoms wherein all the bonds between the atoms in the ring are single bonds. The term “Cx-Cy heterocycloalkyl” refers to a heterocycloalkyl with at least x atoms and no more than y atoms. For example, the term “C5-C6 heterocycloalkyl” includes, without limitation, pyrrolidinyl and 1, 4-dioxanyl. The term “heterocycloalkylene” has the same meaning as heterocycloalkyl, except that the heterocycloalkylene connects to at least two other chemical groups.
The term “x-to y-membered heteroaryl” refers to a cyclic group of atoms with at least x atoms and no more than y atoms. For example, 5-or 6-membered heteroaryl includes, without limitation, pyridinyl and furanyl.
As used herein, the term “carbocycle” refers to a cycloalkyl or an aryl group. Likewise, the term “heterocycle” refers to a heterocycloalkyl or a heteroaryl group.
Possible atoms that make up the ring in heterocycloalkyl and heteroaryl groups, as well as derivatives thereof, include, without limitation, carbon, nitrogen, oxygen, and sulfur.
As used herein, the term “optionally substituted” means the indicated group may be substituted or unsubstituted. The term substituted refers to another chemical moiety that decorates the indicated group by replacement of one H atom. For example, ethanol is an example of ethane substituted with OH. In some embodiments, a group that is optionally substituted is optionally substituted by chloro, fluoro, bromo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C10 aryl, or 5-or 6-membered heteroaryl.
The terms “individual, ” “subject, ” and “patient” are used interchangeably herein to describe a mammal, including humans. In some embodiments, the individual is in need of treatment, for example, the individual may have been diagnosed with, or is suspected of having, a cancer.
It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of” embodiments.
Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X" . In some embodiments, the term “about” a value or parameter means a range within 20%, in either direction, of the value or parameter recited.
As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter.
As used herein and in the appended claims, the singular forms "a, " "an, " and "the" include plural referents unless the context clearly dictates otherwise.
As used herein and in the appended claims, the mole percentages of lipids and lipid stabilizers in lipid nanoparticles are calculated based on the total mole number of components in the lipid nanoparticles.
II. Lipid Nanoparticles (LNPs)
The lipid nanoparticles (LNPs) herein comprise a phospholipid containing a sterol moiety and optionally one or more of an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer. In some embodiments, the LNPs comprise a phospholipid containing a sterol moiety, an ionizable lipid, and a polymer conjugated lipid. In some embodiments, the LNPs comprise a  phospholipid containing a sterol moiety, a polymer conjugated lipid, and a lipid stabilizer. In some embodiments, the LNPs comprise a phospholipid containing a sterol moiety, an ionizable lipid, and a lipid stabilizer. In some embodiments, the LNPs comprise a phospholipid containing a sterol moiety, an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer.
In some embodiments, the phospholipid comprises from 1 to 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 2 to 25 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 3 to 20 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 10, 15, 20, 25, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 10 to 15, 20, 25, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 15 to 20, 25, or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 20 to 25 or 30 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 25 to 30 mol%of the total lipids in the LNP.
In some embodiments, the ionizable lipid comprises from 40 to 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 40 to 50, 60, 70, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 45 to 50, 60, 70, 75, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 50 to 60, 70, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 60 to 65, 70 or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipid comprises from 70 to 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipids comprise about 40, 50, 60, 70, or 80 mol%of the total lipids in the LNP. In some embodiments, the ionizable lipids comprise about 50, 60, or 70 mol%of the total lipids in the LNP.
In some embodiments, the LNP has a molar ratio of the ionizable lipid to the phospholipid of from 20∶1 to 2∶1. In some embodiments, the molar ratio is from 20∶1 to 15∶1, 10∶1, 5∶1, or 2∶1. In some embodiments, the molar ratio is from 18∶1 to 2.5∶1. In some embodiments, the molar ratio is from 16∶1 to 4∶1. In some embodiments, the molar ratio is from 15∶1 to 10∶1, 5∶1, or 2∶1.  In some embodiments, the molar ratio is from 10∶1 to 5∶1 or 2∶1. In some embodiments, the ratio is from 5∶1 to 2∶1. In some embodiments, the ratio is from 15∶1 to 5∶1.
In some embodiments, the polymer conjugated lipid has a molar ratio of 0.5 to 5 mol%of the total lipids in the LNP. In some embodiments, the polymer conjugated lipid has a molar ratio of 1 to 2 mol%of the total lipids in the LNP. In some embodiments, the polymer conjugated lipid has a molar ratio of 1.5 mol%of the total lipids in the LNP.
In some embodiments, the molar ratio of the polymer conjugated lipid to the phospholipid from 1∶2 to 1∶20. In some embodiments, the molar ratio of the polymer conjugated lipid to the phospholipid from 1∶3 to 1∶18. In some embodiments, the molar ratio of the polymer conjugated lipid to the phospholipid from 1∶5 to 1∶10.
In some embodiments, the lipid stabilizer comprises from 5 to 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 5 to 10, 20, 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 8 to 20, 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 10 to 20, 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 20 to 30, 40, or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 30 to 40 or 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises from 40 to 50 mol%of the total lipids in the LNP. In some embodiments, the lipid stabilizer comprises about 5, 10, 20, 30, 40, or 50 mol%of the total lipids in the LNP.
It is to be understood that any of the molar percentages or molar ratios described above for the sterol-containing phospholipid, the ionizable lipid, the polymer conjugated lipid, and the lipid stabilizer may be combined with each other in any embodiment describing the lipid composition of the LNPs described herein, such that every combination is contemplated as though each and every combination were specifically and individually disclosed.
In some embodiments, the phospholipid comprises from 1 to 30 mol%, the ionizable lipid comprises from 40 to 80 mol%, the polymer conjugated lipid comprises from 1 to 2 mol%, and the lipid stabilizer comprises from 5 to 50 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises from 5 to 25 mol%, the ionizable lipid comprises from 45 to 75 mol%, the polymer conjugated lipid comprises from 1 to 2 mol%, and the lipid stabilizer comprises from 20 to 40%of the total lipids in the LNP. In some embodiments, the  phospholipid comprises from 5 to 15 mol%, the ionizable lipid comprises from 40 to 60 mol%, the polymer conjugated lipid comprises from 1 to 2 mol%, and the lipid stabilizer comprises from 20 to 40 mol%of the total lipids in the LNP. In some embodiments, the phospholipid comprises about 10 mol%, the ionizable lipid comprises about 50 mol%, the polymer conjugated lipid comprises about 38.5 mol%, and the lipid stabilizer comprises about 1.5 mol%of the total lipids in the LNP.
In some embodiments, the LNP has a size from 20 to 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 40 to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 50 to 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 60 to 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 70 to 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 80 to 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 90 to 100, 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 100 to 110, 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 110 to 120, 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 120 to 130, 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 130 to 140, 150, 200, 250, or 300 nm. In some embodiments, the size is from 140 to 150, 200, 250, or 300 nm. In some embodiments, the size is from 150 to 200, 250, or 300 nm. In some embodiments, the size is from 200 to 300 nm. In some embodiments, the size is from 60 to 150 nm. In some embodiments, the size is from 65 to 90 nm. In some embodiments, the size is from 70 to 80 nm. In some embodiments, the size is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 nm. In some embodiments, the size is about 85 or about 90 nm. In some embodiments, the size is about 85 nm. In some embodiments, the size is about 90 nm.
A. Phospholipid containing a sterol moiety
The LNPs herein comprise a phospholipid containing a sterol moiety. The phospholipid containing a sterol moiety is any phospholipid that incorporates a sterol moiety into the lipid structure. In some embodiments, the sterol moiety is incorporated in place of one or more alkyl chains on the phospholipid. In some embodiments, the sterol moiety is incorporated into one alkyl chain of the phospholipid. In some embodiments, the sterol moiety is connected through the O atom of the sterol (e.g. converting the sterol moiety into the -O-atom of an ester  connection to the remainder of the phospholipid) . In some embodiments, the sterol moiety is cholesterol. In some embodiments, the sterol moiety is a cholesterol moiety connected through O atom of the sterol (e.g. by converting the sterol O atom of cholesterol into the -O-atom of an ester connection to the remainder of the phospholipid) .
In some embodiments, the phospholipid has a structure of selected from:
B. Ionizable lipids
In some embodiments, the LNPs comprise an ionizable lipid. In some embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the cationic lipid is a cationic lipid described in International Patent Publication No. WO 2021/204175, the entirety of which is incorporated herein by reference.
In some embodiments, the cationic lipid is a compound of Formula (01-I) :
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene, wherein one or more -CH2-in the alkylene or alkenylene is optionally replaced by -O-;
L1 is-OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NRaC (=O) R1, -C (=O) NRbRc, -NRaC (=O) NRbRc, -OC (=O) NRbRc, -NRaC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (ORb) (ORc) , - (C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, or R1;
L2 is-OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NReRf, -NRdC (=O) NReRf, -OC (=O) NReRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (ORe) (ORf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, or R2;
R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;
Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;
Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;
G3 is C2-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene;
R3 is -N (R4) R5;
R4 is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4-to 8-membered heterocyclyl, or C6-C10 aryl; or R4, G3 or part of G3, together with the nitrogen to which they are attached form a cyclic moiety;
R5 is C1-C12 alkyl or C3-C8 cycloalkyl; or R4, R5, together with the nitrogen to which they are attached form a cyclic moiety;
x is 0, 1, or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of Formula (01-II) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
is a single bond or a double bond;
G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene, wherein one or more -CH2-in the alkylene or alkenylene is optionally replaced by -O-;
L1 is-OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NRaC (=O) R1, -C (=O) NRbRc, -NRaC (=O) NRbRc, -OC (=O) NRbRc, -NRaC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (ORb) (ORc) , - (C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, or R1;
L2 is-OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NReRf, -NRdC (=O) NReRf, -OC (=O) NReRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (ORe) (ORf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, or R2;
R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;
Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;
Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;
G4 is a bond, C1-C23 alkylene, C2-C23 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene;
R3 is -N (R4) R5;
R4 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4-to 8-membered heterocyclyl, or C6-C10 aryl; or R4, G3 or part of G3, together with the nitrogen to which they are attached form a cyclic moiety;
R5 is C1-C12 alkyl or C3-C8 cycloalkyl; or R4 and R5, together with the nitrogen to which they are attached form a cyclic moiety;
x is 0, 1, or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of Formula (01-I-B) , (01-I-B’) , (01-I-B”) , (01-I-C) , (01-I-D) , or (01-I-E) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, G1 and G2 are each independently C3-C7 alkylene. In some embodiments, G1 and G2 are each independently C5 alkylene. In some embodiments, G3 is C2-C4 alkylene. In some embodiments, G3 is C2 alkylene. In some embodiments, G3 is C4 alkylene.
In some embodiments, R3 has one of the following structures:
In some embodiments, R1, R2, Rc, and Rf are each independently branched C6-C32 alkyl or branched C6-C32 alkenyl. In some embodiments, R1, R2, Rc, and Rf are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. In some embodiments, R1, R2, Rc, and Rf are each independently -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl. In some embodiments, R1, R2, Rc, and Rf are each independently -R7-CH (R8) (R9) , wherein R7 is C0-C1 alkylene, and R8 and R9 are independently C4-C8 alkyl.
In some embodiments, the cationic lipid is a compound in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 1.

In some embodiments, the cationic lipid is a cationic lipid described in International Patent Application No. PCT/CN2022/072694, the entirety of which is incorporated herein by reference. In some embodiments, the cationic lipid is a compound of Formula (02-I) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more -CH2-in G1 and G2 is optionally replaced by -O-, -C (=O) O-, or -OC (=O) -;
each L1 is independently -OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NRaC (=O) R1, -C (=O) NRbRc, -NRaC (=O) NRbRc, -OC (=O) NRbRc, -NRaC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (ORb) (ORc) , -NRap (=O) (ORb) (ORc) ;
each L2 is independently-OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NReRf, -NRdC (=O) NReRf, -OC (=O) NReRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (ORe) (ORf) , -NRdp (=O) (ORe) (ORf) ;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;
Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by a C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
R3 is -N (R4) R5, -OR6, or-SR6;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl;
x is 0, 1, or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of Formula (02-II) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more -CH2-in G1 and G2 is optionally replaced by -O-, -C (=O) O-, or -OC (=O) -;
each L1 is independently -OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NRaC (=O) R1, -C (=O) NRbRc, -NRaC (=O) NRbRc, OC (=O) NRbRc, -NRaC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (ORb) (ORc) , -NRap (=O) (ORb) (ORc) ;
each L2 is independently-OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NReRf, -NRdC (=O) NReRf, -OC (=O) NReRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (ORe) (ORf) , -NRdp (=O) (ORe) (ORf) ;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;
Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by a C3-C8 cycloalkylene or C3-C8 cycloalkenylene;
R3 is -N (R4) R5, -OR6, or-SR6;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl;
x is 0, 1, or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.
In some embodiments, the compound is a compound of Formula (02-V-A) , (02-V-B) , (02-V-C) , (02-V-D) , (02-V-E) , (02-V-F) :
wherein z is an integer from 2 to 12,
x0 is an integer from 1 to 11;
y0 is an integer from 1 to 11;
x1 is an integer from 0 to 9;
y1 is an integer from 0 to 9;
x2 is an integer from 2 to 9;
x3 is an integer from 1 to 5;
x4 is an integer from 0 to 3;
y2 is an integer from 2 to 9;
y3 is an integer from 1 to 5; and
y4 is an integer from 0 to 3;
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
In some embodiments, z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 and y0 are independently 2 to 6. In some embodiments, x0 and y0 are independently 4 or 5. In some embodiments, x1 and y1 are independently 2 to 6. In some embodiments, x1 and y1 are independently 4 or 5. In some embodiments, x2 and y2 are independently an integer from 2 to 8. In some embodiments, x2 and y2 are independently 3, 5, or 7. In some embodiments, x3 and y3 are both 1. In some embodiments, x4 and y4 are independently 0 or 1.
In some embodiments, each L1 is independently -OR1, -OC (=O) R1, or-C (=O) OR1, and each L2 is independently -OR2, -OC (=O) R2, or -C (=O) OR2. In some embodiments, R1 and R2 are independently straight C6-C10 alkyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl.
In some embodiments, the compound is a compound of formula (02-VI-A) , (02-VI-B) , (02-VI-C) , (02-VI-D) , (02-VI-E) , or (02-VI-F) :
wherein z is an integer from 2 to 12;
y is an integer from 2 to 12;
x0 is an integer from 1 to 11;
x1 is an integer from 0 to 9;
x2 is an integer from 2 to 5;
x3 is an integer from 1 to 5; and
x4 is an integer from 0 to 3;
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, z is an integer from 2 to 6. In some embodiments, z is 2, 4, or 5. In some embodiments, x0 is 4 or 5. In some embodiments, x1 is 4 or 5. In some embodiments, x2 is an integer from 2 to 5. In some embodiments, x2 is 3 or 5. In some embodiments, x3 is 0 or 1. In some embodiments, y is an integer from 2 to 6. In some embodiments, y is 5.
In some embodiments, each L1 is independently -OR1, -OC (=O) R1 or -C (=O) OR1, and L2 is -OC (=O) R2 or -C (=O) OR2, -NRdC (=O) R2, or -C (=O) NReRf. In some embodiments, R1 is straight C6-C10 alkyl or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, R2 and Rf are each independently straight C6-C18 alkyl, C6-C18 alkenyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, Rd and Re are each independently H.
In some embodiments, the compound is a compound in Table 2, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 2.


In some embodiments, the cationic lipid described herein is a cationic lipid described in International Patent Publication No. WO 2022/152109, the entirety of which is incorporated herein by reference.
In some embodiments, the cationic lipid is a compound of Formula (03-I) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene, wherein one or more -CH2-in G1 and G2 is optionally replaced by -O-;
each L1 is independently-OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NRaC (=O) R1, -C (=O) NRbRc, -NRaC (=O) NRbRc, -OC (=O) NRbRc, -NRaC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (ORb) (ORc) , -NRap (=O) (ORb) (ORc) , - (C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, - (4-to 8-membered heterocyclylene) -R1, or R1;
each L2 is independently -OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NReRf, -NRdC (=O) NReRf, -OC (=O) NReRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (ORe) (ORf) , -NRdp (=O) (ORe) (ORf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, - (4-to 8-membered heterocyclylene) -R2, or R2;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;
Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene, C3-C8 cycloalkenylene, C3-C8 cycloalkynylene, 4-to 8-membered heterocyclylene, C6-C10 arylene, or 5-to 10-membered heteroarylene;
R3 is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4-to 8-membered heterocyclyl, C6-C10 aryl, or 5-to 10-membered heteroaryl; or R3 and G1, or part of G1, together with the nitrogen to which they are attached form a cyclic moiety; or R3 and G3 or part ofG3, together with the nitrogen to which they are attached form a cyclic moiety;
R4 is C1-C12 alkyl or C3-C8 cycloalkyl;
x is 0, 1, or 2;
n is I or 2;
m is 1 or 2; and
wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of Formula (03-II-A) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, the cationic lipid is a compound of Formula (03-II-B) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, the cationic lipid is a compound of Formula (03-II-C) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, the cationic lipid is a compound of Formula (03-II-D) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, G1 and G2 are each independently C2-C12 alkylene. In some embodiments, G1 and G2 are each independently C5 alkylene. In some embodiments, G3 is C2-C6 alkylene.
In some embodiments, R3 is C1-C12 alkyl, C2-C12 alkenyl, or C3-C8 cycloalkyl. In some embodiments, R3 is C3-C8 cycloalkyl. In some embodiments, R3 is unsubstituted. In some embodiments, R4 is substituted C1-C12 alkyl. In some embodiments, R4 is-CH2CH2OH.
In some embodiments, L1 is-OC (=O) R1, -C (=O) OR1, -NRaC (=O) R1, or -C (=O) NRbRc; and L2 is -OC (=O) R2, -C (=O) OR2, -NRdC (=O) R2, or -C (=O) NReRf. In some embodiments, R1, R2, Rc, and Rf are each independently straight C6-C18 alkyl, straight C6-C18 alkenyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, R1, R2, Rc, and Rf are each independently straight C7-C15 alkyl, straight C7-C15 alkenyl, or-R7-CH (R8) (R9) , wherein R7 is C0-C1 alkylene, and R8 and R9 are independently C4-C8 alkyl or C6-C10 alkenyl. In some embodiments, Ra, Rb, Rd, and Re are each independently H.
In some embodiments, the cationic lipid is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 3
In some embodiments, the cationic lipid is a cationic lipid described in International Patent Application No. PCT/CN2022/094227, the entirety of which is incorporated herein by reference.
In some embodiments, the cationic lipid is a compound of Formula (04-I) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene;
L1 is-OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NRaC (=O) R1, -C (=O) NRbRc, -NRaC (=O) NRbRc, -OC (=O) NRbRc, -NRaC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (ORb) (ORc) , - (C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, or R1;
L2 is-OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NReRf, -NRdC (=O) NReRf, -OC (=O) NReRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (ORe) (ORf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, or R2;
R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;
Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;
Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;
R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
x is 0, 1, or 2;
s is 0 or 1; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene, is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of Formula (04-III) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;
R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
G3 is C2-C12 alkylene or C2-C12 alkenylene;
G4 is C2-C12 alkylene or C2-C12 alkenylene;
R3 is -N (R4) R5 or -OR6;
R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;
R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl; or R4 and R5, together with the nitrogen to which they are attached form a cyclic moiety;
R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl; and wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently optionally substituted.
In some embodiments, the cationic lipid is a compound of Formula (04-IV) :
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
In some embodiments, G3 is C2-C4 alkylene. In some embodiments, G4 is C2-C4 alkylene.
In some embodiments, R0 is C1-C6 alkyl. In some embodiments, R3 is -OH. In some embodiments, R3 is -N (R4) R5. In some embodiments, R4 is C3-C8 cycloalkyl. In some embodiments, R4 is unsubstituted. In some embodiments, R5 is -CH2CH2OH.
In some embodiments, L1 is-OC (=O) R1, -C (=O) OR1, -C (=O) R1, -C (=O) NRbRc, or R1; and L2 is -OC (=O) R2, -C (=O) OR2, -C (=O) R2, -C (=O) NReRf, or R2. In some embodiments, R1 and R2 are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. In some embodiments, R1 and R2 are each independently -R7-CH (R8) (R9) , wherein R7 is C1-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In some embodiments, R1 is straight C6-C24 alkyl and R2 is branched C6-C24 alkyl. In some embodiments, R1 is straight C6-C24 alkyl and R2 is -R7-CH (R8) (R9) , wherein R7 is C1-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl.
In some embodiments, the cationic lipid is a compound in Table 4, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 4.

In some embodiments, the cationic lipid contained in the particles or compositions provided herein is a cationic lipid described in U.S. Patent Nos. US 10442756B2, US9868691B2, or US9868692B2, all of which are incorporated herein by reference.
In some embodiments, the cationic lipid is a compound Formula (05-I) :
or a salt or isomer thereof, wherein
1 is selected from 1, 2, 3, 4, and 5;
m is selected from 5, 6, 7, 8, and 9;
M1 is a bond or M′;
R4 is unsubstituted C1-C3 alkyl, or - (CH2) nOH, -NHC (S) N (R) 2, -NHC (O) N (R) 2, -N (R) C (O) R, -N (R) S (O) 2R, -N (R) R8, -NHC (=NR9) N (R) 2, -NHC (=CHR9) N (R) 2, -OC (O) N (R) 2, -N (R) C (O) OR, -N (OR) C (O) R, -N (OR) S (O) 2R, -N (OR) C (O) OR, -N (OR) C (O) N (R) 2, -N (OR) C (S) N (R) 2, -N (OR) C (=NR9) N (R) 2, -N (OR) C (=CHR9) N (R) 2, or heteroaryl, and each n is selected from 1, 2, 3, 4, or 5;
M and M′are independently selected from -C (O) O-, -OC (O) -, -C (O) N (R′) -, -P (O) (OR′) O-, -S-S-, an aryl group, and a heteroaryl group; and
R2 and R3 are both C1-C14 alkyl, or C2-C14 alkenyl,
R8 is selected from the group consisting of C3-C6 carbocycle and heterocycle;
R9 is selected from the group consisting of H, CN, NO2, C1-C6 alkyl, -OR, -S (O) 2R, -S (O) 2N (R) 2, C2-C6 alkenyl, C3-C6 carbocycle and heterocycle;
each R is independently selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl, and H; and
R′is a linear alkyl.
In some embodiments, the cationic lipid is SM102 or Lipid 5:
In some embodiments, the cationic lipid is a cationic lipid described in U.S. Patent No. US 10166298B2, the entire teachings of which are incorporated herein by reference.
In some embodiments, the cationic lipid is a compound of Formula (06-I) :
or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:
one of L1 or L2 is -O (C=O) -, - (C=O) O-, -C (=O) -, -O-, -S (O) x-, -S-S-, -C (=O) S-, SC (=O) -, -NRaC (=O) -, -C (=O) NRa-, NRaC (=O) NRa-, -OC (=O) NRa-or -NRaC (=O) O-, and the other of L1 or L2 is -O (C=O) -, - (C=O) O-, -C (=O) -, -O-, -S (O) x-, -S-S-, -C (=O) S-, SC (=O) -, -NRaC (=O) -, -C (=O) NRa-, NRaC (=O) NRa-, -OC (=O) NRa-or -NRaC (=O) O-, or a direct bond;
G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
Ra is H or C1-C12 alkyl;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
R3 is H, OR5, CN, -C (=O) OR4, -OC (=O) R4, or -NR5C (=O) R4;
R4 is C1-C12 alkyl;
R5 is H or C1-C6 alkyl; and
xis0, 1, or 2.
In some embodiments, the cationic lipid is a compound of Table 5, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.
Table 5.
In some embodiments, the cationic lipids of the present disclosure are the same as those disclosed in International Application Publication No. WO 2010/144740, the entire teachings of  which are incorporated herein by reference. For example, the cationic lipid is a compound represented by Formula (07-I) , also named as compound 07-I:
Preferably, the ionizable lipid used in the LNPs according to the present invention is selected from
compounds of Formula (01-I-O) :
wherein y and z are each independently an integer from 4 to 6,
s is an integer from 2 to 4,
t is an integer from 1 to 3, and
R1 and R2 are each independently C12-C22 alkyl;
R4 is C3-C8 cycloalkyl;
R6 is hydrogen or hydroxyl,
compounds of Formula 05-I:
wherein
1 is selected from 1, 2, 3, 4, and 5;
m is selected from 5, 6, 7, 8, and 9;
M1 is -C (O) O-;
R4 is - (CH2) nOH, and n is selected from 1, 2, 3, 4, or 5;
M is -OC (O) -; and
R2 and R3 are both C6-10 alkyl,
compounds of Formula (06-I) :
wherein
L1 and L2 is -O (C=O) -;
G1 and G2 are each independently unsubstituted C4-C8 alkylene;
G3 is C3-C8 alkylene;
R1 and R2 are each independently C12-C22 alkyl;
R3 is H or OH,
compounds of Formula (02-V-B)
wherein
each L1 is independently-OC (=O) R1;
each L2 is independently -OC (=O) R2;
R1 and R2 are each independently C6-C24 alky;
R3 is-OR6;
R6 is hydrogen;
z is an integer from 2 to 12;
x1 is an integer from 0 to 9;
y1 is an integer from 0 to 9;
Compounds of Formula (02-VI-F)
wherein
each L1 is independently -OC (=O) R1;
each L2 is independently-OC (=O) R2;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
R3 is -OR6;
R6 is hydrogen;
z is an integer from 2 to 12;
y is an integer from 2 to 12;
x1 is an integer from 2 to 5;
compounds of Formula (02-V-F)
wherein
each L1 is independently-OC (=O) R1;
each L2 is independently-OC (=O) R2;
R1 and R2 are each independently C6-C24 alkyl;
R3 is -OR6;
R6 is hydrogen;
z is an integer from 2 to 12;
x2 is an integer from 2 to 9;
x4 is an integer from 0 to 3;
y2 is an integer from 2 to 9;
y4 is an integer from 0 to 3,
compounds of formula (03-I)
wherein
G1 and G2 are each independently C3-C8 alkylene;
each L1 is independently-OC (=O) R1 or-C (=O) OR1;
each L2 is independently -C (=O) OR2 or -OC (=O) R2;
R1 is independently C6-C24 alkyl;
R2 is independently C6-C24 alkyl;
G3 is C2-C12 alkylene;
R3 is C3-C8 cycloalkyl;
R4 is C1-C4 hydroxylalkyl;
n is 1 or 2;
m is 1 or 2, and
compound 07-I:
More preferably, the ionizable lipid used in the LNPs according to the present invention is selected from the following compounds:


C. Polymer Conjugated lipids
In some embodiments, the LNP comprises a polymer conjugated lipid. Polymers that can be incorporated into polymer conjugated lipids include polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, a polymer  may include poly (caprolactone) (PCL) , ethylene vinyl acetate polymer (EVA) , poly (lactic acid) (PLA) , poly (L-lactic acid) (PLLA) , poly (glycolic acid) (PGA) , poly (lactic acid-co-glycolic acid) (PLGA) , poly (L-lactic acid-co-glycolic acid) (PLLGA) , poly (D, L-lactide) (PDLA) , poly (L-lactide) (PLLA) , poly (D, L-lactide-co-caprolactone) , poly (D, L-lactide-co-caprolactone-co-glycolide) , poly (D, L-lactide-co-PEO-co-D, L-lactide) , poly (D, L-lactide-co-PPO-co-D, L-lactide) , polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL) , hydroxypropyl methacrylate (HPMA) , polyethyleneglycol, poly-L-glutamic acid, poly (hydroxy acids) , polyanhydrides, polyorthoesters, poly (ester amides) , polyamides, poly (ester ethers) , polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol) (PEG) , polyalkylene oxides (PEO) , polyalkylene terephthalates such as poly (ethylene terephthalate) , polyvinyl alcohols (PVA) , polyvinyl ethers, polyvinyl esters such as poly (vinyl acetate) , polyvinyl halides such as poly (vinyl chloride) (PVC) , polyvinylpyrrolidone, polysiloxanes, polystyrene (PS) , polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellu lose, carboxymethylcellulose, polymers of acrylic acids, such as poly (methyl (meth) acrylate) (PMMA) , poly (ethyl (meth) acrylate) , poly (butyl (meth) acrylate) , poly (isobutyl (meth) acrylate) , poly (hexyl (meth) acrylate) , poly (isodecyl (meth) acrylate) , poly (lauryl (meth) acrylate) , poly (phenyl (meth) acrylate) , poly (methyl acrylate) , poly (isopropyl acrylate) , poly (isobutyl acrylate) , poly (octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, polyoxamines, poly (ortho) esters, poly (butyric acid) , poly (valeric acid) , poly (lactide-co-caprolactone) , trimethylene carbonate, or polyvinylpyrrolidone.
In some embodiments, the polymer conjugated lipid is a PEGylated lipid (PEG lipids) . Without being bound by the theory, it is contemplated that a polymer conjugated lipid component in an LNP can improve colloidal stability and/or reduce protein absorption of the nanoparticles. Exemplary PEGylated lipids that can be used in connection with the present disclosure include but are not limited to PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, the PEG-conjugated lipid may be a PEG-modifiedphosphatidylethanolamine,  PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or a PEG-modified dialkylglycerol. In some embodiments, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, Ceramide-PEG2000, or ChoI-PEG2000.
In some embodiments, the PEGylated lipid is a PEGylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a pegylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2’, 3’-di (tetradecanoyloxy) propyl-1-O- (ω-methoxy (polyethoxy) ethyl) butanedioate (PEG-S-DMG) , a pegylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as ω-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecanoxy) propyl) carbamate or 2, 3-di(tetradecanoxy) propyl-N- (ω-methoxy (polyethoxy) ethyl) carbamate.
In some embodiments, the PEGylated lipid is present in a concentration ranging from 1.0 to 2.5 molar percent. In some embodiments, the polymer conjugated lipid is present in a concentration of about 1.7 molar percent. In some embodiments, the polymer conjugated lipid is present in a concentration of about 1.5 molar percent.
In some embodiments, the molar ratio of the ionizable lipid to the polymer conjugated lipid ranges from about 20∶1 to about 100∶1. In some embodiments, the molar ratio of the ionizable lipid to polymer conjugated lipid ranges from about 25∶1 to about 80∶1 . In some embodiments, the molar ratio of the ionizable lipid to polymer conjugated lipid ranges from about 30∶1 to about 60∶1. In some embodiments, the molar ratio of the ionizable lipid to polymer conjugated lipid ranges from about 30∶1 to about 50∶1.
In some embodiments, the PEGylated lipid has the following Formula:
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
w has a mean value ranging from 30 to 60.
In some embodiments, R12 and R13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average w ranges from 42 to 55, for example, the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some embodiments, the average w is about 49.
In some embodiments, the PEGylated lipid has the following Formula:
or a pharmaceutically salt thereof, wherein the average w is about 49.
D. Lipid stabilizer
In some embodiments, the LNPs comprise a lipid stabilizer. In some embodiments, the lipid stabilizer comprises a sterol. In some embodiments, the lipid stabilizer comprises a corticosteroid. In some embodiments, the lipid stabilizer comprises two or more components. In some embodiments, the lipid stabilizer comprises a corticosteroid and a sterol. In some embodiments, the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, and brassicasterol. In some embodiments, the corticosteroid is selected from the group consisting ofprednisolone, dexamethasone, prednisone, and hydrocortisone. In some embodiments, the lipid stabilizer comprises tomatidine, tomatine, ursolic acid, or alpha-tocopherol. In some embodiments, the lipid stabilizer comprises one or more compounds selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, prednisolone, dexamethasone, prednisone, hydrocortisone, tomatidine, tomatine, ursolic acid, and alpha-tocopherol. In some embodiments, the lipid stabilizer is cholesterol.
E. LNPs comprising mRNA
In some embodiments, the LNPs disclosed herein further comprise a therapeutic payload. The payload can be any substance or compound that has a therapeutic or prophylactic effect. In some embodiments, the therapeutic payload is a small molecule, a cytotoxin, a radioactive ion, a chemotherapeutic compound, a vaccine, or a compound that elicits an immune response.
In some embodiments, the LNPs disclosed herein comprise a nucleic acid. In some embodiments, the nucleic acid is a DNA. In some embodiments, the DNA is catalytic DNA, plasmid DNA, aptamer, or complementary DNA (cDNA) . In some embodiments, the nucleic  acid is an RNA. In some embodiments, the RNA is a messenger RNA (mRNA) , antisense oligonucleotide, microRNA (miRNA) , miRNA inhibitor (e.g., antagomir or antimir) , messenger-RNA-interfering complementary RNA (micRNA) , multivalent RNA, dicer substrate RNA (dsRNA) , small hairpin RNA (shRNA) , antisense RNA, transfer RNA (tRNA) , asymmetrical interfering RNA (aiRNA) , a ribozyme, an aptamer, or a vector. In some embodiments, the RNA is an mRNA hybrid. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA encodes a protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the LNPs comprise an RNAi agent or RNAi-inducing agent. Preferably, the weight ratio of the ionizable lipid to the therapeutic payload is from 5∶1 to 20∶1.
F. Compositions comprising Lipid Nanoparticles (LNPs)
The present disclosure is inclusive with respect to compositions comprising the LNPs described herein. In some embodiments, the composition comprises a plurality of LNPs. In some embodiments, the plurality of LNPs have a polydispersity index (PDI) of 0.001 to 0.2. In some embodiments, the plurality of LNPs have a polydispersity index (PDI) of 0.001 to 0.1. In some embodiments, the LNPs have a PDI of 0.005 to 0.05.
In some embodiments, some of the LNPs of the LNP composition comprise mRNA. In some embodiments, the mRNA encapsulation efficiency (EE%-i.e., the percentage of individual LNPs in the composition that encapsulate the mRNA) of the LNP composition is from 70%to 100%. In some embodiments, the EE%of the LNP composition is from 80 to 95%. In some embodiments, the EE%of the LNP composition is from 85 to 95%. In some embodiments, the EE%of the LNP composition is from 90 to 95%. In some embodiments, the EE%is 80%or greater. In some embodiments, the EE%is 85%or greater. In some embodiments, the EE%is 90%or greater. In some embodiments, the EE%is 95%or greater. In some embodiments, the EE%is 80%-100%. In some embodiments, the EE%is 85%-100%. In some embodiments, the EE%is 90%-100%. In some embodiments, the EE%is 95%-100%.
In some embodiments, the LNP composition is able to increase protein expression compared to comparable LNP compositions that do not comprise a steroid-containing phospholipid.
It is to be understood that any of the molar percentages or molar ratios described above for the sterol-containing phospholipid, the ionizable lipid, the polymer conjugated lipid, and the lipid  stabilizer may be combined with each other in any embodiment describing the composition of LNPs described herein, such that every combination is contemplated as though each and every combination were specifically and individually disclosed.
G. Methods of Making the LNPs and compositions thereof
The LNPs here can be made according to the methods that are well known in the art.
In some embodiments, the method comprises solubilizing the lipid components (e.g., a phospholipid, an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer) in a solvent. In some embodiments, the method comprises the steps:
(a) solubilizing the lipid components (e.g., a phospholipid, an ionizable lipid, a polymer conjugated lipid, and optionally a lipid stabilizer) in a solvent to produce a lipid mixture;
(b) diluting the mRNA in a solvent to produce an mRNA mixture; and
(c) mixing the lipid mixture and mRNA mixture to obtain an mRNA LNP mixture.
In some embodiments, the solvent in step (a) is a polar solvent. In some embodiments, the solvent in step (a) is an alcohol solvent. In some embodiments, the solvent in step (a) is methanol, ethanol, n-propanol, or isopropanol. In some embodiments, the solvent in step (a) is ethanol.
In some embodiments, the solvent in step (b) is an aqueous solvent. In some embodiments, the solvent in step (b) is an aqueous buffer. In some embodiments, the solvent in step (b) is a citrate buffer. In some embodiments, the citrate buffer has a citrate concentration of 5 to 100 mM.In some embodiments, the citrate buffer has a citrate concentration of 10 to 50 mM. In some embodiments, the aqueous solvent of step (b) has a pH of 2-6. In some embodiments, the aqueous solvent of step (b) has a pH of 3-5.
In some embodiments, the mixing of step (c) occurs with a weight ratio of lipid∶mRNA of 10∶1 to 30∶1. In some embodiments, the mixing of step (c) occurs at a volume ratio of lipid∶mRNA of 1∶1 to 1∶5. In some embodiments, the volume ratio is 1∶2 to 1∶4. In some embodiments, the volume ratio is about 1∶3. In some embodiments, the mixing of step (c) is performed with a microfluidic apparatus. In some embodiments, the microfluidic apparatus has a flow rate of 9 to 30 mL/min.
In some embodiments, the mRNA mixture has an mRNA concentration from 1 to 3, 5, 7, 10, 12, 15, 20, 30, 40 or 50 mM. In some embodiments, the concentration is from 3 to 5, 7, 10, 12, 15, 20, or 30mM. In some embodiments, the concentration is from 5 to 7, 10, 12, 15, 20, or  30 mM. In some embodiments, the concentration is from 7 to 10, 12, 15, 20, or 30 mM. In some embodiments, the concentration is from 10 to 12, 15, 20, or 30 mM. In some embodiments, the concentration is from 12 to 15, 20, or 30 mM. In some embodiments, the concentration is from 15 to 20 or 30 mM. In some embodiments, the concentration is from 20 to 30 mM. In some embodiments, the concentration is about 5, 7, 10, 12, 15, or 20 mM. In some embodiments, the concentration is about 5, 7, 10, 12, or 15 mM. In some embodiments, the concentration is about 10 mM. In some embodiments, the concentration is about 12 mM. In some embodiments, the concentration is about 15 mM. In some embodiments, the concentration is about 20 mM.
In some embodiments, the method further comprises a step (d) :
(d) filtering the lipid nanoparticle through a sterile filter.
In some embodiments, the sterile filter is a 0.2 μm sterile filter.
IV. Methods Related to the Lipid Nanoparticles (LNPs)
The present disclosure is inclusive with respect to potentially useful methods that make use of the LNPs described herein.
In some embodiments, a method for expressing protein in a cell, wherein the method comprises introducing an LNP or composition thereof, as described above, to a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the LNP or composition thereof is administered systemically to a mammal. In some embodiments, the mammal is a human.
In some embodiments, the LNP composition is able to increase protein expression compared to comparable LNP compositions that do not comprise a steroid-containing phospholipid.
V. Kits comprising a phospholipid containing a sterol moiety
The present disclosure includes kits comprising a phospholipid containing a sterol moiety and packaging for said phospholipid. In some embodiments, the kit further comprises an ionizable lipid. In some embodiments, the kit further comprises a polymer conjugated lipid. In some embodiments, the kit further comprises a lipid stabilizer. In some embodiments, the kit further comprises an ionizable lipid, a polymer conjugated lipid, and a lipid stabilizer. In some embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the polymer conjugated lipid is a PEGylated lipid. In some embodiments, the lipid stabilizer is cholesterol. In some embodiments, the kit further comprises a cationic lipid, a PEGylated lipid, and cholesterol.
It is understood that any embodiment of the compounds provided herein, as set forth above, and any specific substituent and/or variable in the compound provided herein, as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of the compounds to form embodiments not specifically set forth above. In addition, in the event that a list of substituents and/or variables is listed for any particular group or variable, it is understood that each individual substituent and/or variable may be deleted from the particular embodiment and/or claim and that the remaining list of substituents and/or variables will be considered to be within the scope of embodiments provided herein.
It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compounds.
EXEMPLARY EMBODIMENTS
The following exemplary embodiments are provided herein:
Embodiment 1. A lipid nanoparticle (LNP) comprising
a phospholipid containing a sterol moiety;
an ionizable lipid; and
a polymer conjugated lipid.
Embodiment 2. The LNP of embodiment 1, wherein the phospholipid has a structure selected from:

Embodiment 3. The LNP of embodiment 1 or 2, wherein the phospholipid has the structure:
Embodiment 4. The LNP of embodiment 1 or 2, wherein the phospholipid has the structure:
Embodiment 5. The LNP of any one of embodiments 1 to 4, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20∶1 to 2∶1.
Embodiment 6. The LNP of embodiment 5, wherein the molar ratio of the ionizable lipid to the phospholipid is from 15∶1 to 5∶1.
Embodiment 7. The LNP of any one of embodiments 1 to 4, wherein the ionizable lipid comprises from 40 to 80 mol%of a total amount of lipids in the LNP.
Embodiment 8. The LNP of embodiment 7, wherein the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP.
Embodiment 9. The LNP of any one of embodiments 1 to 8, wherein the ionizable lipid is a cationic lipid.
Embodiment 10. The LNP of any one of embodiments 1 to 8, wherein the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
Embodiment 11. The LNP of any one of embodiments 1 to 10, wherein the polymer conjugated lipid comprises from 1 to 2%of a total amount of lipids in the LNP.
Embodiment 12. The LNP of embodiment 11, wherein the polymer conjugated lipid comprises 1.5%of the total amount of lipids in the LNP.
Embodiment 13. The LNP of any one of embodiments 1 to 12, wherein the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1∶5 to 1∶10.
Embodiment 14. The LNP of any one of embodiments 1 to 13, wherein the polymer conjugated lipid is a PEGylated lipid.
Embodiment 15. The LNP of any one of embodiments 1 to 13, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
w is an integer ranging from 30 to 60.
Embodiment 16. The LNP of any one of embodiments 1 to 15, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
w is an integer ranging from 30 to 60.
Embodiment 17. The LNP of embodiment 15 or 16, wherein w is an integer ranging from 45 to 55.
Embodiment 18. The LNP of embodiment 15 or 16, wherein w is about 49.
Embodiment 19. The LNP of any one of embodiments 1 to 14, wherein the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
Embodiment 20. The LNP of any one of embodiments 1 to 19, further comprising a lipid stabilizer.
Embodiment 21. The LNP of embodiment 20, wherein the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10∶1 to 1∶4.
Embodiment 22. The LNP of embodiment 21, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 5∶1 to 1∶1.
Embodiment 23. The LNP of embodiment 21, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 4∶1 to 3∶1.
Embodiment 24. The LNP of any one of embodiments 20 to 23, wherein the lipid stabilizer comprises from 5 to 50 mol%of a total amount of lipids in the LNP.
Embodiment 25. The LNP of embodiment 24, wherein the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP.
Embodiment 26. The LNP of embodiment 24, wherein the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
Embodiment 27. The LNP of any one of embodiments 1 to 26, wherein the phospholipid comprises from 1 to 30 mol%of a total amount of lipids in the LNP.
Embodiment 28. The LNP of embodiment 27, wherein the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP.
Embodiment 29. The LNP of embodiment 27, wherein the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP.
Embodiment 30. The LNP of embodiment 27, wherein the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP.
Embodiment 31. The LNP of embodiment 27, wherein the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP.
Embodiment 32. The LNP of any one of embodiments 1 to 31, wherein the LNP has a size from 50 nm to 150 nm, as determined using dynamic light scattering.
Embodiment 33. The LNP of embodiment 32, wherein the size is from 60 nm to 140 nm.
Embodiment 34. The LNP of embodiment 32, wherein the size is from 80 nm to 100 nm.
Embodiment 35. The LNP of embodiment 32, wherein the size is from 85 nm to 95 nm.
Embodiment 36. The LNP of any one of embodiments 1 to 35, wherein the LNP encapsulates mRNA.
Embodiment 37. A composition comprising lipid nanoparticles (LNPs) , wherein each LNP is an LNP of any one of embodiments 1 to 36.
Embodiment 38. The composition of embodiment 37, wherein at least 80%of the LNPs encapsulate mRNA.
Embodiment 39. The composition of embodiment 37, wherein at least 85%of the LNPs encapsulate mRNA.
Embodiment 40. A method for expressing protein in a cell, comprising introducing the LNP of embodiment 36 or the composition of embodiment 38 or 39, to the cell.
Embodiment 41. The method of embodiment 40, wherein the cell is a mammalian cell.
Embodiment 42. A method for delivering a protein to a subject, comprising administering the LNP of embodiment 36 or the composition of embodiment 38 or 39 to the individual, wherein the mRNA encodes the protein.
Embodiment 43. The method of embodiment 42, wherein the LNP or the composition is administered systemically.
Embodiment 44. The method of embodiment 42, wherein the subject is a mammal.
Embodiment 45. The method of embodiment 42, wherein the subject is a human.
Embodiment 46. A lipid nanoparticle (LNP) comprising a phospholipid, wherein the phospholipid has a structure selected from:
Embodiment 47. The LNP of embodiment 46, wherein the phospholipid has the structure:
Embodiment 48. The LNP of embodiment 46, wherein the phospholipid has the structure:
Embodiment 49. The LNP of any one of embodiments 46-48, further comprising an ionizable lipid.
Embodiment 50. The LNP of embodiment 49, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20∶1 to 2∶1.
Embodiment 51. The LNP of embodiment 50, wherein the molar ratio of ionizable lipid to phospholipid is from 15∶1 to 5∶1.
Embodiment 52. The LNP of embodiment 49, wherein the ionizable lipid comprises from 40 to 80 mol%of a total amount oflipids in the LNP.
Embodiment 53. The LNP of embodiment 52, wherein the ionizable lipid comprises from 50 to 70 mol%of the total amount of lipids in the LNP.
Embodiment 54. The LNP of any one of embodiments 49 to 53, wherein the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, or wherein the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
Embodiment 55. The LNP of any one of embodiments 49 to 54, wherein the ionizable lipid is a cationic lipid.
Embodiment 56. The LNP of any one of embodiments 46 to 55, further comprising a polymer conjugated lipid.
Embodiment 57. The LNP of embodiment 56, wherein the polymer conjugated lipid comprises from 1 to 2%of a total amount oflipids in the LNP.
Embodiment 58. The LNP of embodiment 57, wherein the polymer conjugated lipid comprises 1.5%of the total amount of lipids in the LNP.
Embodiment 59. The LNP of any one of embodiments 46 to 58, wherein the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid from 1∶5 to 1∶10.
Embodiment 60. The LNP of any one of embodiments 56 to 59, wherein the polymer conjugated lipid is a PEGylated lipid.
Embodiment 61. The LNP of any one of embodiments 56 to 60, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
w is an integer ranging from 30 to 60.
Embodiment 62. The LNP of any one of embodiments 56 to 61, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
or a pharmaceutically acceptable salt thereof, wherein
w is an integer ranging from 30 to 60.
Embodiment 63. The LNP of embodiment 61 or 62, wherein w is an integer ranging from 45 to 55.
Embodiment 64. The LNP of embodiment 61 or 62, wherein w is about 49.
Embodiment 65. The LNP of any one of embodiments 56 to 60, wherein the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
Embodiment 66. The LNP of any one of embodiments 46 to 65, further comprising a lipid stabilizer.
Embodiment 67. The LNP of embodiment 66, wherein the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10∶1 to 1∶4.
Embodiment 68. The LNP of embodiment 67, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 5∶1 to 1∶1.
Embodiment 69. The LNP of embodiment 67, wherein the molar ratio of the lipid stabilizer to the phospholipid is from 4∶1 to 3∶1.
Embodiment 70. The LNP of any one of embodiments 66 to 69, wherein the lipid stabilizer comprises from 5 to 50 mol%of a total amount of lipids in the LNP.
Embodiment 71. The LNP of embodiment 70, wherein the lipid stabilizer comprises from 8 to 40 mol%of the total amount of lipids in the LNP.
Embodiment 72. The LNP of embodiment 70, wherein the lipid stabilizer comprises from 10 to 30 mol%of the total amount of lipids in the LNP.
Embodiment 73. The LNP of any one of embodiments 46 to 72, wherein the phospholipid comprises from 1 to 30 mol%of a total amount of lipids in the LNP.
Embodiment 74. The LNP of embodiment 73, wherein the phospholipid comprises from 2 to 25 mol%of the total amount of lipids in the LNP.
Embodiment 75. The LNP of embodiment 73, wherein the phospholipid comprises from 3 to 20 mol%of the total amount of lipids in the LNP.
Embodiment 76. The LNP of embodiment 73, wherein the phospholipid comprises from 5 to 15 mol%of the total amount of lipids in the LNP.
Embodiment 77. The LNP of embodiment 73, wherein the phospholipid comprises about 10 mol%of the total amount of lipids in the LNP.
Embodiment 78. The LNP of any one of embodiments 46 to 77, wherein the LNP has a size from 50 nm to 150 nm, as determined using dynamic light scattering.
Embodiment 79. The LNP of embodiment 78, wherein the size is from 60 nm to 140 nm.
Embodiment 80. The LNP of embodiment 78, wherein the size is from 80 nm to 100 nm.
Embodiment 81. The LNP of embodiment 78, wherein the size is from 85 nm to 95 nm.
Embodiment 82. The LNP of any one of embodiments 46 to 81, wherein the LNP encapsulates mRNA.
Embodiment 83. A composition comprising lipid nanoparticles (LNPs) , wherein each LNP is an LNP of any one of embodiments 46 to 82.
Embodiment 84. The composition of embodiment 83, wherein at least 80%of the LNPs encapsulate mRNA.
Embodiment 85. The composition of embodiment 83, wherein at least 85%of the LNPs encapsulate mRNA.
Embodiment 86. A method for expressing protein in a cell, comprising introducing the LNP of embodiment 82 or the composition of embodiment 84 or 85, to the cell.
Embodiment 87. The method of embodiment 86, wherein the cell is a mammalian cell.
Embodiment 88. A method for delivering a protein to a subject, comprising administering the LNP of embodiment 82 or the composition of embodiment 84 or 85 to the individual, wherein the mRNA encodes the protein.
Embodiment 89. The method of embodiment 88, wherein the LNP or the composition is administered systemically.
Embodiment 90. The method of embodiment 88, wherein the subject is a mammal.
Embodiment 91. The method of embodiment 88, wherein the subject is a human.
EXAMPLES
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
General preparative High Performance Liquid Chromatography (HPLC) method: HPLC purification is carried out on a Waters 2767 equipped with a diode array detector (DAD) on an Inertsil Pre-C8 OBD column, generally with water containing 0.1%trifluoroacetic acid (TFA) as solvent A and acetonitrile as solvent B.
General Liquid Chromatography-Mass Spectrometry (LCMS) method: LCMS analysis is conducted on a Shimadzu (LC-MS2020) System. Chromatography is performed on a SunFire C18, generally with water containing 0.1%formic acid as solvent A and acetonitrile containing 0.1%formic acid as solvent B.
The Abbreviation OChemsPC refers to the following compound:
The Abbreviation PChemsPC refers to the following compound:
The Abbreviation DChemsPC refers to the following compound:
“DSPC” refers to distearoylphosphatidylcholine. “compound 01-1” refers to compound 01-1 in Table 1. “Chol” is an abbreviation for cholesterol. “DMG-PEG” refers to 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. ALC-0315 refers to compound 06-1, MC3 refers to compound 07-I.
Example 1. Preparation of Compound 02-1 and Compound 02-3
Compound 02-1 was prepared according to the scheme below.
Compound 02-1: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H) , 1.27-1.63 (m, 53H) , 1.97-2.01 (m, 2H) , 2.28-2.64 (m, 14H) , 3.52-3.58 (m, 2H) , 4.00-4.10 (m, 8H) . LCMS: Rt: 1.080 min; MS m/z (ESI) : 826.0 [M+H] +.
Compound 02-3 was prepared in analogous fashion as Compound 02-1, using corresponding starting material.
Example 2. Preparation of Compound 02-2
Compound 02-2 was prepared according to the scheme below.
Compound 02-2: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H) , 1.28-1.67 (m, 54H) , 1.88-2.01 (m, 7H) , 2.28-2.56 (m, 18H) , 3.16-3.20 (m, 1H) , 3.52-3.54 (m, 2H) , 4.00-4.10 (m, 8H) . LCMS: Rt: 1.060 min; MS m/z (ESI) : 923.0 [M+H] +.
Example 3. Preparation of Compound 02-4
Compound 02-4 was prepared according to the scheme below.
Compound 02-4: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H) , 1.26-1.32 (m, 34H) , 1.41-1.49 (m, 4H) , 1.61-1.66 (m, 15H) , 2.00-2.03 (m, 1H) , 2.21-2.38 (m, 8H) , 2.43-2.47 (m, 4H) , 2.56-2.60 (m, 2H) , 3.50-3.54 (m, 2H) , 4.03-4.14 (m, 8H) . LCMS: Rt: 1.030 min; MS m/z (ESI) : 798.0 [M+H] +.
Example 4. Preparation of Compound 02-9 and Compound 02-14
Compound 02-9 was prepared according to the scheme below.
Compound 02-9: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H) , 1.28-1.30 (m, 33H) , 1.58-2.01 (m, 18H) , 2.30-2.54 (m, 18H) , 3.10-3.19 (m, 1H) , 3.52-3.68 (m, 8H) , 4.09-4.20 (m, 8H) . LCMS: Rt: 1.677 min; MS m/z (ESI) : 927.7 [M+H] +.
Compound 02-14 was prepared in analogous fashion as Compound 02-9, using corresponding starting material.
Example 5. Preparation of Compound 02-10 and Compound 02-11
Compound 02-10 was prepared according to the scheme below.
Compound 02-10: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H) , 1.26-1.41 (m, 48H) , 1.51-1.72 (m, 11H) , 1.94-2.03 (m, 1H) , 2.29-2.32 (m, 6H) , 2.41-2.91 (m, 5H) , 3.51-3.76 (m, 2H) , 3.96-4.10 (m, 6H) . LCMS: Rt: 1.327 min; MS m/z (ESI) : 782.6 [M+H] +.
Compound 02-11 was prepared in analogous fashion as Compound 02-10, using corresponding starting material.
Example 6. Preparation of Compound 02-12
Compound 02-12 was prepared according to the scheme below.
Compound 02-12: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.89 (m, 18H) , 1.25-1.35 (m, 53H) , 1.41-1.48 (m, 8H) , 1.56-1.61 (m, 20H) , 1.95-2.01 (m, 2H) , 2.28-2.35 (m, 6H) , 2.43-2.46 (m, 4H) , 2.56-2.58 (m, 2H) , 3.51-3.54 (m, 2H) , 4.00-4.10 (m, 8H) . LCMS: Rt: 0.080 min; MS m/z (ESI) : 1050.8 [M+H] +.
Example 7. Preparation of Compound 02-20
Compound 02-20 was prepared according to the scheme below.
Compound 02-20: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H) , 1.25-1.36 (m, 48H) , 1.41-1.48 (m, 5H) , 1.60-1.62 (m, 8H) , 1.97-2.00 (m, 1H) , 2.27-2.32 (m, 6H) , 2.43-2.46 (m, 4H) , 2.56-2.59 (m, 2H) , 3.52-3.54 (m, 2H) , 4.01-4.10 (m, 6H) . LCMS: Rt: 0.093 min; MS m/z (ESI) : 782.6 [M+H] +.
Example 8. Preparation of Compound 04-1
Compound 04-1 was prepared according to the scheme below.
LCMS for compound 1-1 of Example 8 -Rt: 0.750 min; MS m/z (ESI) : 206.2 [M+H] +.
LCMS for compound 1-2 of Example 8 -Rt: 0.870 min; MS m/z (ESI) : 448.3 [M+H] +.
LCMS for compound 1-3 of Example 8 -Rt: 1.360 min; MS m/z (ESI) : 616.5 [M+H] +.
Compound 04-1: 1H NMR (400 MHz, CDCl3) δ: 0.79-0.83 (m, 6H) , 1.14-1.26 (m, 38H) , 1.47-1.61 (m, 6H) , 1.86-1.96 (m, 4H) , 2.51-2.58 (m, 4H) , 3.17 (s, 1H) , 3.32-3.44 (m, 5H) , 3.51-3.66 (m, 3H) . LCMS: Rt: 0.94 min; MS m/z (ESI) : 526.5 [M+H] +.
Example 9. Preparation of Compound 04-2
Compound 04-2 was prepared according to the scheme below.
LCMS for compound 2-1 of Example 9 -Rt: 1.340 min; MS m/z (ESI) : 630.5 [M+H] +.
Compound 04-2: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 6H) , 1.25-1.33 (m, 35H) , 1.50-1.69 (m, 7H) , 1.87-1.99 (m, 1H) , 2.00-2.08 (m, 2H) , 2.33 (t, J=7.6 Hz, 2H) , 2.56-2.81 (m, 4H) , 3.17-3.27 (m, 1H) , 3.38-3.48 (m, 3H) , 3.50-3.65 (m, 3H) , 5.08-5.14 (m, 1H) . LCMS: Rt: 1.180 min; MS m/z (ESI) : 540.4 [M+H] +.
Example 10. Preparation of Compound 04-7
Compound 04-7 was prepared according to the scheme below.
LCMS for compound 7-1 of Example 10 -LCMS: Rt: 0.780 min; MS m/z (ESI) : 427.4 [M+H] +.
Compound 04-7: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H) , 1.26-1.35 (m, 45H) , 1.41-1.67 (m, 7H) , 2.28-2.32 (m, 3H) , 2.36-2.70 (m, 11H) , 2.79-2.83 (m, 2H) , 3.35-3.46 (m, 4H) , 3.77-3.85 (m, 1H) , 3.96-3.97 (m, 2H) . LCMS: Rt: 1.220 min; MS m/z (ESI) : 669.6 [M+H] +.
Example 11. Preparation of Compound 04-8
Compound 04-8 was prepared according to the scheme below.
LCMS for compound 8-1 of Example 11 -LCMS: Rt: 0.730 min; MS m/z (ESI) : 371.3 [M+H] +.
Compound 04-8: 1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 9H) , 1.25-1.27 (m, 47H) , 1.40-1.49 (m, 4H) , 1.56-1.73 (m, 8H) , 2.30 (t, J=7.6 Hz, 3H) , 2.40-2.82 (m, 10H) , 3.32-3.38 (m, 1H) , 3.43-3.46 (m, 3H) , 3.70-3.80 (m, 1H) , 3.92-3.97 (m, 2H) . LCMS: Rt: 1.090 min; MS m/z (ESI) : 709.6 [M+H] +.
Example 12. Preparation of Compound 04-65, Compound 04-66 and Compound 04-67
Compound 04-65 was prepared according to the scheme below.
Compound 04-65: 1H NMR (400 MHz, CCl3D) : δ: 0.79-0.83 (m, 12H) , 1.23-1.27 (m, 62H) , 1.29-1.37 (m, 2H) , 1.51-1.61 (m, 2H) , 1.76-1.93 (m, 7H) , 2.13-2.16 (m, 4H) , 2.17-2.25 (m, 3H) , 2.41-2.51 (m, 7H) , 3.05-3.06 (m, 1H) , 3.52-3.54 (m. 2H) , 3.92-4.03 (m, 4H) . LCMS: Rt: 0.588 min; MS m/z (ESI) : 863.6 [M+H] +.
Compounds 04-66 and 04-67 were prepared in analogous fashion as Compound 04-65, using corresponding starting material.
Example 13. Preparation of Compound 04-68
Compound 04-68 was prepared according to the scheme below.
1H NMR for compound 68-2 of Example 13 -1H NMR (400 MHz, CDCl3) δ: 0.86-0.90 (m, 12H) , 1.26-1.46 (m, 53H) , 1.56-1.62 (m, 2H) , 1.83 (s, 2H) , 1.96-2.02 (m, 1H) , 2.23-2.24 (m, 4H) , 3.64 (s, 2H) , 4.02-4.11 (m, 4H) .
Compound 04-68: 1H NMR (400 MHz, CDCl3) δ: 0.83-0.92 (m, 12H) , 1.17-1.37 (m, 56H) , 1.38-1.45 (m, 2H) , 1.64-1.67 (m, 2H) , 1.70-1.86 (m, 6H) , 1.92-2.04 (m, 2H) , 2.19-2.26 (m, 4H) , 2.40-2.49 (m, 3H) , 2.57-2.65 (m, 2H) , 3.41-3.51 (m, 2H) , 3.97-4.12 (m, 4H) . LCMS: Rt: 0.080 min; MS m/z (ESI) : 778.5 [M+H] +.
Example 14. Preparation of Compound 04-69, Compound 04-79 and Compound 04-80
Compound 04-69 was prepared according to the scheme below.
LCMS of compound 69-1 of Example 14 -Rt: 1.290 min; MS m/z (ESI) : 750.7 [M+H] +.
Compound 04-69: 1H NMR (400 MHz, CDCl3) δ: 0.83-0.92 (m, 12H) , 0.98-1.06 (m, 3H) , 1.17-1.47 (m, 52H) , 1.54-1.72 (m, 5H) , 1.78-2.06 (m, 8H) , 2.20-2.27 (m, 4H) , 2.37-2.46 (m, 4H) , 2.49-2.66 (m, 5H) , 3.01-3.12 (m, 1H) , 3.52-3.59 (m, 2H) , 3.98-4.11 (m, 4H) . LCMS: Rt: 0.093 min; MS m/z (ESI) : 821.6 [M+H] +.
Compounds 04-79 and 04-80 were prepared in an analogous fashion as compound 04-69, using corresponding starting material.

Example 15. Initial screening of LNPs comprising a steroid containing phospholipid
Briefly, the specified amounts of the lipid components were solubilized in ethanol at the specified molar ratios (see Table 6) . The mRNA was diluted in 10 to 50 mM citrate buffer, pH = 3-5. The LNPs were prepared at a total lipid to mRNA weight ratio of approximately 10: 1 to 30: 1 by mixing the ethanolic lipid solution with the aqueous mRNA solution at a volume ratio of 1: 3 using a microfluidic apparatus, total flow rate ranging from 9-30 mL/min. Ethanol was thereby removed and replaced by Dulbecco′sphosphate-buffered saline (DPBS) using dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter.
Lipid nanoparticle size were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) using a 173° backscatter detection mode. The encapsulation efficiency of lipid nanoparticles was determined using a Quant-it Ribogreen RNA quantification assay kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.
To measure the size and polydispersibility index (PDI) of lipid nanoparticle formulations were diluted 20-fold in PBS and transferred 1 mL in measurement cuvette. The LNP encapsulation efficiency (EE%) was determined using a Quant-it RiboGreen RNA assay kit, LNP formulations were diluted to 0.5 μg/mL in Tris-EDTA and 0.1%Triton respectively. In order to determine free RNA and total RNA fluorescence intensity, ribogreen reagent were diluted 200-fold with Tris-EDTA buffer and mix at the same volume as diluted LNP formulation. Fluorescence intensity was measured at room temperature in a Molecular Devices Spectramax iD3 spectrometer using excitation and emission wavelengths of 488 nm and 525 nm. EE%was calculated based on the ratio of encapsulated to total RNA fluorescence intensity.
Table 6: Expression levels of LNPs comprising a steroid containing phospholipid
Lipid nanoparticles encapsulating human erythropoietin (hEPO) mRNA were prepared as described above, and systemically administered to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at 0.5mg/kg dose by tail vein injection. Mice were euthanized by CO2 overdoses at 6 hours post administration, and blood samples were taken for hEPO measurement. Particularly, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 ℃, snap-frozen and stored at -80 ℃ for analysis. The serum hEPO level was measured using an ELISA assay carried out using a commercial kit (DEP00, R&D systems) according to manufacturer's instructions. The hEPO expression levels (μg/ml) measured from the tested group are plotted in FIG. 1 and summarized in Table 6.
As shown, replacing DSPC with PChemsPC and adjustment of the molar ratio significantly increased EPO-LNP in-vivo protein expression by 2-3 fold.
Example 16. Expression levels of loaded LNPs comprising a steroid containing phospholipid
This study examined in vivo expression levels of LNP formulations containing 45%-75%compound 01-1 with DSPC or PChemsPC. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15. Results are shown in FIG. 2 and Table 7.
Of the DSPC LNPs tested, LNPs containing 45-60 mol%compound 01-1 resulted in the highest protein expression level. For PChemsPC LNPs, 60 -75 mol%compound 01-1 resulted in the highest protein expression level.
Table 7. Further expression levels of LNPs comprising a steroid containing phospholipid
Example 17. Screen of %PChemsPC for optimal protein expression
A further screen was conducted to examine in vivo expression levels of LNP formulations containing 5-25 mol%PChemsPC with different amounts of compound 01-1. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15. Results are shown in FIG. 3 and Table 8.
Of the PChemsPC LNPs tested, 5-10 mol%PChemsPC had the highest protein expression level. The optimal amount of PChemsPC varied with the amount of compound 01-1. When the mol%of PChemsPC exceeded 15%, protein expression appeared to decrease.
Table 8. Additional expression levels of LNPs comprising a steroid containing phospholipid.
Example 18. Screen of LNP compositions with a steroid containing phospholipid for optimal protein expression
This study examined in-vivo EPO expression level of PChemsPC LNPs with different compositions. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15. In this study, a molar ratio of 50%-75%compound 01-1, 18.5%-38.5%cholesterol, 5-15%PChemsPC were tested to achieve acceptable protein expression levels. Corresponding LNP characterizations were listed in Table 9.
Table 9. Additional expression levels of LNPs comprising a steroid containing phospholipid with different molar ratios.
It can be seen from Table 9 that within LNP formulations comprsing 60-75 mol%cationic lipids, 5-10 mol%PChemsPC, and 18.5-28.5 mol%cholesterol can result in a high protein expression and the physicochemical properties thereof such as sizes, polydispersibility index and encapsulation efficiency are acceptable.
Example 19. Tissue-specific Expression of Nucleic Acid Molecules Delivered in LNP formulations.
To study the tissue biodistribution of LNPs in mice, LNP formulations listed in Table 10 containing mRNA encoding luciferase were prepared as described in Example 15.
Table 10. LNP compositions and physical characterizations

Each formulation was systematically administered to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at a 0.25 mg/kg dose by tail vein injection. After 6 hours, the mice were subcutaneously administered with XenoLight D-luciferin (potassium salt) , a substrate ofluciferase that catalyze the production of luminescence. The mice were subsequently euthanized by CO2 overdoses 15 min thereafter. Mice tissues were harvested and placed in a luminescence imaging scanner to measure the expression level of luciferase in each tissue. The luminescence levels measured from harvest liver tissues were plotted in FIG. 4, showing the mean value and standard deviation (SD) of at least five repeated tested animals for each group.
As shown in FIG. 4, LNPs composed of a steroid containing phospholipid (e.g. PChemsPC, OChemsPC, DChemsPC) yield higher liver signal, in comparison to DSPC control. A molar ratio of 50%-75%compound 01-1 with PChemsPC yields higher liver luminescence signal.
The percentage of luminescence intensity in different tissues were calculated and plotted in FIG. 5.
As shown in FIG. 5, after injection, DSPC LNP leads to 96%liver distribution with around 3%spleen distribution, while steroid containing phospholipid LNP shows 98 -99%liver distribution, with 0.4%spleen distribution. It can be seen that steroid-modified phospholipid LNP reveals better liver-tropism.
Example 20. Characterization of sterol-modified phospholipid LNP with different ionizable lipids
LNP formulations containing PChemsPC were prepared with different ionizable lipids. The LNP formulations were composed of a ionizable lipid at a molar ratio of 65%, a PChemsPC lipid at a molar ratio of 10%, a cholesterol-based lipid at a molar ratio of 23.5%and a PEGylated lipid at a molar ratio of 1.5%. Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15.
Compound 01-1with DSPC control was used as a comparison group here in this study. Corresponding LNP characterizations with different ionizable lipids were listed in Table 11.
Table 11. LNP characterizations with different ionizable lipids.
As shown in Table 11, most of the ionizable lipids show better protein expression level compared to compound 01-1 control, indicates that PChemsPC is widely compatible with most  of the ionizable lipids. The combination of PChemsPC and Compound 02-3 can yield at most 2.8-fold protein expression level escalation.
Additionally, for commercial lipids ALC-0315 and MC3, the effect of replacement of DSPC with PChemsPC on in-vivo protein expression level was also studied. In this study, the LNP formulations were composed of an ionizable lipid at a molar ratio of 50%-65%, a phospholipid at a molar ratio of 10%, a cholesterol-based lipid at a molar ratio of 23.5%-38.5%and a PEGylated lipid at a molar ratio of 1.5%. LNP characterizations were listed in Table 12.
Table 12. Physical characterizations of ALC-0315 and MC3 LNPs.
In vivo hEPO expression levels fold change were depicted in FIG. 6.
As shown in FIG. 6, replacement of DSPC with PChemsPC can significantly improve in-vivo protein expression level by 1.6-1.8 fold.
Example 21. Characterization of in vivo serum cytokines post injection of a steroid containing phospholipid LNP
As an allogenic substance, LNP injection will lead to significant increase of pro-inflammatory cytokines, such as inteleukin-6 (IL-6) , tumor necrosis factor-alpha (TNF-α) , interferon-gamma (INF-γ) , interferon-alpha (IFN-α) , which can cause innate immune response and leads to undesirable side effects. This study evaluates serum cytokine levels after LNP systematically administration, comparing the serum cytokine boosted by DSPC LNP and PChemsPC LNP.
Lipid nanoparticles containing human erythropoietin (hEPO) mRNA were prepared as described in Example 15, and systemically administered to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) at 0.5mg/kg dose by tail vein injection. Mice were euthanized by CO2 overdoses at 6 hours post administration, and blood samples were taken for cytokines measurement.  Particularly, sera were separated from total blood by centrifugation at 5000g for 10 minutes at 4 ℃, snap-frozen and stored at -80 ℃ for analysis.
DSPC LNPs were composed of ionizable lipid with a molar ratio of 50%, DSPC with a molar ratio of 10%, cholesterol with a molar ratio of 38.5%and a molar ratio of 1.5%for PEGylated lipid. PChemsPC LNPs were composed of ionizable lipid with a molar ratio of 65%, PChemsPC with a molar ratio of 10%, a molar ratio of 23.5%and 1.5%for cholesterol and PEGylated lipids respectively. Several ionizable lipids (e.g. compound 01-1, lipid 5, SM-102, ALC-0315, Compound 03-135) were tested in this study.
The results were shown in FIGs. 7-11. As shown in FIGs. 7-11, PChemsPC LNP can singnificantly deminish IL-6 levels compared to corresponding DSPC control. Especially for ALC-0315 LNP, around 9-fold drop of IL-6 level were observed, indicating that PChemsPC LNPs have better safety post administration.
Example 22. Characterization of in vivo serum cytokines after administration of a steroid containing phospholipid LNP with self-amplifying mRNA (saRNA)
In this study, lipid nanoparticles containing human erythropoietin (hEPO) self-amplifying mRNA were prepared as described in Example 15. After tail vein injection, mice were euthanized by CO2 overdoses at 6 hours post administration, and blood samples were taken for cytokines measurement. Cytokine levels were measured and plotted in FIG. 12 and FIG. 13.
As shown in FIG. 12 and FIG. 13, PChemsPC LNPs lead to significantly lower IL-6, IFN-α, TNF-α levels.
Example 23. Characterization of sterol-modified phospholipid LNP with CD3-CD19 mRNA
In this study, DSPC LNPs were composed of compound 01-1 with a molar ratio of 50%, DSPC with a molar ratio of 10%, cholesterol with a molar ratio of 38.5%and a molar ratio of 1.5%for PEGylated lipid. PChemsPC LNPs were composed of compound 01-1 with a molar ratio of 65%, PChemsPC with a molar ratio of 10%, a molar ratio of 23.5%and 1.5%for cholesterol and PEGylated lipids respectively.
Lipid nanoparticles encapsulating CD19-CD3 mRNA were prepared as described in Example 15, and systemically administered to 6-8 week old female Balb/c mice (Xipuer-Bikai, Shanghai) at 0.3mg/kg dose by tail vein injection. Mice were euthanized by CO2 overdoses at 6  hours post administration, and blood samples were taken for antibody measurement. Particularly, serum was separated from total blood by centrifugation at 5000g for 10 minutes at 4 ℃, snap-frozen and stored at -80 ℃ for analysis. The serum antibody level was shown in FIG. 14.
As shown in FIG. 14, PChemsPC LNP can significantly improve CD3-CD19 antibody expression level post administration.

Claims (53)

  1. A lipid nanoparticle (LNP) comprising
    a phospholipid containing a sterol moiety;
    an ionizable lipid; and
    a polymer conjugated lipid.
  2. The LNP of claim 1, wherein the phospholipid has a structure selected from:
  3. The LNP of claim 1 or 2, wherein the phospholipid comprises from 1 to 30 mol%, preferably from 2 to 25 mol%, preferably from 3 to 20 mol%, more preferably from 5 to 15 mol%of the total amount of lipids in the LNP.
  4. The LNP of any one of claims 1 to 3, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20∶1 to 2∶1, preferably from 18∶1 to 2.5∶1, preferably from 16∶1 to 4∶1, more preferably from 15∶1 to 5∶1.
  5. The LNP of any one of claims 1 to 4, wherein the ionizable lipid is a cationic lipid, preferably the ionizable lipid is a compound according to any one of the formula selected from  01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, more preferably the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
  6. The LNP of any one of claims 1 to 4, wherein the ionizable lipid is selected from
    compounds of Formula (01-I-O) :
    wherein y and z are each independently an integer from 4 to 6,
    s is an integer from 2 to 4,
    t is an integer from 1 to 3, and
    R1 and R2 are each independently C12-C22 alkyl;
    R4 is C3-C8 cycloalkyl;
    R6 is hydrogen or hydroxyl,
    compounds of Formula 05-I:
    wherein
    l is selected from 1, 2, 3, 4, and 5;
    m is selected from 5, 6, 7, 8, and 9;
    M1 is -C (O) O-;
    R4 is - (CH2) nOH, and n is selected from 1, 2, 3, 4, or 5;
    M is -OC (O) -; and
    R2 and R3 are both C6-10 alkyl,
    compounds of Formula (06-I) :
    wherein
    L1 and L2 is -O (C=O) -;
    G1 and G2 are each independently unsubstituted C4-C8 alkylene;
    G3 is C3-C8 alkylene;
    R1 and R2 are each independently C12-C22 alkyl;
    R3 is H or OH,
    Compounds of Formula (02-V-B)
    wherein
    each L1 is independently -OC (=O) R1;
    each L2 is independently -OC (=O) R2;
    R1 and R2 are each independently C6-C24 alky;
    R3 is -OR6;
    R6 is hydrogen;
    z is an integer from 2 to 12;
    x1 is an integer from 0 to 9;
    y1 is an integer from 0 to 9;
    Compounds of Formula (02-VI-F)
    wherein
    each L1 is independently -OC (=O) R1;
    each L2 is independently -OC (=O) R2;
    R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
    R3 is -OR6;
    R6 is hydrogen;
    z is an integer from 2 to 12;
    y is an integer from 2 to 12;
    x1 is an integer from 2 to 5;
    Compounds of Formula (02-V-F)
    wherein
    each L1 is independently -OC (=O) R1;
    each L2 is independently -OC (=O) R2;
    R1 and R2 are each independently C6-C24 alkyl;
    R3 is -OR6;
    R6 is hydrogen;
    z is an integer from 2 to 12;
    x2 is an integer from 2 to 9;
    x4 is an integer from 0 to 3;
    y2 is an integer from 2 to 9;
    y4 is an integer from 0 to 3,
    Compounds of formula (03-I)
    wherein
    G1 and G2 are each independently C3-C8 alkylene;
    each L1 is independently -OC (=O) R1 or -C (=O) OR1;
    each L2 is independently -C (=O) OR2 or -OC (=O) R2;
    R1 is independently C6-C24 alkyl;
    R2 is independently C6-C24 alkyl;
    G3 is C2-C12 alkylene;
    R3 is C3-C8 cycloalkyl;
    R4 is C1-C4 hydroxylalkyl;
    n is 1 or 2;
    m is 1 or 2, and
    compound 07-I:
  7. The LNP of claim 6, wherein the ionizable lipid is selected from the following compounds:


  8. The LNP of any one of claims 1 to 7, wherein the ionizable lipid comprises from 40 to 80 mol%, from 45 to 75 mol%, from 50 to 70 mol%, or from 60 to 65 mol%of a total amount of lipids in the LNP.
  9. The LNP of any one of claims 1 to 8, wherein the polymer conjugated lipid is a PEGylated lipid, preferably with the structure:
    or a pharmaceutically acceptable salt thereof, wherein
    R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
    w is an integer ranging from 30 to 60, preferably from 45 to 55, more preferably w is 49.
  10. The LNP of any one of claims 1 to 9, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
    or a pharmaceutically acceptable salt thereof, wherein
    w is an integer ranging from 30 to 60, preferably from 45 to 55, more preferably w is 49,
    preferably the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
  11. The LNP of any one of claims 1 to 10, wherein the polymer conjugated lipid comprises from 0.5 to 5 mol%, preferably from 1 to 2 mol%, more preferably 1.5 mol%of the total amount of lipids in the LNP.
  12. The LNP of any one of claims 1 to 11, wherein the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid of from 1∶2 to 1∶20, preferably from 1∶3 to 1∶18 or from 1∶5 to 1∶10.
  13. The LNP of any one of claims 1 to 12, further comprising a lipid stabilizer.
  14. The LNP of claim 13, wherein the lipid stabilizer is selected from sterols, corticosteroids, tomatidine, tomatine, ursolic acid, and alpha-tocopherol, preferably selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, prednisolone, dexamethasone, prednisone, hydrocortisone, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.
  15. The LNP of claim 13 or 14, wherein the lipid stabilizer comprises from 5 to 50 mol%, preferably from 8 to 40 mol%, more preferably from 10 to 30 mol%of a total amount of lipids in the LNP.
  16. The LNP of any of claims 13 to 15, wherein the LNP has a molar ratio of the lipid stabilizer to the phospholipid of from 10∶1 to 1∶4, preferably from 5∶1 to 1∶3.
  17. The LNP of any one of claims 1 to 16, wherein the LNP has a size of from 20 nm to 300 nm, preferably from 50 nm to 150 nm, preferably from 60 nm to 140 nm, more preferably from 80 nm to 100 nm, even more preferably from 85 nm to 95 nm, as determined using dynamic light scattering.
  18. A composition comprising lipid nanoparticles (LNPs) and a therapeutic payload, wherein each LNP is an LNP of any one of claims 1 to 17.
  19. The composition of claim 18, wherein at least 80%, preferably at least 85%of the LNPs encapsulate the therapeutic payload.
  20. The composition of claim 18 or 19, wherein the therapeutic payload is selected from nucleic acids, preferably selected from DNAs and RNAs, more preferably selected from catalytic DNA, plasmid DNA, aptamer, complementary DNA, mRNA, antisense oligonucleotide, miRNA, miRNA inhibitor, micRNA, multivalent RNA, dsRNA, shRNA, antisense RNA, tRNA, aiRNA, a ribozyme, an aptamer, and a vector.
  21. The composition of claim 18 or 19, wherein the therapeutic payload is a mRNA.
  22. A method for expressing protein in a cell, comprising introducing the composition of claim 21 to the cell.
  23. The method of claim 22, wherein the cell is a mammalian cell.
  24. A method for delivering a protein to a subject, comprising administering the composition of claim 21 to the subject, wherein the mRNA encodes the protein.
  25. The method of claim 24, wherein the composition is administered systemically.
  26. The method of claim 24 or 25, wherein the subject is a mammal, in particular, a human.
  27. A lipid nanoparticle (LNP) comprising a phospholipid, wherein the phospholipid has a structure selected from:

  28. The LNP of claim 27, further comprising an ionizable lipid.
  29. The LNP of claim 28, wherein the LNP has a molar ratio of the ionizable lipid to the phospholipid from 20∶1 to 2∶1, preferably from 18∶1 to 2.5∶1, preferably from 16∶1 to 4∶1, more preferably from 15∶1 to 5∶1.
  30. The LNP of claim 27 or 28, wherein the ionizable lipid comprises from 40 to 80 mol%, from 45 to 75 mol%, from 50 to 70 mol%, or from 60 to 65 mol%of a total amount of lipids in the LNP.
  31. The LNP of any one of claims 28 to 30, wherein the ionizable lipid is a cationic lipid, preferably the ionizable lipid is a compound according to any one of the formula selected from 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV, 05-I, 06-I, and sub-formulas thereof, more preferably the ionizable lipid is a cationic lipid selected from the compounds listed in any one of Tables 1 to 5.
  32. The LNP of any one of claims 28 to 30, wherein the ionizable lipid is selected from compounds of Formula (01-I-O) :
    wherein y and z are each independently an integer from 4 to 6,
    s is an integer from 2 to 4,
    t is an integer from 1 to 3, and
    R1 and R2 are each independently C12-C22 alkyl;
    R4 is C3-C8 cycloalkyl;
    R6 is hydrogen or hydroxyl,
    compounds of Formula 05-I:
    wherein
    l is selected from 1, 2, 3, 4, and 5;
    m is selected from 5, 6, 7, 8, and 9;
    M1 is -C (O) O-;
    R4 is - (CH2) nOH, and n is selected from 1, 2, 3, 4, or 5;
    M is -OC (O) -; and
    R2 and R3 are both C6-10 alkyl,
    compounds of Formula (06-I) :
    wherein
    L1 and L2 is -O (C=O) -;
    G1 and G2 are each independently unsubstituted C4-C8 alkylene;
    G3 is C3-C8 alkylene;
    R1 and R2 are each independently C12-C22 alkyl;
    R3 is H or OH,
    Compounds of Formula (02-V-B)
    wherein
    each L1 is independently -OC (=O) R1;
    each L2 is independently -OC (=O) R2;
    R1 and R2 are each independently C6-C24 alky;
    R3 is -OR6;
    R6 is hydrogen;
    z is an integer from 2 to 12;
    x1 is an integer from 0 to 9;
    y1 is an integer from 0 to 9;
    Compounds of Formula (02-VI-F)
    wherein
    each L1 is independently -OC (=O) R1;
    each L2 is independently -OC (=O) R2;
    R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
    R3 is -OR6;
    R6 is hydrogen;
    z is an integer from 2 to 12;
    y is an integer from 2 to 12;
    x1 is an integer from 2 to 5;
    Compounds of Formula (02-V-F)
    wherein
    each L1 is independently -OC (=O) R1;
    each L2 is independently -OC (=O) R2;
    R1 and R2 are each independently C6-C24 alkyl;
    R3 is -OR6;
    R6 is hydrogen;
    z is an integer from 2 to 12;
    x2 is an integer from 2 to 9;
    x4 is an integer from 0 to 3;
    y2 is an integer from 2 to 9;
    y4 is an integer from 0 to 3,
    Compounds of formula (03-I)
    wherein
    G1 and G2 are each independently C3-C8 alkylene;
    each L1 is independently -OC (=O) R1 or -C (=O) OR1;
    each L2 is independently -C (=O) OR2 or -OC (=O) R2;
    R1 is independently C6-C24 alkyl;
    R2 is independently C6-C24 alkyl;
    G3 is C2-C12 alkylene;
    R3 is C3-C8 cycloalkyl;
    R4 is C1-C4 hydroxylalkyl;
    n is 1 or 2;
    m is 1 or 2, and
    compound 07-I:
  33. The LNP of any one of claims 28 to 30, wherein the ionizable lipid is selected from the following compounds:


  34. The LNP of any one of claims 27 to 33, further comprising a polymer conjugated lipid.
  35. The LNP of claim 34, wherein the polymer conjugated lipid comprises from 0.5 to 5 mol%, preferably from 1 to 2 mol%, more preferably 1.5 mol%of the total amount of lipids in the LNP.
  36. The LNP of claim 34 or 35, wherein the LNP has a molar ratio of the polymer conjugated lipid to the phospholipid of from 1∶2 to 1∶20, preferably from 1∶3 to 1∶18 or from 1∶5 to 1∶10.
  37. The LNP of any one of claims 34 to 36, wherein the polymer conjugated lipid is a PEGylated lipid, preferably with the structure:
    or a pharmaceutically acceptable salt thereof, wherein
    R12 and R13 are each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and
    w is an integer ranging from 30 to 60, preferably from 45 to 55, more preferably w is 49.
  38. The LNP of any one of claims 34 to 36, wherein the polymer conjugated lipid is a PEGylated lipid with the structure:
    or a pharmaceutically acceptable salt thereof, wherein
    w is an integer ranging from 30 to 60, preferably from 45 to 55, more preferably w is 49preferably the polymer conjugated lipid is DMG-PEG or DMPE-PEG.
  39. The LNP of any one of claims 27 to 38, further comprising a lipid stabilizer.
  40. The LNP of claim 39, wherein the lipid stabilizer is selected from sterols, corticosteroids, tomatidine, tomatine, ursolic acid, and alpha-tocopherol, preferably selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, prednisolone, dexamethasone, prednisone, hydrocortisone, tomatidine, tomatine, ursolic acid, and alpha-tocopherol.
  41. The LNP of claim 39 or 40, wherein the LNP has a molar ratio of the lipid stabilizer to the phospholipid from 10∶1 to 1∶4, preferably from 5∶1 to 1∶3.
  42. The LNP of any one of claims 39 to 41, wherein the lipid stabilizer comprises from 5 to 50 mol%, preferably from 8 to 40 mol%, more preferably from 10 to 30 mol%of a total amount of lipids in the LNP.
  43. The LNP of any one of claims 27 to 42, wherein the phospholipid comprises from 1 to 30 mol%, preferably from 2 to 25 mol%, preferably from 3 to 20 mol%, more preferably from 5 to 15 mol%of a total amount of lipids in the LNP.
  44. The LNP of any one of claims 27 to 43, wherein the LNP has a size of from 20 nm to 300 nm, preferably from 50 nm to 150 nm, preferably from 60 nm to 140 nm, more preferably from 80 nm to 100 nm, even more preferably from 85 nm to 95 nm, as determined using dynamic light scattering.
  45. A composition comprising lipid nanoparticles (LNPs) and a therapeutic payload, wherein each LNP is an LNP of any one of claims 27 to 44.
  46. The composition of claim 45, wherein at least 80%, preferably at least 85%of the LNPs encapsulate the therapeutic payload.
  47. The composition of claim 45 or 46, wherein wherein the therapeutic payload is selected from nucleic acids, preferably selected from DNAs and RNAs, more preferably selected from  catalytic DNA, plasmid DNA, aptamer, complementary DNA, mRNA, antisense oligonucleotide, miRNA, miRNA inhibitor, micRNA, multivalent RNA, dsRNA, shRNA, antisense RNA, tRNA, aiRNA, a ribozyme, an aptamer, and a vector.
  48. The composition of claim 45 or 46, wherein the therapeutic payload is a mRNA.
  49. A method for expressing protein in a cell, comprising introducing the composition of claim 48, to the cell.
  50. The method of claim 48, wherein the cell is a mammalian cell.
  51. A method for delivering a protein to a subject, comprising administering the composition of claim 48 to the subject, wherein the mRNA encodes the protein.
  52. The method of claim 51, wherein the composition is administered systemically.
  53. The method of claim 51 or 52, wherein the subject is a mammal, in particular, a human.
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Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010144740A1 (en) * 2009-06-10 2010-12-16 Alnylam Pharmaceuticals, Inc. Improved lipid formulation
US9868691B2 (en) * 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2018081480A1 (en) * 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
CN108368028A (en) * 2015-10-28 2018-08-03 爱康泰生治疗公司 Novel lipid and lipid nanoparticle preparation for delivering nucleic acid
WO2019089828A1 (en) * 2017-10-31 2019-05-09 Acuitas Therapeutics, Inc. Lamellar lipid nanoparticles
WO2019141814A1 (en) * 2018-01-18 2019-07-25 Etherna Immunotherapies Nv Lipid nanoparticles
WO2020219941A1 (en) * 2019-04-26 2020-10-29 Genevant Sciences Gmbh Lipid nanoparticles
WO2021022173A1 (en) * 2019-07-31 2021-02-04 Modernatx, Inc. Compositions and methods for delivery of rna interference agents to immune cells
WO2021030701A1 (en) * 2019-08-14 2021-02-18 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
WO2021250263A1 (en) * 2020-06-11 2021-12-16 Etherna Immunotherapies Nv Lipid nanoparticles
WO2022112855A1 (en) * 2020-11-27 2022-06-02 Guangzhou Ribobio Co., Ltd Lipid compound and the composition thereof
WO2022152109A2 (en) * 2021-01-14 2022-07-21 Suzhou Abogen Biosciences Co., Ltd. Lipid compounds and lipid nanoparticle compositions
WO2023031392A2 (en) * 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031394A1 (en) * 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010144740A1 (en) * 2009-06-10 2010-12-16 Alnylam Pharmaceuticals, Inc. Improved lipid formulation
US9868691B2 (en) * 2015-09-17 2018-01-16 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
CN108368028A (en) * 2015-10-28 2018-08-03 爱康泰生治疗公司 Novel lipid and lipid nanoparticle preparation for delivering nucleic acid
WO2018081480A1 (en) * 2016-10-26 2018-05-03 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
WO2019089828A1 (en) * 2017-10-31 2019-05-09 Acuitas Therapeutics, Inc. Lamellar lipid nanoparticles
WO2019141814A1 (en) * 2018-01-18 2019-07-25 Etherna Immunotherapies Nv Lipid nanoparticles
WO2020219941A1 (en) * 2019-04-26 2020-10-29 Genevant Sciences Gmbh Lipid nanoparticles
CN114026233A (en) * 2019-04-26 2022-02-08 盖纳万科学有限公司 Lipid nanoparticles
WO2021022173A1 (en) * 2019-07-31 2021-02-04 Modernatx, Inc. Compositions and methods for delivery of rna interference agents to immune cells
WO2021030701A1 (en) * 2019-08-14 2021-02-18 Acuitas Therapeutics, Inc. Improved lipid nanoparticles for delivery of nucleic acids
WO2021250263A1 (en) * 2020-06-11 2021-12-16 Etherna Immunotherapies Nv Lipid nanoparticles
WO2022112855A1 (en) * 2020-11-27 2022-06-02 Guangzhou Ribobio Co., Ltd Lipid compound and the composition thereof
WO2022152109A2 (en) * 2021-01-14 2022-07-21 Suzhou Abogen Biosciences Co., Ltd. Lipid compounds and lipid nanoparticle compositions
WO2023031392A2 (en) * 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031394A1 (en) * 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FLASIŃSKI MICHAŁ, KONDERLA BEATA, BRONIATOWSKI MARCIN, WYDRO PAWEŁ: "Sterol–Phospholipid Hybrids at the Air/Water Interface: Studies on Properties and Interactions with Parent Lipid Molecules", LANGMUIR, AMERICAN CHEMICAL SOCIETY, US, vol. 32, no. 16, 26 April 2016 (2016-04-26), US , pages 4095 - 4102, XP093187646, ISSN: 0743-7463, DOI: 10.1021/acs.langmuir.5b04311 *

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