WO2024083172A1 - Lipid compound and lipid nanoparticle composition - Google Patents

Abstract

The present invention relates to a lipid compound and a lipid nanoparticle composition. The present invention specifically relates to a lipid compound, which can be used in combination with other lipid components such as neutral lipids, cholesterol, and polymer conjugated lipids to form lipid nanoparticles to deliver therapeutic agents, such as nucleic acid molecules, for therapeutic or prophylactic purposes. Further provided herein are a lipid nanoparticle composition comprising the lipid compound.

Description

Lipid compounds and lipid nanoparticle compositions Technical Field

The present invention generally relates to a lipid compound that can be used to combine with other lipid components (e.g., neutral lipids, cholesterol and polymer-conjugated lipids) to form lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, including nucleic acid mimetics such as locked (LNA), peptide nucleic acid (PNA) and morpholino oligonucleotides) intracellularly and extracellularly for therapeutic or preventive purposes including vaccination.

Background technique

Therapeutic nucleic acids have the potential to revolutionize vaccination, gene therapy, protein replacement therapy, and other genetic disease therapies. Since the first clinical studies of therapeutic nucleic acids began in the 2000s, significant progress has been made through the design of nucleic acid molecules and improvements in their delivery methods. However, nucleic acid therapeutics still face several challenges, including low cell permeability and high sensitivity to degradation of certain nucleic acid molecules (including RNA). Therefore, there is a need to develop new nucleic acid molecules and related methods and compositions to facilitate their delivery outside or inside cells for therapeutic and/or preventive purposes.

Summary of the invention

In one embodiment, lipid compounds are provided herein, including pharmaceutically acceptable salts or stereoisomers thereof, which can be used alone, or with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and/or their analogs, and/or with polymer-conjugated lipids, and/or polymers in combination, to form lipid nanoparticles for delivery of therapeutic agents (e.g., nucleic acid molecules, including nucleic acid mimetics such as locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino ring oligonucleotides). In some cases, lipid nanoparticles are used to deliver nucleic acids, such as antisense and/or messenger RNA. It also provides methods for treating various diseases or conditions (such as diseases or conditions caused by infectious entities and/or protein deficiencies) using such lipid nanoparticles.

In one embodiment, provided herein is a compound represented by formula (I):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein L ₁ , L ₂ , R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are as defined herein or elsewhere.

In one embodiment, the invention provides a nanoparticle composition comprising a compound provided herein and a therapeutic or prophylactic agent. In one embodiment, the therapeutic or prophylactic agent comprises at least one mRNA encoding an antigen or a fragment or epitope thereof.

It is obvious to those skilled in the art that the present invention has been described in detail for specific embodiments, however, it should be understood that it is only given in an illustrative manner rather than a restrictive manner, and various changes and modifications within the scope of the present invention will be obvious to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the total fluorescence intensity of the compound in mice;

FIG2 is a graph showing the fluorescence intensity of the compound in the liver;

FIG3 is a graph showing the fluorescence intensity of the compounds in the spleen.

Detailed ways

common technology

The techniques and methods described or referenced in the present invention include conventional methods that are generally easily understood or commonly used by those skilled in the art, such as the methods described in Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), etc.

the term

Unless otherwise described, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following terminology will be used for description, and where appropriate, terms used in the singular will also include the plural, and vice versa. The disclosures of all patents and other publications cited herein are incorporated herein by reference in their entirety. In the event that any description and interpretation of a term herein conflicts with any document incorporated herein by reference, the description and interpretation of the following terminology shall prevail.

Unless otherwise specified herein, the term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by generally having poor solubility in water but being soluble in many non-polar organic substances. Although lipids generally have poor solubility in water, certain classes of lipids (e.g., lipids modified with polar groups such as DMG-PEG2000) have limited water solubility and can be dissolved in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can generally be divided into at least three categories: (1) "simple lipids", including fats and oils as well as waxes; (2) "compound lipids", including phospholipids and glycolipids (such as DMPE-PEG2000); (3) "derivatized lipids", such as steroids, etc. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound", also referred to as "lipid-like", refers to lipid-like compounds such as amphiphilic compounds having the physical properties of lipids.

The term "lipid nanoparticle" or "LNP" refers to a particle with a nanometer scale (nm) (e.g., 1nm to 1,000nm) comprising one or more types of lipid molecules. LNP provided herein may further include at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, LNP comprises a non-lipid payload molecule partially or completely encapsulated inside a lipid shell. In particular, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. It is contemplated that cationic lipids can interact with negatively charged payload molecules and promote the incorporation and/or encapsulation of payloads into LNPs during LNP formation. As provided herein, other lipids that can form a part of LNP include, but are not limited to, neutral lipids and charged lipids, such as steroids, polymer-conjugated lipids and various zwitterionic lipids. In certain embodiments, LNP according to the present invention comprises one or more lipids of formula (I) (and its subformula) as described herein.

The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or a lipid that can respond to the pH value or hydrogen ion activity of its environment (e.g., its intended use environment) and is positively charged. Therefore, the term "cation" covers the scope of "permanent cation" and "cationizable". In certain embodiments, the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid includes a zwitterionic lipid that is positively charged in the environment in which it is intended to be used (e.g., at physiological pH). In certain embodiments, the cationic lipid is a lipid of one or more formulas (I) (and its subformulas) described herein.

The term "polymer-conjugated lipid" or "polymer-conjugated lipid" refers to a molecule that comprises both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid (PEG-lipid), wherein the polymer portion comprises polyethylene glycol.

The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or a neutral zwitterionic form at a selected pH. In some embodiments, the selected useful pH value or range corresponds to the pH conditions of the environment in which the lipid is intended to be used, such as physiological pH. As non-limiting examples, neutral lipids that can be used in conjunction with the disclosure herein include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 11,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-(((2,3-bis(oleyloxy)propyl))dimethylammoniumphosphate)ethylhydrogen (DOCP), sphingomyelin (SM), ceramides, steroids such as sterols and derivatives thereof. Neutral lipids can be synthetic or derived (isolated or modified) from natural sources or compounds.

The term "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form in a selected pH value or range. In some embodiments, the selected pH value or range corresponds to the pH conditions of the intended use environment of the lipid, such as physiological pH. As a non-limiting example, the charged lipid that can be used in conjunction with the disclosure herein includes but is not limited to phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG-Na) and 1,2-dioleoyl-sn-glycero-3-phospho-sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived (isolated or modified) from natural sources or compounds.

As used herein, unless otherwise specified, the term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting only of saturated carbon and hydrogen atoms. In one embodiment, the alkyl group has, for example, 1 to 24 carbon atoms (C ₁ -C ₂₄ alkyl), 4 to 20 carbon atoms (C ₄ -C ₂₀ alkyl), 10 to 20 carbon atoms (C ₁₀ -C ₂₀ alkyl), 6 to 16 carbon atoms (C ₆ -C ₁₆ alkyl), six to nine carbon atoms (C ₆ -C ₉ alkyl), one to fifteen carbon atoms (C ₁ -C ₁₅ alkyl), one to twelve carbon atoms (C ₁ -C ₁₂ alkyl), one to eight carbon atoms (C ₁ -C ₈ alkyl), or one to six carbon atoms (C ₁ -C ₆ alkyl), and is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified, alkyl groups are optionally substituted.

As described herein, unless otherwise indicated, the term "alkenyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds. As understood by those of ordinary skill in the art, the term "alkenyl" also includes groups with "cis" and "trans" configurations, or "E" and "Z" configurations. In one embodiment, the alkenyl group has, for example, 2 to 24 carbon atoms ( _C2 - _C24 alkenyl), 4 to 20 carbon atoms ( _C4 - _C20 alkenyl), 6 to 16 carbon atoms ( _C6 - _C16 alkenyl), six to nine carbon atoms ( _C6 - _C9 alkenyl), two to fifteen carbon atoms ( _C2 - _C15 alkenyl), two to twelve carbon atoms ( _C2 - _C12 alkenyl), two to eight carbon atoms ( _C2 - _C8 alkenyl) or 2 to 6 carbon atoms ( _C2 - _C6 alkenyl), and is connected to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, etc. Unless otherwise specified, alkenyl groups are optionally substituted.

As used herein, unless otherwise specified, the term "alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon triple bonds. In one embodiment, the alkynyl group has, for example, 2 to 24 The alkynyl radicals are alkynyl radicals having 4 to 20 carbon atoms (C ₄ -C ₂₀ alkynyl radicals), 6 to 16 carbon atoms (C _{6 -C 16 alkynyl} radicals), 6 to 9 carbon atoms (C ₆ -C ₉ alkynyl radicals), 2 to 15 carbon atoms (C ₂ -C ₁₅ alkynyl radicals), 2 to 12 carbon atoms (C ₂ -C ₁₂ alkynyl radicals), 2 to 8 carbon atoms (C ₂ -C ₈ alkynyl radicals), or 2 to 6 carbon atoms (C ₂ -C ₆ alkynyl radicals), and are connected to the rest of the molecule by a single bond. Examples of alkynyl radicals include, but are not limited to, ethynyl radicals, propynyl radicals, butynyl radicals, pentynyl radicals, and the like. Unless otherwise indicated, alkynyl radicals are optionally substituted.

As described herein, unless otherwise indicated, the term "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a group consisting only of saturated carbon and hydrogen. In one embodiment, the alkylene group has, for example, 1 to 24 carbon atoms (C ₁ -C ₂₄ alkylene), 1 to 15 carbon atoms (C ₁ -C ₁₅ alkylene), 1 to 12 carbon atoms (C ₁ -C ₁₂ alkylene), 1 to 8 carbon atoms (C ₁ -C ₈ alkylene), 1 to 6 carbon atoms (C ₁ -C ₆ alkylene), 2 to 4 carbon atoms (C ₂ -C ₄ alkylene), 1 to 2 carbon atoms (C ₁ -C ₂ alkylene). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butene, etc. The alkylene chain is connected to the rest of the molecule by a single bond and is connected to the free radical group by a single bond. The connection of the alkylene chain to the rest of the molecule and to the free radical group can be through one carbon or any two carbons in the chain. Unless otherwise stated, alkylene chains are optionally substituted.

As described herein, unless otherwise indicated, the term "alkenylene" refers to a straight or branched divalent hydrocarbon chain that connects the remainder of a molecule to a radical group consisting only of carbon and hydrogen, the radical group comprising one or more carbon-carbon double bonds. In one embodiment, alkenylene has, for example, 2 to 24 carbon atoms ( _C2 - _C24 alkenylene), 2 to 15 carbon atoms ( _C2 - _C15 alkenylene), 2 to 12 carbon atoms ( _C2 - _C12 alkenylene), 2 to 8 carbon atoms ( _C2 - _C8 alkenylene), 2 to 6 carbon atoms ( _C2 - _C6 alkenylene) or 2 to 4 carbon atoms ( _C2 - _C4 alkenylene). Examples of alkenylene include, but are not limited to, vinylene, propenylene, n-butenyl, etc. Alkenylene is connected to the remainder of the molecule by a single bond or double bond, and is connected to the radical group by a single bond or double bond. The connection of alkenylene to the remainder of the molecule and to the radical group can be through one carbon or any two carbons in the chain. Unless otherwise specified, an alkenylene group is optionally substituted.

As described herein, unless otherwise indicated, the term "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms and being saturated. Cycloalkyl can include condensed rings or bridged ring systems. In one embodiment, cycloalkyl has, for example, 3 to 15 ring carbon atoms (C ₃ -C ₁₅ cycloalkyl), 3 to 10 ring carbon atoms (C ₃ -C ₁₀ cycloalkyl) or 3 to 8 ring carbon atoms (C ₃ -C ₈ cycloalkyl). Cycloalkyl is connected to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, decahydroalkyl, 7,7-dimethyl-bicyclo [2.2.1] heptyl, etc. Unless otherwise indicated, cycloalkyl is optionally substituted.

As used herein, unless otherwise specified, the term "cycloalkylene" is a divalent cycloalkyl group. Unless otherwise specified, a cycloalkylene group is optionally substituted.

As described herein, unless otherwise indicated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms and including one or more carbon-carbon double bonds. The cycloalkenyl group may include a condensed ring or a bridged ring system. In one embodiment, the cycloalkenyl group has, for example, 3 to 15 ring carbon atoms ( _C3 - _C15 cycloalkenyl), 3 to 10 ring carbon atoms ( _C3 - _C10 cycloalkenyl) or 3 to 8 ring carbon atoms ( _C3 - _C8 cycloalkenyl). The cycloalkenyl group is connected to the remainder of the molecule by a single bond. The example of a monocyclic cycloalkenyl group includes but is not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl etc. Unless otherwise indicated, the cycloalkenyl group is optionally substituted.

As used herein, unless otherwise specified, the term "cycloalkenylene" is a divalent cycloalkenyl group. Unless otherwise specified, a cycloalkenylene group is optionally substituted.

As used herein, unless otherwise indicated, the term "heterocyclyl" refers to a non-aromatic radical containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus and sulfur. Aromatic group monocyclic or polycyclic part. The heterocyclic group can be connected to the main structure at any heteroatom or carbon atom. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, tetracyclic or other polycyclic system, wherein the polycyclic system can be a fused ring, a bridged ring or a spirocyclic system. The heterocyclic polycyclic system can include one or more heteroatoms in one or more rings. The heterocyclic group can be saturated or partially unsaturated. Saturated heterocycloalkyl can be referred to as "heterocycloalkyl". If the heterocyclic group contains at least one double bond, the partially unsaturated heterocycloalkyl can be referred to as "heterocycloalkenyl"; if the heterocyclic group contains at least one triple bond, it can be referred to as "heterocycloalkynyl". In one embodiment, the heterocyclic group has, for example, 3 to 18 ring atoms (3 to 18-membered heterocyclic group), 4 to 18 ring atoms (4 to 18-membered heterocyclic group), 5 to 18 ring atoms (5 to 18-membered heterocyclic group), 4 to 8 ring atoms (4 to 8-membered heterocyclic group) or 5 to 8 ring atoms (5 to 8-membered heterocyclic group). In this article, whenever it appears, a numerical range such as "3 to 18" refers to each integer in a given range. For example, "3 to 18-membered heterocyclic radical" means that the heterocyclic radical can be composed of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, up to 18 ring atoms, etc. Examples of heterocyclic radicals include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinylpyrrolyl, isothiazolyl, furanyl, furanyl, piperidinyl, quinolyl and isoquinolyl. Unless otherwise indicated, heterocyclic radicals are optionally substituted.

As used herein, unless otherwise specified, the term "heterocyclylene" is a divalent heterocyclyl group. Unless otherwise specified, a heterocyclylene group is optionally substituted.

As described herein, unless otherwise indicated, the term "aryl" refers to a monocyclic aromatic group and/or a polycyclic monovalent aromatic group comprising at least one aromatic hydrocarbon ring. In certain embodiments, the aryl group has 6 to 18 ring carbon atoms (C6-C18 aryl), 6 to 14 ring carbon atoms (C6-C14 aryl) or 6 to 10 ring carbon atoms (C6-C10 aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl and terphenyl. The term "aryl" also refers to a bicyclic, tricyclic or other polycyclic hydrocarbon ring, wherein at least one ring is aromatic, and the other rings may be saturated, partially unsaturated or aromatic, such as dihydronaphthyl, indenyl, indanyl or tetrahydronaphthyl (tetrahydronaphthyl). Unless otherwise indicated, aryl is optionally substituted.

As used herein, unless otherwise specified, the term "arylene" is a divalent aromatic group. Unless otherwise specified, an arylene group is optionally substituted.

As described herein, unless otherwise indicated, the term "heteroaryl" refers to a monocyclic aromatic group and/or polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more independently selected from O, S and N, one to three or one to four heteroatoms. Heteroatoms in heteroaryl can be connected to the main structure at any carbon atom. In certain embodiments, heteroaryl has 5 to 20, 5 to 15 or 5 to 10 ring atoms. The term "heteroaryl" also refers to bicyclic, tricyclic or other polycyclic rings, wherein at least one ring is aromatic, and the other rings can be saturated, partially unsaturated or aromatic, wherein at least one aromatic ring contains one or more monocyclic heteroaryl examples, including but not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl. The example of bicyclic heteroaryl includes but is not limited to indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolyl, tetrahydroisoquinolyl, isoquinolyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, oxynaphthyl, furanpyridinyl, thienopyridinyl, dihydroisoindolyl and tetrahydroquinolyl. The example of tricyclic heteroaryl includes but is not limited to carbazolyl, benzindolyl, phenanthroline base, acridinyl, phenanthridinyl and xanthine. Unless otherwise indicated, heteroaryl is optionally substituted.

As used herein, unless otherwise specified, the term "heteroarylene" is a divalent heteroaryl group. Unless otherwise specified, a heteroarylene group is optionally substituted.

When groups described herein are "substituted," they may be substituted with any suitable substituent(s). Illustrative examples of substituents include, but are not limited to, those shown in the exemplary compounds and embodiments provided herein, as well as: halogen atoms such as F, Cl, Br or I; cyano; oxo (=O); hydroxyl (-OH); alkyl; alkenylalkynylcycloalkylaryl-(C=O)OR';-O(C=O)R';-C(=O)R'; -S(O) _xR ';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O)_xNR'R';-NR'S(O) _xR '; and -S(O) _xNR'R ', wherein R' at each occurrence is independently H, C 1 -C ₁ -C In some embodiments, the substituent is C ₁ -C ₁₂ alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments, the substituent is C ₁ -C 12 alkyl. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halogen group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is hydroxy. In other embodiments, the substituent is alkoxy (-OR'). In other embodiments, the substituent is carboxyl. In other embodiments, the substituent is amino (-NR'R').

As used herein, unless otherwise indicated, the term "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstances may or may not occur, and that the description includes instances where the event or circumstances occur and instances where the event or circumstances do not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes substituted alkyl groups and unsubstituted alkyl groups.

"Prodrug" refers to a compound that can be converted into a biologically active compound under physiological conditions or by solvolysis. Therefore, the term "prodrug" refers to a metabolic precursor of a pharmaceutically acceptable biologically active compound. When administered to a subject in need, the prodrug may be inactive, but is converted into the biologically active compound of the present invention in vivo. The prodrug is generally rapidly converted in vivo to the parent biologically active compound of the present invention, for example, by hydrolysis in the blood. Prodrug compounds generally provide advantages of solubility, tissue compatibility or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

In some embodiments, the term "prodrug" is also meant to include any carriers that are covalently bonded, and when such prodrugs are administered to mammalian subjects, they release the active compounds of the present invention in vivo. Prodrugs of the compounds of the present invention can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved into the parent compounds of the present invention by conventional manipulation or in vivo. Prodrugs include compounds of the present invention wherein a hydroxyl, amino or thiol group is bonded to any of the following groups, and when the prodrugs of the compounds of the present invention are administered to mammalian subjects, the groups are cleaved to form free hydroxyls, free aminos or free thiol groups, respectively.

Examples of "prodrugs" include, but are not limited to, acetate, formate and benzoate derivatives of amide derivatives of alcohol or amine functional groups in the compounds provided herein, and the like.

As used herein, unless otherwise stated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

Examples of "pharmaceutically acceptable acid addition salts" include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, glucuronic acid, glutamic acid, Acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-dicarboxylic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

Examples of "pharmaceutically acceptable base addition salts" include, but are not limited to, salts prepared by adding an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferably, the inorganic salt is an ammonium salt, a sodium salt, a potassium salt, a calcium salt, and a magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydraziniline, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Compounds provided herein may contain one or more asymmetric centers, and thus may produce enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)-, or as (D)- or (L)- in terms of absolute stereochemistry for amino acids. Unless otherwise indicated, compounds provided herein are intended to include all of these possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing/separating single enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When compounds described herein contain olefin double bonds or other geometric asymmetric centers, unless otherwise indicated, the compounds are intended to include E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

As used herein, unless otherwise specified, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space. "Atropisomers" are stereoisomers that are hindered in the rotation of atoms about single bonds. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio may be referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

"Stereoisomers" may also include E and Z isomers or mixtures thereof, and cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

"Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The difference in concentration of the isomeric forms will depend on the environment in which the compound is located, and may depend on whether the compound is a solid or in an organic or aqueous solution.

The compounds described herein may contain isotopes of atoms that are not part of the natural order at one or more atoms. For example, the compound may be radiolabeled with a radioactive isotope, such as tritium-3 ( ³ H), iodine-125 ( ¹²⁵ I), sulfur-35 ( ³⁵ S) or carbon-14 ( ¹⁴ C), or may be isotopically enriched with deuterium ( ² H), carbon-13 ( ¹³ C) or nitrogen-15 ( ¹⁵ N). As used herein, an "isotope" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition that is different from the natural isotopic composition of the atom. "Isotopically enriched" may also refer to a compound containing at least one atom that is isotopically enriched. The isotopic composition of an atom is different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents, such as cancer therapeutics, research reagents (such as binding assay reagents) and diagnostic agents (such as in vivo imaging agents). All isotopic variants of the compounds described herein, whether or not radioactive, are intended to be included within the scope of the embodiments provided herein. In some embodiments, isotopes of compounds described herein are provided, for example, isotopes are enriched in deuterium, carbon-13 and/or nitrogen-15. As used herein, "deuterated" refers to a compound in which at least one hydrogen (H) is replaced by deuterium (represented by D or ² H), that is, the compound is enriched in deuterium at least one position.

It should be noted that if there is a discrepancy between a structure described herein and the name of the structure, the described structure should have greater weight.

As used herein, unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or livestock.

The term "composition" is intended to encompass a product containing specified ingredients (eg, mRNA molecules), optionally in specified amounts.

The terms "polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including, for example, DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or may be any substrate incorporated into a polymer by DNA polymerase or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and analogs thereof. Nucleic acids may be in single-stranded or double-stranded form. As described herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimetics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino oligonucleotides. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, which is generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise indicated, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition of nascent RNA transcripts from 5' to 3' is called the transcription direction; the sequence region on the DNA chain with the same sequence as the RNA transcript located from 5' to the 5' end of the RNA transcript is called the "upstream sequence"; the sequence region on the DNA chain with the same sequence as the RNA transcript located from 3' to the 3' end is called the "downstream sequence".

"Isolated nucleic acid" refers to nucleic acid, for example, it can be RNA, DNA or mixed nucleic acid, which is basically separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) by nature, including native sequences. "Isolated" nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules in natural sources. In addition, when produced by recombinant technology, "isolated" nucleic acid molecules (such as mRNA molecules) can be substantially free of other cell materials or culture medium, or can be substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding antigens described herein are separated or purified. The term includes nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogs or analogs synthesized by heterologous systems. Substantially pure molecules can include isolated forms of molecules.

The term "coding nucleic acid" or its grammatical equivalents includes: (a) nucleic acid molecules that can be transcribed to produce mRNA that can be translated into peptides and/or polypeptides when in their natural state or by methods well known to those skilled in the art, and (b) mRNA molecules themselves. The antisense strand is the complementary sequence of the nucleic acid molecule, and the coding sequence can be inferred therefrom. The term "coding region" refers to the portion of the coding nucleic acid sequence that can be translated into a peptide or polypeptide. The term "untranslated region" refers to the portion of the coding nucleic acid sequence that can be translated into a peptide or polypeptide. Or "UTR" refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. This depends on the orientation of the UTR relative to the coding region of the nucleic acid molecule. If the UTR is located at the 5' end of the coding region, the UTR is called a 5'-UTR; if it is located at the 3' end of the coding region, the UTR is called a 3'-UTR.

As described herein, the term "mRNA" refers to a messenger RNA molecule comprising one or more open reading frames (ORFs), which can be translated by a cell or organism to produce one or more peptides or protein products. The region comprising one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule also comprises one or more untranslated regions (UTRs).

In certain embodiments, mRNA is a monocistronic mRNA comprising only one ORF. In certain embodiments, monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, mRNA is a polycistronic mRNA comprising two or more ORFs. In certain embodiments, polycistronic mRNA encodes two or more peptides or proteins that are identical or different from each other. In certain embodiments, each peptide or protein encoded by polycistronic mRNA comprises at least one epitope of a selected antigen. In certain embodiments, different peptides or proteins encoded by polycistronic mRNA each comprise at least one epitope of a different antigen. In any embodiment described herein, at least one epitope can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 epitopes of an antigen.

The term "nucleobase" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

As used herein, the term "functional nucleotide analogue" refers to a modified form of a canonical nucleotide A, G, C, U or T, which (a) retains the base pairing properties of the corresponding canonical nucleotide, and (b) comprises at least one chemical modification of any combination of (i) to (iii) of (i) a nucleobase, (ii) a sugar group, (iii) a phosphate group or (iv) a corresponding natural nucleotide. As described herein, base pairs not only cover standard Watson-Crick A-T, A-U or C-G base pairs, but also include base pairs formed between canonical nucleotides and functional nucleotide analogs or between a pair of functional nucleotide analogs, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows the formation of hydrogen bonds between the modified nucleobase and the standard nucleobase or between two complementary modified nucleobase structures. For example, a functional analogue of guanosine (G) retains the ability to base pair with a functional analogue of cytosine (C) or cytosine. An example of this non-canonical base pairing is the base pairing between a modified nucleotide inosine and adenine, cytosine or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-naturally occurring. Therefore, nucleic acid molecules comprising functional nucleotide analogs can have at least one modified nucleobase, sugar group or internucleoside bond. Exemplary chemical modifications to the nucleobase, sugar group or internucleoside bond of nucleic acid molecules are provided herein.

As used herein, the terms "translation enhancing element," "TEE," and "translation enhancer" refer to a region in a nucleic acid molecule whose function is to promote the translation of a nucleic acid coding sequence into a protein or peptide product, such as through cap-dependent or cap-independent translation. TEEs are typically located in the UTR region of a nucleic acid molecule (such as mRNA) and are capable of enhancing the translation level of a coding sequence located upstream or downstream. For example, a TEE in the 5'-UTR of a nucleic acid molecule can be located between the promoter and the start codon of the nucleic acid molecule. Various TEE sequences are known in the art (Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10(8): 747–750; Chappell et al. PNAS June 29, 2004101(26)9590-9594). It is known that certain TEEs are conserved across multiple species (Pánek et al. Nucleic Acids Research, Volume 41, Issue 16, 1 September 2013, Pages 7625–7634).

As used herein, the term "stem-loop sequence" refers to a single-stranded polynucleotide sequence having at least two regions that are complementary or substantially complementary to each other when read in opposite directions to form at least one double helix and no double helix. The complementary loops and the resulting loop structure are called stem-loop structures, hairpins or hairpin loops, which are also secondary structures present in many RNA molecules.

As used herein, the term "peptide" refers to a polymer containing 2-50 amino acid residues linked by one or more covalent peptide bonds. The term is applicable to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs or non-natural amino acids).

The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers having more than fifty amino acid residues linked by covalent peptide bonds. That is, the description of polypeptides is equally applicable to the description of proteins, and vice versa. The term is applicable to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). As used herein, the term encompasses amino acid chains of any length, including full-length proteins (e.g., antigens).

The term "antigen" refers to a substance that can be recognized by the subject's immune system (including the adaptive immune system) and can produce an immune response (including an antigen-specific immune response) in the subject's body in contact with the antigen. In certain embodiments, the antigen is a protein associated with a diseased cell (e.g., a cell infected by a pathogen or a neoplastic cell) (e.g., a tumor-associated antigen (TAA)).

In the context of a peptide or polypeptide, the term "fragment" refers to a peptide or polypeptide that contains less than the full-length amino acid sequence. Such fragments can come from truncation of the N-terminus, truncation of the C-terminus and/or deletion of residues within the amino acid sequence. Fragments can be produced by alternative RNA splicing or in vivo proteases. In certain embodiments, a fragment refers to a polypeptide comprising at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, or at least 950 consecutive amino acid residues. In a specific embodiment, a fragment of a polypeptide retains at least 1, at least 2, at least 3 or more functions of the polypeptide.

"Epitope" is a site on the surface of an antigen molecule to which a specific antibody molecule binds, for example, a local region on the surface of an antigen that can bind to one or more antigen binding regions of an antibody, has antigenic or immunogenic activity in animals such as mammals (e.g., humans), and can induce an immune response. An epitope with immunogenic activity is a part of a polypeptide that triggers an antibody response in an animal. An epitope with antigenic activity is a part of a polypeptide that an antibody binds to, as determined by any method known in the art, including, for example, by immunoassay. Antigenic epitopes do not necessarily have to be immunogenic. Epitopes are usually composed of a collection of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed by continuous sequences of amino acids in proteins. Conformational epitopes are formed by discontinuous amino acids in a protein sequence, but are bound together when the protein is folded into its three-dimensional structure. When the three-dimensional structure of a protein is in a changed configuration, such as after activation or binding of another protein or ligand, an induced epitope is formed. In certain embodiments, an epitope is a three-dimensional surface feature of a polypeptide. In other embodiments, an epitope is a linear feature of a polypeptide. Typically, an antigen has several or many different epitopes and can react with many different antibodies.

As used herein, the term "gene vaccine" refers to a therapeutic or preventive composition comprising at least one nucleic acid molecule encoding an antigen associated with a target disease (such as an infectious disease or a neoplastic disease). Peptides or proteins are encoded by administering a vaccine (vaccination) to a subject, thereby inducing an immune response to the target disease in the subject. In certain embodiments, the immune response includes an adaptive immune response, such as the production of antibodies to the encoded antigen, and/or immune cells that can activate and proliferate for the specific elimination of diseased cells expressing the antigen. In certain embodiments, the immune response also includes an innate immune response. According to the present invention, the vaccine can be administered to the subject before or after the onset of clinical symptoms of the target disease. In some embodiments, vaccination of healthy or asymptomatic subjects makes the vaccinated subject immune or less sensitive to the target disease process. In some embodiments, vaccination of subjects with disease symptoms can improve the disease condition of the vaccinated subject or treat the disease.

The terms "innate immune response" and "innate immunity" are well known in the art and refer to non-specific defense mechanisms initiated by the human immune system upon recognition of pathogen-associated molecules, which involve different forms of cellular activity, including cytokine production and cell death in various pathways. As described herein, the innate immune response includes, but is not limited to, increased production of inflammatory cytokines (e.g., type I interferon or IL-10 production), activation of the NFκB pathway, increased proliferation, maturation, differentiation and/or survival of immune cells, and in some cases induced apoptosis. The activation of innate immunity can be detected using methods known in the art, such as by measuring the activation of (NF)-κB.

The terms "adaptive immune response" and "adaptive immunity" are well known in the art and refer to antigen-specific defense mechanisms initiated by the human immune system after recognizing a specific antigen, including humoral responses and cell-mediated responses. As described herein, the adaptive immune response includes a cellular response triggered and/or enhanced by a vaccine composition (such as a genetic composition described herein). In some embodiments, the vaccine composition comprises an antigen that is a target of an antigen-specific adaptive immune response. In other embodiments, the vaccine composition allows the production of an antigen in an immunized subject after administration, which antigen is a target of an antigen-specific adaptive immune response. The activation of an adaptive immune response can be detected using methods known in the art, such as by monitoring the production of antigen-specific antibodies or monitoring the level of antigen-specific cell-mediated cytotoxicity.

The term "antibody" is intended to include polypeptide products secreted by effector B cells, which are composed of two pairs of identical polypeptide chains, wherein each pair of polypeptide chains has a heavy chain (about 50-70 kDa) and a light chain (about 25 kDa), the N-terminal portion of each chain contains a variable region of about 100 to about 130 or more amino acids, and the C-terminal portion of each chain includes a constant region, which can bind to a specific molecular antigen. Immunoglobulins are not just antibodies. For example, see Antibody Engineering (Borrebaeck ed., 2d ed. 1995) and Kuby, Immunology (3d ed. 1997). In a specific embodiment, the specific molecular antigen includes a polypeptide, a fragment or an epitope thereof, which can bind to the antibodies described herein. Antibodies also include, but are not limited to, synthetic antibodies, antibodies produced by recombination, camelized antibodies, intracelluar antibodies, anti-Id antibodies and functional fragments of these antibodies. The functional fragment of an antibody refers to a functional polypeptide fragment separated from the heavy chain or light chain of the aforementioned antibody that can retain a part or all of the binding activity. Some non-limiting examples of functional fragments include single-chain antibodies (scFv) (including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-stabilized antibodies (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies and minibodies. In particular, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that can be antigen binding domains or contain antigen binding sites (such as one or more CDRs of an antibody). Such antibody fragments can be found in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2d ed. The antibodies provided by the present invention can be any type of immunoglobulin molecule (such as IgG, IgE, IgM, IgD and IgA, etc.) or any subclass (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, etc.).

The term "administering" refers to the act of delivering an in vitro substance (such as a lipid nanoparticle composition described herein) to a patient, for example, by mucosal, intramuscular/subcutaneous injection, intravenous injection, or other physical means known in the art. When used to treat a disease, disorder, condition, or symptom thereof, the administration of the substance is usually performed after the onset of the disease, disorder, condition, or symptom thereof. When used to prevent a disease, disorder, condition, or symptom, the administration of the substance is usually performed before the onset of the disease, disorder, condition, or symptom.

"Chronic" administration refers to administration in a continuous mode (e.g., over a period of time such as days, weeks, months or years) as opposed to an acute mode of administration to maintain the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration is not continuous but rather periodic, without interruption of treatment.

The term "targeted delivery" or verb form of "target" refers to the process of promoting the delivery of agents (e.g., therapeutic payload molecules in lipid nanoparticle compositions described herein) to reach specific organs, tissues, cells and/or intracellular compartments (referred to as target locations), making the target location more than any other organ, tissue, cell or intracellular compartment (referred to as non-target location) delivered. Targeted delivery can be detected by methods known in the art, such as by comparing the concentration of the agent delivered in the target cell population after systemic administration with the concentration of the agent delivered in the non-target cell population. In certain embodiments, compared to non-target locations, targeted delivery results in a concentration at least 2 times higher at the target location.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their causes, and/or improve or remedy damage. Diseases caused by or associated with a disease, disorder or condition include infection and tumor formation, etc. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

As described herein, the term "therapeutically effective amount" refers to an amount of an agent (such as a vaccine composition) sufficient to reduce and/or improve the severity and/or duration of symptoms associated with a given disease, disorder or condition (such as an infectious disease caused by a viral infection, or a neoplastic disease of cancer, etc.). The "therapeutically effective amount" of a substance/molecule/agent of the present disclosure (such as a lipid nanoparticle composition described herein) may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount includes an amount in which any toxic or deleterious effects of the substance/molecule/agent are offset by the beneficial effects of the treatment. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition or a therapeutic or preventive agent (such as a therapeutic mRNA) contained therein that is effective in "treating" a disease, disorder, or condition in a subject or mammal.

A "prophylactic effective amount" is an amount that will have the expected prophylactic effect when administered to a subject, for example, the amount of a pharmaceutical composition that prevents, delays or reduces the likelihood of onset (or recurrence) of a disease, disorder, and related symptoms (such as infectious diseases caused by viral infection or neoplastic diseases such as cancer). Conditions or related symptoms. Usually, but not necessarily, because a prophylactic dose is used in a subject before or at an earlier stage of a disease, disorder, or condition, the prophylactic effective amount may be less than the therapeutic effective amount. A complete therapeutic or prophylactic effect does not necessarily occur by administering one dose, but may only occur after a series of doses are administered. Therefore, a therapeutic or prophylactic effective amount may be administered in one or more administrations.

The term "prevent" or "preventing" refers to reducing the likelihood of developing a disease, disorder, condition or associated symptoms, such as an infectious disease, such as infection by a virus, or a neoplastic disease, such as cancer.

The term "management" refers to the beneficial effects that a subject obtains from a treatment (e.g., a prophylactic or therapeutic agent) that does not result in a cure of the disease. In certain embodiments, one or more therapies (e.g., a prophylactic or therapeutic agent, such as a lipid nanoparticle composition described herein) are administered to a subject to "manage" one or more symptoms of an infectious or neoplastic disease, thereby preventing the progression or worsening of the disease.

The term "prophylactic agent" refers to any agent that can completely or partially inhibit the development, recurrence, onset, or spread of a disease and/or symptoms associated therewith in a subject.

The term "therapeutic agent" refers to any drug useful in treating, preventing or alleviating a disease, disorder or condition, including any drug useful in treating, preventing or alleviating one or more symptoms of a disease, disorder or condition and associated symptoms.

The term "therapy" refers to any regimen, method and/or agent that can be used to prevent, manage, treat and/or improve a disease, disorder or condition. In certain embodiments, the term "therapy" refers to biological therapy, supportive therapy and/or other therapies known to those skilled in the art, such as medical personnel, that can be used to prevent, control, treat and/or improve a known disease, disorder or condition.

A "prophylactically effective serum titer" is a serum titer of antibodies in a subject (eg, a human) that fully or partially inhibits the development, recurrence, onset or spread of a disease, disorder or condition and symptoms associated therewith.

In certain embodiments, a "therapeutically effective serum titer" is a serum titer of antibodies in a subject (eg, a human) that reduces the severity, duration, and/or symptoms associated with a disease, disorder, or condition.

The term "serum titer" refers to the average serum titer from a subject from multiple samples (e.g., at multiple time points) or in a population of at least 10, at least 20, at least 40 subjects, up to about 100, 1000 or more subjects.

The term "side effect" encompasses unwanted and/or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Adverse effects are not necessarily unfavorable. Adverse effects of a therapy (e.g., a prophylactic or therapeutic agent) may be harmful, uncomfortable, or risky. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, hair loss, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rash or swelling at the site of administration, flu-like symptoms such as fever, chills, tiredness, digestive problems, and allergic reactions. Other undesirable effects experienced by patients are known in the art and are described in Physician’s Desk Reference (68th ed. 2014).

The terms "subject" and "patient" can be used interchangeably. As described herein, in certain embodiments, the subject is a mammal, such as a non-primate (such as a cow, pig, horse, cat, dog, rat, etc.) or a primate (such as a monkey and a human). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (such as a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (such as a human) at risk of developing an infectious disease or a neoplastic disease.

The term "detectable probe" refers to a composition that provides a detectable signal. The term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, etc. that provides a detectable signal through its activity.

The term "detectable agent" refers to a substance that can be used to determine the presence of a desired molecule in a sample or subject, such as an antigen encoded by an mRNA molecule as described herein. A detectable agent can be a substance that can be visualized, or a substance that can be determined and/or measured (such as by quantification).

By "substantially all" is meant at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

As used herein, unless otherwise indicated, the term "about" or "approximately" refers to an acceptable error for a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" refers to within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.05%, or less of a given value or range.

As used herein, the singular terms "a,""an," and "the" include plural forms unless the context clearly indicates otherwise.

All publications, patent applications, accession numbers and other references cited in this specification are incorporated herein by reference in their entirety, and each individual publication or patent application is expressly and individually incorporated by reference. The disclosed publications discussed herein are publications disclosed prior to the filing date of this application. Anything herein shall not be construed as admitting that the present invention is not entitled to be earlier than such publications by virtue of prior invention. In addition, the release date provided herein may be different from the actual release date, and the actual release date may require independent confirmation.

The present invention has been described in a number of embodiments. However, it will be appreciated that various modifications may be made without departing from the spirit, concept and scope of the present invention. Therefore, the descriptions in the experimental section and the examples are intended to illustrate rather than limit the scope of the invention described in the claims.

Lipid compounds

In one embodiment, provided herein is a compound represented by formula (I):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

_L1 is selected from _C1 , _C2 , _C3 , C4, _C5 , _C6 , C7, _C8 , _C9 , _C10 , _C11 or _C12 alkylene, or _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 or _C12 alkenylene or alkynylene, _wherein one or more methylene groups _are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

_L2 is selected from _C1 , _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 or _C20 alkylene, or _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _{C13 , C14} , _C15 , _C16 , _C17 , _C18 , _C19 or C20 _alkylene . ₂₀ alkenylene or alkynylene, wherein one or more methylene groups are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

_R1 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl or heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl or heteroaryl is optionally substituted independently by hydroxy, hydroxy( _C1 - _C6 )alkyl, mercapto, amino, halogen, cyano, aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or _Rb -X-; optionally, two substituents on the same carbon atom of the alkyl, alkenyl or alkynyl together with the carbon atom form a cycloalkyl; the alkyl is _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , C7 _, C8, _C9 or _C10 alkyl _, the alkenyl or alkynyl is _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 or C10 ₁₀ alkenyl or alkynyl, the cycloalkyl or cycloalkenyl is C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ or C ₁₀ cycloalkyl or cycloalkenyl;

_R2 is selected from _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 , _C20 , _C21 , _C22 , _C23 , _C24 , _C25 , _C26 , _C27 , _C28 , _C29 or _{C30 alkyl} , alkenyl or alkynyl;

_R3 is selected from _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 , _C20 , _C21 , _C22 , _C23 , _C24 , _C25 , _C26 , _C27 , _C28 , _C29 or _{C30 alkyl} , alkenyl or alkynyl;

_R4 is selected from H, _C4 , _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _{C16 , C17, C18, C19, C20, C21, C22, C23 or C24 alkyl , alkenyl or alkynyl , wherein one} or more methylene groups are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

_R5 is selected from H, _C4 , _C5 , _C6 , _C7 , C8, _C9 , _{C10 , C11} , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 , _C20 , _C21 , _C22 , _C23 or _C24 alkyl, alkenyl or alkynyl, wherein one or more methylene groups are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

and _R4 and _R5 are not H at the same time;

X is selected from O, S or N(R _c );

_Ra is selected from H, ( _C1 - _C10 ) alkyl (preferably ( _C1 - _C6 ) alkyl), ( _C2 - _C6 ) alkenyl, ( _C2 - _C6 ) alkynyl, ( _C3 - _C10 ) cycloalkyl, hydroxyl or mercapto;

_Rb is selected from H, ( _C1 - _C6 )alkyl, ( _C2 - _C6 )alkenyl, ( _C2 - _C6 )alkynyl, wherein the ( _C1 - _C6 )alkyl, ( _C2 - _C6 )alkenyl or ( _C2 - _C6 )alkynyl is optionally substituted with hydroxy, thiol, amino, halogen, cyano, aryl _, heteroaryl or (C3-C10)cycloalkyl; ( _C3 - _C10 )cycloalkyl, aryl or heteroaryl optionally substituted with ( _C1 - _C6 )alkyl, ( _C2 - _C6 )alkenyl, ( _C2 -C6)alkynyl, hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or ( _C3 - _C10 )cycloalkyl; ( _C4 - _C10 )cycloalkenyl optionally substituted with oxo or _{-N ( Rd} ) ₂ , R _d is independently selected from H, or (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl or (C ₂ -C ₆ )alkynyl optionally substituted with hydroxy; or

R _c is selected from H, (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, wherein the (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl or (C ₂ -C ₆ )alkynyl is optionally substituted with hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or (C ₃ -C ₁₀ )cycloalkyl; (C 3 -C 10 )cycloalkyl, aryl or heteroaryl optionally substituted with (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or (C ₃ -C ₁₀ )cycloalkyl; (C ₄ -C ₁₀ )cycloalkenyl optionally substituted with oxo or _-N (R _{d ) 2} , R _d is independently selected from H, or (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl or (C ₂ -C ₆ )alkynyl optionally substituted with hydroxy; or

wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, aryl, heteroaryl, phenyl, phenylene, naphthylene or amino group is optionally substituted independently by one or more substituents; preferably, the substituents are selected from (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, (C ₃ -C ₁₀ ) cycloalkyl, (C ₃ -C ₁₀ ) cycloalkenyl, hydroxyl, thiol, amino, halogen, cyano, heterocyclyl, aryl or heteroaryl.

In one embodiment, _L1 is selected from _L1a - _OL1a , _L1a - _SL1a , _L1a -S(O) _-L1a , _L1a -S(O) ₂ - _L1a , _L1a -C(O) _-L1a , L1a-C(S) _-L1a , L1a _{- NRa - L1a} , _L1a- OC(O)-L1a, _L1a - _C (O) _-OL1a , _L1a -NRa _-C (O) _-L1a , _L1a -C(O)-NRa _{- L1a} , L _1a -SC(O)-L _1a or L _1a -OC(S)-L _1a , L _1a at each occurrence is independently selected from _a chemical bond, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ or C ₁₂ alkylene, or C ₂ , C 3 , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ or C ₁₂ alkenylene or alkynylene.

In one embodiment, _L2a is selected from _L2a - _OL2a , _L2a - _SL2a , _L2a -S(O) _-L2a , _L2a -S(O) ₂ - _L2a , L2a-C(O) _-L2a , _L2a -C(S) _-L2a , _L2a - _{NRa - L2a} , _L2a -OC(O)-L2a, _L2a - _C (O) _-OL2a , _L2a -NRa _- C(O) _-L2a , _L2a -C(O)-NRa _{- L2a} , _L2a -SC(O) _-L2a or _L2a -OC(S) _-L2a , _L2a at each occurrence is independently selected from a chemical bond, _C1 , _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _{C13, C14 , C15} , _C16 , _C17 , _C18 , _C19 or _C20 alkylene, or _C2 , _C3 , _C4 , _C5 , _C6 , C7, _C8 , _C9 , _C10 , _C11 , _C12 , _{C13 ,} C14, _C15 , _C16 , _C17 , _C18 , _C19 or C20 alkylene _{. 19} or C ₂₀ alkenylene or alkynylene.

In one embodiment, R ₄ is selected from the group consisting of: H, R _4a -OR _4b , R _4a -SR _4b , R _4a -S(O)-R _4b , R _4a -S(O) ₂ -R _4b , R _4a -C(O)-R _4b , R _4a -C(S)-R _4b , R _4a -NR _a -R _4b , R _4a -OC(O)-R _4b , R _4a -C(O)-OR _4b , R _4a -NR _a -C(O)-R _4b , R _4a -C(O)-NR _a -R _4b , R _4a -SC(O)-R _4b or R _4a -OC(S)-R _4b , R _4a is selected from C ₄ , C ₅ , C ₆ , C ₇ , C 8, C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ , C ₁₉ , C ₂₀ , _{C 21 ,} C ₂₂ , C ₂₃ or C ₂₄ alkyl, alkenyl or alkynyl, and R _4b is selected from a chemical bond, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ or C ₆ alkylene, or C ₂ , C ₃ , C ₄ , C ₅ or C ₆ alkenylene or alkynylene.

In one embodiment, R _5b is selected from the group consisting of H, R _5a -OR _5b , R _5a -SR _5b , R _5a -S(O)-R _5b , R _5a -S(O) ₂ -R _5b , R _5a -C(O)-R _5b , R _5a -C(S)-R _5b , R _5a -NR _a -R _5b , R _5a -OC(O)-R _5b , R _5a -C(O)-OR _5b , R _5a -NR _a -C(O)-R _5b , R _5a -C(O)-NR _a -R _5b , _R5a -SC(O) _-R5b or _R5a -OC(S) _-R5b , _R5a is selected from _C4 , _C5 , _C6 , _C7 , C8, _C9 , _C10 , C11, _C12 , _C13 , _C14 , _C15 , _C16 , _{C17 , C18, C19, C20, C21, C22, C23 or C24 alkyl , alkenyl or alkynyl , and R5b is} selected from a chemical bond, _C1 , _C2 , _C3 , _C4 , _C5 or _C6 alkylene, or _C2 , _C3 , _C4 , _C5 or _C6 alkenylene or alkynylene.

In one embodiment, the aryl group is phenyl, and/or the heteroaryl group is selected from pyridyl, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyrazinyl, pyrimidinyl, pyridazinyl or triazinyl, and/or the heterocyclic group is selected from (C ₃ -C ₁₀ ) monocyclic cycloalkyl containing one or two heteroatoms selected from oxygen, nitrogen or sulfur.

In one embodiment, the alkyl, alkenyl, alkynyl, alkylene, alkenylene or alkynylene group is linear or branched, and the cycloalkyl, cycloalkylene, cycloalkenyl group is monocyclic.

In one embodiment, neither R ₄ nor R ₅ is H.

In one embodiment, the compound is selected from:

It should be understood that any embodiment of the compound provided herein as described above, and any specific substituent and/or variable of the compound provided herein as described above can be independently combined with other embodiments and/or substituents and/or various variables of the compound to form an embodiment that is not specifically set forth. In addition, in the case of listing substituents and/or variables for any particular group or variable, it should be understood that each individual substituent and/or variable can be deleted from a specific embodiment and/or claim, and the remaining substituent and/or variable list will be considered to be within the scope of the embodiment provided herein.

It is to be understood that in this specification, combinations of substituents and/or variables of the described chemical formulas are permissible only if such combinations result in stable compounds.

Nanoparticle composition

In one aspect, described herein are nanoparticle compositions comprising a lipid compound described herein. In a specific embodiment, the nanoparticle composition comprises a compound according to formula (I) (and subformulae thereof) described herein.

In some embodiments, the maximum size of the nanoparticle composition provided herein is 1 μm or shorter (e.g., ≤1 μm, ≤900nm, ≤800nm, ≤700nm, ≤600nm, ≤500nm, ≤400nm, ≤300nm, ≤200nm, ≤175nm, ≤150nm, ≤125nm, ≤100nm, ≤75nm, ≤50nm or shorter), when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy or other methods. In one embodiment, the lipid nanoparticles provided herein have at least one dimension in the range of about 40 to about 200nm. In one embodiment, at least one dimension is in the range of about 40 to about 100nm.

Nanoparticle compositions that can be used in conjunction with the present invention include lipid nanoparticles (LNP), nanolipoprotein particles, liposomes, lipid vesicles and lipid complexes, etc. In some embodiments, the nanoparticle composition comprises a vesicle of one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and/or cross-linked to each other. The lipid bilayer can include one or more ligands, proteins or channels.

The properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different properties than a nanoparticle composition comprising a different structural lipid. Similarly, the properties of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher mole fraction of phospholipids can have different properties than a nanoparticle composition comprising a lower mole fraction of phospholipids. The properties can also vary depending on the method and conditions of preparation of the nanoparticle composition.

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (transmission electron microscopy or scanning electron microscopy, etc.) can be used to detect the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, and Worcestershire, UK) can also be used to measure multiple characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

Dh (size): The average size of the nanoparticle composition can be between 10s nm and 100s nm. For example, the average size can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the average size of the nanoparticle composition can be about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 70nm 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm, or about 90nm to about 100nm. In certain embodiments, the average size of the nanoparticle composition can be about 70nm to about 100nm. In some embodiments, the average size can be about 80nm. In other embodiments, the average size can be about 100nm.

PDI: The composition of the nanoparticles can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

Encapsulation efficiency: The encapsulation efficiency of the therapeutic and/or prophylactic agent represents the amount of the therapeutic and/or prophylactic agent encapsulated or combined with the nanoparticle composition after preparation, relative to the amount initially provided. High encapsulation efficiency (e.g., close to 100%) is desired. Encapsulation efficiency can be measured by comparing the amount of the therapeutic and/or prophylactic agent comprising the nanoparticle composition before decomposing the nanoparticle composition with one or more organic solvents or detergents and after decomposition in solution. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agent (e.g., RNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and/or prophylactic agent can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

Apparent pKa: The zeta potential of a nanoparticle composition can be used to indicate the electromotive force of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. It is generally desirable for nanoparticle compositions to have relatively low positive or negative charges, as higher charged materials may interact adversely with cells, tissues, and other elements of the human body. In some embodiments, the zeta potential of a nanoparticle composition can be from about -10 mV to about +20 mV, from about -10 mV to about +20 mV. To about +15mV, about -10mV to about +10mV, about -10mV. To about +5mV, about -10mV to about 0mV, about -10mV to about -5mV, about -5mV to about +20mV, about -5mV to about +15mV, about -5mV to about +10mV, about -5mV to about +5mV, about -5mV to about 0mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about +5mV to about +15mV or about +5mV to about +10mV.

In another embodiment, the RNA of self-replication can be prepared in liposomes. As a non-limiting example, the RNA of self-replication can be prepared in liposomes as described in International Publication No. WO20120067378, which is incorporated herein by reference in its entirety. On the one hand, liposomes may include lipids that are conducive to the delivery pKa value of mRNA. On the other hand, liposomes may have a substantially neutral surface charge at physiological pH, and therefore can be effectively used for immunization (see, for example, the liposomes described in International Publication No. WO20120067378, which is incorporated herein by reference in its entirety).

In some embodiments, the nanoparticle composition comprises a lipid component comprising at least one lipid, such as a compound according to formula (I) (and subformulae thereof) as described herein. For example, in some embodiments, the nanoparticle composition may include a lipid component comprising one of the compounds provided herein. The nanoparticle composition may also include one or more other lipid or non-lipid components as described below.

Cationic/ionizable lipids

As described herein, in some embodiments, the nanoparticle compositions provided herein include, in addition to the lipids according to formula (I) (and subformulae thereof), one or more charged or ionizable lipids. It is contemplated that certain charged or zwitterionic lipid components of the nanoparticle compositions are similar to lipid components in cell membranes, thereby improving cellular uptake of the nanoparticles. Exemplary charged or ionizable lipids that can form part of the nanoparticle compositions of the present invention include, but are not limited to, 3-(docosylamino)-N1,N1,4-triacontyl-1-piperazineethylamine (KL10), N1-[2-(docosylamino)ethyl]-N1,N4,N4-triacontyl-1,4-piperazinedienamide (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaconane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptahedralidol (heptaconitol ester (heptaconitol ester) and 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA). ptatriaconta)-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine 1-yloxy]propan-1-amine (octyl-CLinDMA (2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylethyl-3-[((9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2S)), (12Z,15Z)-N,N-dimethyl-2-nonyldocosyl-12,15-den-1-amine, N,N-dimethyl-1-{((1S,2R)-2-octylcyclopropyl}heptadecan-8-amine. Additional exemplary charged or ionizable lipids (such as lipid 5) that form part of the nanoparticle compositions of the present invention include those described in Sabnis et al. "A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates", Molecular Therapy Vol. 26 No 6, 2018, the entire contents of which are incorporated herein by reference.

In some embodiments, suitable cationic lipids include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1); N1-[2-((1S)-1-[(3- =Aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylformamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); dioctadecylamino-glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); SAINT-2, N-methyl-4-(dioleyl)methylpyridine; 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); di-alkylated amino acids (DILA ² )(e.g., C18:1-norArg-C16); dioleyl dimethyl ammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC); (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl); (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G); and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen). Cationic lipids with charged head groups at physiological pH, such as primary amines (e.g., DODAG N', N'-dioctadecyl-N-4,8-diaza-10-aminodecanoyl glycine amide) and guanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol (BGSC), bis-guanidinium trisaminoethylamine-cholesterol (BGTC), PONA, and (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G)) are also suitable. Another suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl). In certain embodiments, the cationic lipid is a specific enantiomer or racemic form, and includes various salt forms (e.g., chlorides or sulfates) of the above cationic lipids. For example, in some embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl) or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate). In some embodiments, the cationic lipid is an ionizable cationic lipid, such as (for example) dioctadecyl dimethyl ammonium bromide (DDAB); 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA); 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP); 1,2-dioleyloxy-3-dimethylaminopropane (DODMA); and morpholino cholesterol (Mo-CHOL). In certain embodiments, the lipid nanoparticle includes a combination of two or more cationic lipids (e.g., two or more cationic lipids as above).

In addition, in some embodiments, the charged or ionizable lipid that can form part of the present nanoparticle composition is a lipid comprising a cyclic amine group. Additional cationic lipids suitable for use in the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330 and WO2015011633, the entire contents of which are incorporated herein by reference in their entirety.

Polymer-conjugated lipids

In some embodiments, the lipid component of nanoparticle composition may include one or more polymer-conjugated lipids (polymer conjugated lipids), such as PEGylated lipids (PEG lipids). It is contemplated that the polymer conjugated lipid component in the nanoparticle composition can improve colloidal stability and/or reduce the protein absorption of nanoparticles. Exemplary cationic lipids that can be used in conjunction with the present disclosure include but are not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000 or Chol-PEG2000.

In one embodiment, the polymer-conjugated lipid is a pegylated lipid. Some embodiments include pegylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate.

In one embodiment, the polymer-conjugated lipid is present at a molar concentration of 1.0 to 2.5%. In one embodiment, the polymer-conjugated lipid is present at a molar concentration of about 1.7%. In one embodiment, the polymer-conjugated lipid is present at a molar concentration of about 1.5%.

In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is about 35:1 to about 25: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is about 100:1 to about 20:1.

In one embodiment, the PEGylated lipid has the formula:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R ¹² and R ¹³ are each independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and

The average value of w is between 30 and 60.

In one embodiment, R ¹² and R ¹³ are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, w is in the range of 42 to 55 on average, for example, w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 on average. In a specific embodiment, the average w is about 49.

In one embodiment, the PEGylated lipid has the formula:

The average value of w is about 49.

Structural lipids

In some embodiments, the lipid component of the nanoparticle composition may include one or more structural lipids. It is contemplated that the structural lipids can stabilize the amphipathic structure of the nanoparticles, such as, but not limited to, the lipid bilayer structure of the nanoparticles. Exemplary structural lipids that can be used in conjunction with the present disclosure include, but are not limited to, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha-tocopherol and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone and hydrocortisone) or a combination thereof.

In one embodiment, the lipid nanoparticles provided herein include a steroid or a steroid analog. In one embodiment, the steroid or steroid analog is cholesterol. In one embodiment, the molar concentration range of the presence of the steroid is 39-49%, 40-46%, 40-44%, 40-42%, 42-44% or 44-46%. In one embodiment, the steroid is present at a molar concentration of 40, 41, 42, 43, 44, 45 or 46%.

In one embodiment, the molar ratio of cationic lipid to steroid is 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol is about 5:1 to 1:1. In one embodiment, the steroid is present at a molar concentration of 32-40% of the steroid.

Phospholipids

In some embodiments, the lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. It is contemplated that the phospholipids may be assembled into one or more lipid bilayer structures. Exemplary phospholipids that may form part of the present nanoparticle composition include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPSC), 1,2-dioleoyl-sn-glycero-3-phosphocholine ...DPSC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPSC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPSC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPSC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPSC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPSC), 1,2-dioleoyl-sn-glycero- Choline (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-heneicosanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleyl-2-cholesteryl hemisuccinyl-sn-glycero-3 -phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-dihard Fatty acyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl 1-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin. In certain embodiments, the nanoparticle composition includes DSPC. In certain embodiments, the nanoparticle composition includes DOPE. In some embodiments, the nanoparticle composition includes DSPC and DOPE.

Other exemplary neutral lipids include dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylphosphatidylethanolamine (DPPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1,2-divaleryl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

In one embodiment, the neutral lipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA) or phosphatidylglycerol (PG).

Additional phospholipids that may form part of the nanoparticle compositions of the present invention also include those described in WO2017/112865, the entire contents of which are incorporated herein by reference in their entirety.

Treatment payload

Nanoparticle compositions as described herein may further comprise one or more therapeutic and/or prophylactic agents. These therapeutic and/or prophylactic agents are sometimes referred to as "therapeutic payloads" or "payloads" in the present disclosure. In some embodiments, nanoparticles may be used as delivery vehicles to administer therapeutic payloads in vivo or in vitro.

In some embodiments, the nanoparticle composition comprises a small molecule compound (e.g., a small molecule drug) as a therapeutic payload, for example, an antineoplastic agent (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), an antineoplastic agent (e.g., actinomycin D, vincristine, vinblastine, cytarabine, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate, purine and pyrimidine analogs), an anti-infective agent, a local anesthetic (e.g., dibucaine and chlorpromazine), a beta-adrenergic blocker (e.g., propranolol), a , timolol, and labetalol), antihypertensives (such as clonidine and hydralazine), antidepressants (such as imipramine, amitriptyline, and doxepin), anticonvulsants (such as phenytoin), antihistamines (such as diphenhydramine, chlorpheniramine, and promethazine), antibiotics/antibacterials (gentamicin, ciprofloxacin, and cefoxitin, among others), antifungals (such as miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, anesthetics, and imaging agents.

In some embodiments, the therapeutic payload comprises a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that causes an immune response, and/or another therapeutic and/or prophylactic agent. Cytotoxins or cytotoxic agents include any substance that may be harmful to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, ketomethoate, 1-nortestosterone, oryzalin, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, maytansinol, rachemycin (CC-1065) and analogs or homologs thereof. Radioactive ions include, but are not limited to, iodine (such as iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.

In other embodiments, the therapeutic payload of the present nanoparticle compositions can include, but are not limited to, therapeutic and/or prophylactic agents such as antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, racletin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., Dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vinblastine, vincristine, paclitaxel, and maytansine alkaloids).

In some embodiments, the nanoparticle composition comprises biomolecules such as peptides and polypeptides as therapeutic payloads. The biomolecules forming part of the nanoparticle composition can be of natural or synthetic origin. For example, in some embodiments, the therapeutic payload of the nanoparticle composition may include, but is not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing hormone releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, cholera vaccine, and peptides and polypeptides.

Nucleic Acids

In some embodiments, the nanoparticle composition comprises one or more nucleic acid molecules (such as DNA or RNA molecules) as a therapeutic payload. Exemplary forms of nucleic acid molecules that can be included in the present nanoparticle composition as a therapeutic payload include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), and hybrid forms thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, and the like. In certain embodiments, the therapeutic payload comprises RNA. RNA molecules that can be included in the nanoparticle composition of the present invention as a therapeutic payload include, but are not limited to, short isomers, agonists (agomir), antagonists (antagomir), antisense molecules, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of RNA molecules known in the art. In certain embodiments, the RNA is mRNA.

In other embodiments, the nanoparticle composition comprises siRNA molecules as therapeutic payloads. In particular, in some embodiments, the siRNA molecules are capable of selectively interfering with and down-regulating the expression of a target gene. In the embodiment, when the nanoparticle composition comprising siRNA is administered to the subject, the siRNA payload selectively silences genes associated with a specific disease, disorder or condition. In some embodiments, the siRNA molecule comprises a sequence complementary to an mRNA sequence encoding a target protein product. In some embodiments, the siRNA molecule is an immunomodulatory siRNA.

In some embodiments, the nanoparticle composition comprises a shRNA molecule or a vector encoding a shRNA molecule as a therapeutic payload. In particular, in some embodiments, the therapeutic payload produces shRNA inside the target cell after being administered to the target cell. Constructs and mechanisms associated with shRNA are known in the art.

In some embodiments, nanoparticle compositions include mRNA molecules as therapeutic payloads. In particular, in some embodiments, the mRNA molecules encode target polypeptides, including any naturally or non-naturally occurring or modified polypeptides. The polypeptides encoded by the mRNA can have any size and can have any secondary structure or activity. In some embodiments, when expressed in cells, the polypeptides encoded by the mRNA payload can have a therapeutic effect.

In some embodiments, the nucleic acid molecules of the present disclosure include mRNA molecules. In specific embodiments, the nucleic acid molecules include at least one coding region (such as an open reading frame (ORF)) encoding a peptide or polypeptide of interest. In some embodiments, the nucleic acid molecules further include at least one untranslated region (UTR). In specific embodiments, the untranslated region (UTR) is located upstream (5' end) of the coding region, referred to herein as 5'-UTR. In specific embodiments, the untranslated region (UTR) is located downstream (3' end) of the coding region, referred to herein as 3'-UTR. In specific embodiments, the nucleic acid molecules include 5'-UTR and 3'-UTR simultaneously. In some embodiments, 5'-UTR includes a 5'-cap structure. In some embodiments, the nucleic acid molecules include a Kozak sequence (e.g., in 5'-UTR). In some embodiments, the nucleic acid molecules include a poly-A region (e.g., in 3'-UTR). In some embodiments, the nucleic acid molecules include a polyadenylic acid signal (e.g., in 3'-UTR). In some embodiments, the nucleic acid molecules include a conserved region (e.g., in 3'-UTR). In some embodiments, the nucleic acid molecules include a secondary structure. In some embodiments, the secondary structure is a stem loop. In some embodiments, the nucleic acid molecule comprises a stem loop sequence (e.g., in a 5'-UTR and/or a 3'-UTR). In some embodiments, the nucleic acid molecule comprises one or more intron regions that can be excised during splicing. In a specific embodiment, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR and a coding region. In a specific embodiment, the nucleic acid molecule comprises one or more regions selected from a coding region and a 3'-UTR. In a specific embodiment, the nucleic acid molecule comprises one or more regions selected from a 5'-UTR, a coding region, and a 3'-UTR.

Coding region

In some embodiments, nucleic acid molecules of the present disclosure include at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) encoding a single peptide or protein. In some embodiments, the coding region includes at least two ORFs, each ORF encoding a peptide or protein. In embodiments where the coding region includes more than one ORF, the peptides and/or proteins encoded by the ORFs may be identical or different from each other. In certain embodiments, a plurality of ORFs in the coding region are separated by non-coding sequences. In a specific embodiment, the non-coding sequence separating two ORFs includes an internal ribosome entry site (IRES).

It is contemplated that the internal ribosome entry site (IRES) can act as the only ribosome binding site, or as one of multiple ribosome binding sites of an mRNA. An mRNA molecule containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by ribosomes (e.g., polycistronic mRNA). Therefore, in some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise one or more internal ribosome entry sites (IRES). Examples of IRES sequences that can be used in conjunction with the present disclosure include, but are not limited to, those from microtumor viruses (e.g., FMDV), pest viruses (CFFV), polioviruses (PV), encephalomyocarditis virus (ECMV), hand, foot and mouth virus (HFMV), and serotype 2 (SSV). The sequences of the cytokine antigen (CNA), ...

In various embodiments, the nucleic acid molecules of the present invention encode at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 peptides or proteins. The peptides and proteins encoded by the nucleic acid molecules can be the same or different. In some embodiments, the nucleic acid molecules of the present disclosure encode dipeptides (such as carnosine and anserine). In some embodiments, the nucleic acid molecules encode tripeptides. In some embodiments, the nucleic acid molecules encode tetrapeptides. In some embodiments, the nucleic acid molecules encode pentapeptides. In some embodiments, the nucleic acid molecules encode hexapeptides. In some embodiments, the nucleic acid molecules encode heptapeptides. In some embodiments, the nucleic acid molecules encode octapeptides. In some embodiments, the nucleic acid molecules encode nonapeptides. In some embodiments, the nucleic acid molecules encode decapeptides. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 15 amino acids. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 50 amino acids. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 100 amino acids. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 150 amino acids. In some embodiments, the nucleic acid molecules encode peptides or polypeptides having at least about 300 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 500 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 1000 amino acids.

In some embodiments, the length of the nucleic acid molecules of the present disclosure is at least about 30 nucleotides (nt). In some embodiments, the length of the nucleic acid molecules is at least about 35nt. In some embodiments, the length of the nucleic acid molecules is at least about 40nt. In some embodiments, the length of the nucleic acid molecules is at least about 45nt. In some embodiments, the length of the nucleic acid molecules is at least about 50nt. In some embodiments, the length of the nucleic acid molecules is at least about 55nt. In some embodiments, the length of the nucleic acid molecules is at least about 60nt. In some embodiments, the length of the nucleic acid molecules is at least about 65nt. In some embodiments, the length of the nucleic acid molecules is at least about 70nt. In some embodiments, the length of the nucleic acid molecules is at least about 75nt. In some embodiments, the length of the nucleic acid molecules is at least about 80nt. In some embodiments, the length of the nucleic acid molecules is at least about 85nt. In some embodiments, the length of the nucleic acid molecules is at least about 90nt. In some embodiments, the length of the nucleic acid molecules is at least about 95nt. In some embodiments, the length of the nucleic acid molecules is at least about 100nt. In some embodiments, the length of the nucleic acid molecules is at least about 120nt. In some embodiments, the length of the nucleic acid molecules is at least about 140nt. In some embodiments, the length of the nucleic acid molecule is at least about 160nt. In some embodiments, the length of the nucleic acid molecule is at least about 180nt. In some embodiments, the length of the nucleic acid molecule is at least about 200nt. In some embodiments, the length of the nucleic acid molecule is at least about 250nt. In some embodiments, the length of the nucleic acid molecule is at least about 300nt. In some embodiments, the length of the nucleic acid molecule is at least about 400nt. In some embodiments, the length of the nucleic acid molecule is at least about 500nt. In some embodiments, the length of the nucleic acid molecule is at least about 600nt. In some embodiments, the length of the nucleic acid molecule is at least about 700nt. In some embodiments, the length of the nucleic acid molecule is at least about 800nt. In some embodiments, the length of the nucleic acid molecule is at least about 900nt. In some embodiments, the length of the nucleic acid molecule is at least about 1000nt. In some embodiments, the length of the nucleic acid molecule is at least about 1100nt. In some embodiments, the length of the nucleic acid molecule is at least about 1200nt. In some embodiments, the length of the nucleic acid molecule is at least about 1300nt. In some embodiments, the length of the nucleic acid molecule is at least about 1400nt. In some embodiments, the length of the nucleic acid molecule is at least about 1500 nt. In some embodiments, the length of the nucleic acid molecule is at least about 1600 nt. In some embodiments, the length of the nucleic acid molecule is at least about 1700 nt. In some embodiments, the length of the nucleic acid molecule is at least about 1800 nt. In some embodiments, the length of the nucleic acid molecule is at least about 1900 nt. In some embodiments, the length of the nucleic acid molecule is at least about 2000 nt. In some embodiments, the length of the nucleic acid molecule is at least about In some embodiments, the length of the nucleic acid molecule is at least about 2500nt. In some embodiments, the length of the nucleic acid molecule is at least about 3000nt. In some embodiments, the length of the nucleic acid molecule is at least about 3500nt. In some embodiments, the length of the nucleic acid molecule is at least about 4000nt. In some embodiments, the length of the nucleic acid molecule is at least about 4500nt. In some embodiments, the length of the nucleic acid molecule is at least about 5000nt.

In certain embodiments, the therapeutic payload includes a vaccine composition (such as a gene vaccine) as described herein. In some embodiments, the therapeutic payload comprises a compound capable of inducing immunity to one or more target conditions or diseases. In some embodiments, the target symptoms are associated with pathogens such as coronaviruses (such as 2019-nCoV), influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, or infections caused by them. In some embodiments, the therapeutic payload comprises a nucleic acid sequence (such as mRNA) encoding a pathogenic protein characteristic of a pathogen or an antigenic fragment or epitope thereof. After the vaccine is inoculated into the subject, it expresses the encoded pathogenic protein (or its antigenic fragment or epitope), thereby inducing immunity to the pathogen in the subject.

In some embodiments, the target disorder is associated with or caused by the neoplastic growth of cells, such as cancer. In some embodiments, the therapeutic payload comprises a nucleic acid sequence (such as mRNA) encoding a tumor-associated antigen (TAA) or an antigenic fragment or epitope thereof characteristic of cancer. The vaccine, after being administered to a vaccinated subject, expresses the encoded TAA (or its antigenic fragment or epitope), thereby inducing immunity against tumor cells expressing the TAA in the subject.

5`-Cap structure

It is expected that the 5'-cap structure of the polynucleotide participates in nuclear export and improves the stability of the polynucleotide, and binds to the mRNA cap binding protein (CBP) in the cell that is responsible for the stability of the polynucleotide. The binding of CBP to the poly-A binding protein forms a mature circular mRNA, thereby obtaining translation ability. The 5'-cap structure further assists in the removal of the 5'-end intron during the mRNA splicing process. Therefore, in some embodiments, the nucleic acid molecule of the present disclosure comprises a 5'-cap.

Nucleic acid molecules may be 5'-capped by the cell's endogenous transcriptional machinery, thereby generating a 5'-ppp-5'-triphosphate bond between the guanine cap terminal residue and the 5'-transcribed sense nucleotide of the polynucleotide. This 5'-guanylate cap is then methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or pre-terminal transcribed nucleotides at the 5' end of the polynucleotide may also be optionally 2'-O-methylated. 5'-decapping by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations of a native 5'-cap structure produced by an endogenous process. Modifications to the 5'-cap can increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and can improve the translation efficiency of the polynucleotide.

Exemplary changes to the native 5'-Cap structure include creating a non-hydrolyzable cap structure, thereby preventing decapping and increasing the half-life of the polynucleotide. In some embodiments, because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester bond, in some embodiments, modified nucleotides can be used during the capping reaction. For example, in some embodiments, Vaccinium capping enzyme from New England Biolabs can be used with α-thioguanosine nucleotides to generate a phosphorothioate bond in the 5'-ppp-5' according to the manufacturer's instructions. Other modified guanosine nucleotides, such as α-methylphosphonate and selenophosphate nucleotides, can also be used.

Other exemplary alterations of the native 5'-Cap structure include modifications at the 2'- and/or 3'-positions of the capping guanosine triphosphate (GTP), replacement of the sugar ring oxygen (oxygen participating in the carbon ring) with a methylene moiety (CH2), modifications of the triphosphate bridge portion of the cap structure, or modifications of the nucleobase (G) portion.

Other exemplary changes to the natural 5'-cap structure include, but are not limited to, 2'-O-methylation of the 5'-end of the polynucleotide and/or the 5'-terminal nucleic acid on the 2'-hydroxyl group of the ribose, which can generate a plurality of different 5'-cap structures of the polynucleotide (e.g., mRNA molecules). Additional exemplary 5'-cap structures that can be used in conjunction with the present disclosure also include those described in International Patent Publication No. Those described in WO2008127688, WO 2008016473 and WO 2011015347, the entire contents of which are incorporated herein by reference.

In various embodiments, the 5'-cap may include a cap analog. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, are chemically different from the natural (i.e., endogenous, wild-type or physiological) 5'-cap structure while retaining the function of the cap. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or attached to a polynucleotide.

For example, the reverse reverse cap analog (ARCA) cap comprises two guanosines linked by a 5'-5'-triphosphate group, wherein one of the guanosines comprises an N7-methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7G-3'mppp-G, which can be equivalently referred to as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unchanged guanosine is linked to the 5'-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3'-O-methylated guanosines provide the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap structure is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (ie, N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, _m7Gm -ppp-G).

In some embodiments, the cap analog can be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified with a borate phosphate group or a phosphoselenate group at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Pat. No.: 8,519,110, the entire contents of which are incorporated herein by reference.

In some embodiments, the cap analog can be a N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and/or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (e.g., see Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for various cap analogs and methods for synthesizing cap analogs, which are incorporated herein by reference). In other embodiments, the cap analog useful for the nucleic acid molecules of the present disclosure is a 4-chloro/bromophenoxyethyl analog.

In various embodiments, the cap analog may include a guanosine analog. Available guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido.

It is expected that, although cap analogs allow simultaneous capping of polynucleotides in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This structural difference of cap analogs from the natural 5'-cap structure of polynucleotides generated from the cell's endogenous transcription machinery may result in reduced translational capacity and reduced cellular stability.

Therefore, in some embodiments, the nucleic acid molecules of the present disclosure may also be capped after transcription using enzymes to produce a more realistic 5'-cap structure. As used herein, the phrase "more realistic" refers to features that closely reflect or mimic endogenous or wild-type features in structure or function. That is, compared to synthetic or analogs of the prior art, "more realistic" features represent better endogenous, wild-type, natural or physiological cell functions and/or structures, or their performance is better than the corresponding endogenous, wild-type natural type, natural or physiological features in one or more aspects. Non-limiting examples of more realistic 5'-cap structures used in conjunction with nucleic acid molecules of the present disclosure are those with enhanced binding of cap-binding proteins, increased half-life, and reduced sensitivity to 5'. Compared to synthetic 5'-cap structures known in the art (or to wild-type, natural or physiological 5'-cap structures), β-endonuclease reduces 5'-decapping. For example, in some embodiments, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a canonical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide. The cap guanine contains an N7-methylation, while the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl group. This structure is referred to as a Cap1 structure. Compared to other 5' cap analog structures, this cap leads to higher translational capacity, cellular stability and reduced activation of cellular proinflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p(Cap 0), 7mG(5')ppp(5')NlmpNp(Cap 1), 7mG(5')-ppp(5')NlmpN2mp(Cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up(Cap 4).

It is contemplated that the nucleic acid molecules of the present disclosure can be capped after transcription and because the process is more efficient, nearly 100% of the nucleic acid molecules can be capped.

Untranslated regions (UTRs)

In some embodiments, the nucleic acid molecules of the present disclosure include one or more untranslated regions (UTRs). In some embodiments, the UTR is located upstream of the coding region of the nucleic acid molecule, referred to as the 5'-UTR. In some embodiments, the UTR is located downstream of the coding region of the nucleic acid molecule, referred to as the 3'-UTR. The sequence of the UTR may be homologous or heterologous to the sequence of the coding region in the nucleic acid molecule. The nucleic acid molecule may include multiple UTRs, which may have the same or different sequences and/or genetic origins. According to the present disclosure, any part of the UTR in the nucleic acid molecule (including the absence thereof) may be codon optimized, and one or more different structural or chemical modifications may be independently included, before and/or after codon optimization.

In some embodiments, nucleic acid molecules (such as mRNA) of the present disclosure comprise UTRs and coding regions that are homologous to each other. In other embodiments, nucleic acid molecules (such as mRNA) of the present disclosure comprise UTRs and coding regions that are heterologous to each other. In some embodiments, in order to detect the activity of UTR sequences, nucleic acid molecules comprising UTRs and detectable probe coding sequences can be administered in vitro (e.g., in cell or tissue culture) or in vivo (e.g., to a subject). And the effects of UTR sequences (such as regulation of expression levels, cellular localization of the encoded product, or half-life of the encoded product) can be detected using methods known in the art.

In some embodiments, the UTR of the nucleic acid molecule (such as mRNA) of the present disclosure comprises at least one translation enhancer element (TEE), which acts to increase the polypeptide or protein yield produced from the nucleic acid molecule. In some embodiments, the TEE is located in the 5'-UTR of the nucleic acid molecule. In other embodiments, the TEE is located at the 3'-UTR of the nucleic acid molecule. In other embodiments, at least two TEEs are located at the 5'-UTR and 3'-UTR of the nucleic acid molecule, respectively. In some embodiments, the nucleic acid molecule (such as mRNA) of the present disclosure may comprise one or more copies of a TEE sequence or comprise more than one different TEE sequence. In some embodiments, the different TEE sequences in the nucleic acid molecule may be homologous or heterologous to each other.

It is known in the art that there are various TEE sequences that can be used in conjunction with the present disclosure. For example, in some embodiments, the TEE can be an internal ribosome entry site (IRES), HCV-IRES or an IRES element. Chappell et al. Chappell et al. Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004; Zhou et al. Proc. Natl. Acad. Sci. 102:6273-6278, 2005. Additional internal ribosome entry sites (IRES) that can be used in conjunction with the present disclosure include, but are not limited to, those described in U.S. Patent No. 7,468,275, U.S. Patent Publication No. 2007/0048776 and U.S. Patent Publication No. 2011/0124100 and International Patent Publication No. WO2007/025008 and International Patent Publication No. WO2001/055369, the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, TEEs may be those described in Supplementary Tables 1 and 2 of Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10(8): 747–750, the contents of which are incorporated herein by reference in their entirety.

Additional exemplary TEEs that can be used in conjunction with the present disclosure include, but are not limited to, those disclosed in U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, U.S. Patent No. 7,183,395, U.S. Patent Publication No. 2009/0226470, U.S. Patent Publication No. 2013/0177581, U.S. Patent Publication No. 2007/0048776, U.S. Patent Publication No. 2011/0124100, U.S. Patent Publication No. 2009/0093049, International Patent Publication No. 2013/0177581, U.S. ... The TEE sequences disclosed in International Patent Publication No. WO2009/075886, International Patent Publication No. WO2012/009644 and International Patent Publication No. WO1999/024595, International Patent Publication No. WO2007/025008, International Patent Publication No. WO2001/055371, European Patent No. 2610341, and European Patent No. 2610340, all of which are incorporated herein by reference in their entirety.

In various embodiments, the nucleic acid molecules (such as mRNA) disclosed herein include at least one UTR, which includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 67, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In some embodiments, the TEE sequence in the UTR of the nucleic acid molecule is a copy of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of the nucleic acid molecule have different sequences. In some embodiments, a plurality of different TEE sequences are arranged in the UTR region of the nucleic acid molecule in one or more repetitive patterns. For illustration purposes only, the repetition pattern can be, for example, ABABAB, ABABBAABBAABB, ABCABCABC, etc., wherein in these exemplary patterns, each capital letter (A, B or C) represents a different TEE sequence. In some embodiments, at least two TEE sequences are continuous with each other in the UTR of a nucleic acid molecule (i.e., there is no spacer sequence between them). In other embodiments, at least two TEE sequences are separated by a spacer sequence. In some embodiments, UTR can include a TEE sequence-spacer sequence module, which is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or more than 9 times. In any embodiment described in this paragraph, UTR can be a 5'-UTR, a 3'-UTR, or both a 5'-UTR and a 3'-UTR of a nucleic acid molecule.

In some embodiments, the UTR of the nucleic acid molecule (such as mRNA) of the present disclosure comprises at least one translation inhibition element, the function of which is to reduce the amount of the polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the UTR of the nucleic acid molecule comprises one or more miR sequences or fragments thereof (such as miR seed sequences) recognized by one or more microRNAs. In some embodiments, the UTR of the nucleic acid molecule comprises one or more stem-loop structures that downregulate the translation activity of the nucleic acid molecule. Other mechanisms for inhibiting the translation activity associated with the nucleic acid molecule are known in the art. In any embodiment described in this paragraph, the UTR can be the 5'-UTR, 3'-UTR, or both the 5'-UTR and 3'-UTR of the nucleic acid molecule.

Polyadenylation (Poly-A) region

During natural RNA processing, long chains of adenosine nucleotides (poly-A) are often added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3'-end of the transcript is cleaved to release the 3'-hydroxyl. Poly-A polymerase then adds chains of adenosine nucleotides to the RNA. This process is called polyadenylation, and a poly-A region of 100 to 250 residues in length is added. It is contemplated that the poly-A region can confer a variety of advantages to the nucleic acid molecules of the present invention.

Therefore, in some embodiments, nucleic acid molecules (such as mRNA) of the present disclosure comprise polyadenylation signals. In some embodiments, nucleic acid molecules (such as mRNA) of the present disclosure comprise one or more polyadenylation (poly-A) regions. In some embodiments, the poly-A region is entirely composed of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end and at least one poly-A region at its 3' end.

According to the present disclosure, in different embodiments, the poly-A region can have varying lengths. In particular, in some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 30 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 35 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 40 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 45 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 50 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 55 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 60 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 65 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 70 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 75 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecule of the present disclosure is at least 80 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 85 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 90 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 95 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 100 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 110 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 120 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 130 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 140 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 150 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 160 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 170 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 180 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 190 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 200 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 225 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 250 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 275 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 300 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 350 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 400 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 450 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 500 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 600 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 700 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 800 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 900 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1000 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1100 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1200 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1300 nucleotides. In some embodiments, the nucleic acids of the present disclosure In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 1400 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 1500 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 1600 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 1700 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 1800 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 1900 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 2000 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 2250 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 2500 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure has a length of at least 2750 nucleotides. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 3000 nucleotides in length.

In some embodiments, the length of the poly-A region in the nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof (e.g., the length of the coding region or the length of the open reading frame). For example, in some embodiments, the poly-A region accounts for about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total length of the nucleic acid molecule containing the poly-A region.

It can be expected that certain RNA binding proteins can bind to the poly-A region located at the 3' end of the mRNA molecule. These poly-A binding proteins (PABPs) can regulate mRNA expression, for example, interact with the translation initiation machinery in the cell and/or protect the 3'-poly-A tail from degradation. Therefore, in some embodiments, the nucleic acid molecules (such as mRNA) of the present disclosure include at least one binding site for a poly-A binding protein (PABP). In other embodiments, the nucleic acid molecules are conjugated or complexed with PABP before being loaded into a delivery vehicle (e.g., lipid nanoparticle).

In some embodiments, nucleic acid molecules (such as mRNA) of the present disclosure comprise poly-A-G tetramers. G tetramers are cyclic hydrogen-bonded arrays of four guanosine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G tetramers are bound to the ends of the poly-A region. The stability, protein production, and other parameters of the resulting polynucleotides (such as mRNA) can be measured, including half-life at different time points. Studies have shown that the protein yield produced by the polyA-G tetramer structure is at least equal to 75% of the protein yield produced by the poly-A region of 120 nucleotides alone.

In some embodiments, the nucleic acid molecules (such as mRNA) of the present disclosure may include a poly-A region and may be stabilized by adding a 3' stabilizing region. In some embodiments, the 3' stabilizing region that can be used to stabilize nucleic acid molecules (such as mRNA) includes a poly-A or poly-A-G tetramer structure, which is described in International Patent Publication No. WO2013/103659, which is incorporated herein by reference in its entirety.

In other embodiments, 3' stabilizing regions that can be used in conjunction with the nucleic acid molecules of the present disclosure include chain terminating nucleosides, such as, but not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleosides or O-methyl nucleosides, 3'-deoxynucleosides, 2',3'-dideoxynucleosides 3'-O-methyl nucleosides, 3'-O-ethyl nucleosides, 3'-arabinoside, other alternative nucleosides described herein or known in the art.

secondary structure

The stem-loop structure can guide RNA folding, protect the structural stability of nucleic acid molecules (such as mRNA), provide recognition sites for RNA binding proteins, and serve as substrates for enzymatic reactions. For example, the integration of miR sequences and/or TEE sequences will change the shape of the stem-loop region, which may increase and/or decrease translation (Kedde et al. A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221and miR-222accessibility. Nat Cell Biol., 2010 Oct; 12(10): 1014-20, the contents of which are incorporated herein by reference in their entirety).

Thus, in some embodiments, the nucleic acid molecules described herein (such as mRNA) or a portion thereof may adopt a stem-loop structure, such as, but not limited to, a histone stem-loop. In some embodiments, the stem-loop structure is formed by a stem-loop sequence of about 25 or about 26 nucleotides in length, which may be, but not limited to, those described in International Patent Publication No. WO2013/103659, the entire contents of which are incorporated herein by reference. Other examples of stem-loop sequences include those described in International Patent Publication No. WO2012/019780 and International Patent Publication No. WO201502667, the contents of which are incorporated herein by reference. In some embodiments, the stem-loop sequence includes TEE as described herein. In some embodiments, the stem-loop sequence includes a miR sequence as described herein. In a specific embodiment, the stem-loop sequence may include a miR-122 seed sequence. In a specific embodiment, the nucleic acid molecule includes the stem-loop sequence CAAAGGCTCTTTTCAGAGCCACCA (SEQ ID NO: 1). In other embodiments, the nucleic acid molecule comprises the stem-loop sequence CAAAGGCUCUUUUCAGAGCCACCA (SEQ ID NO: 2).

In some embodiments, the nucleic acid molecules (such as mRNA) of the present disclosure include a stem-loop sequence located upstream (5' end) of the nucleic acid molecule coding region. In some embodiments, the stem-loop sequence is located within the 5'-UTR of the nucleic acid molecule. In some embodiments, the nucleic acid molecules (such as mRNA) of the present disclosure include a stem-loop sequence located downstream (3' end) of the nucleic acid molecule coding region. In some embodiments, the stem-loop sequence is located within the 3'-UTR of the nucleic acid molecule. In some cases, the nucleic acid molecule may include more than one stem-loop sequence. In some embodiments, the nucleic acid molecule includes at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3'-UTR.

In some embodiments, the nucleic acid molecule comprising the stem-loop structure further comprises a stabilization region. In some embodiments, the stabilization region comprises at least one chain termination nucleoside, which acts to slow down degradation and thus increases the half-life of the nucleic acid molecule. Exemplary chain termination nucleoside that can be used in conjunction with the present disclosure include, but are not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymidine, 2', 3'-dideoxynucleoside, 2', 3'-dideoxyadenosine, 2', 3'-dideoxyuridine, 2', 3'-dideoxycytosine, 2', 3'-dideoxyguanosine, 2', 3'-dideoxythymidine, 2'-deoxynucleoside or O-methyl nucleoside, 3'-deoxynucleoside, 2', 3'-dideoxynucleoside 3'-O-methyl nucleoside, 3'-O-ethyl nucleoside, 3'-arabinoside, other alternative nucleoside described herein or known in the art. In other embodiments, the stem-loop structure can be stabilized by altering the 3' region of the polynucleotide, which alterations can prevent and/or inhibit the addition of oligio(U) (International Patent Publication No. WO2013/103659, which is incorporated herein by reference in its entirety).

In some embodiments, the nucleic acid molecules of the present disclosure include at least one stem-loop sequence and a poly-A region or a polyadenylation signal. Non-limiting examples of polynucleotide sequences including at least one stem-loop sequence and a poly-A region or a polyadenylation signal are included in International Patent Publication No. WO2013/120497, International Patent Publication No. WO2013/120629, International Patent Publication No. WO2013/120500, International Patent No. WO2013/120627, International Patent No. WO2013/120498, International Patent Publication No. WO2013/120626, International Patent Publication No. WO2013/120499 and International Patent Publication No. WO2013/120628, the entire contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a pathogen antigen or a fragment thereof, as described in International Patent Publication No. WO2013/120499 and International Patent Publication No. WO2013/120628, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a therapeutic protein, as described in International Patent Publication No. WO2013/120497 and International Patent Publication No. WO2013/120629, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a tumor antigen or a fragment thereof, as described in International Patent Publication No. WO2013/120500 and International Patent Publication No. WO2013/120627, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode an allergenic antigen or an autoimmune autoantigen, as described in International Patent Publication No. WO2013/120498 and International Patent Publication No. WO2013/120626, the contents of which are incorporated herein by reference in their entirety.

Functional nucleotide analogs

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode an allergenic antigen or an autoimmune autoantigen, as described in International Patent Publication No. WO2013/120498 and International Patent Publication No. WO2013/120626, the contents of which are incorporated herein by reference in their entirety.

Therefore, in some embodiments, the payload nucleic acid molecule comprises at least one functional nucleotide analog as described herein. In some embodiments, the functional nucleotide analog comprises at least one chemical modification to a nucleobase, a sugar group and/or a phosphate group. Therefore, the payload nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to a nucleobase, a sugar group and/or a nucleoside bond. Exemplary chemical modifications to a nucleobase, a sugar group or a nucleoside bond of a nucleic acid molecule are provided herein.

As described herein, all nucleotides in the payload nucleic acid molecule can range from 0% to 100% as functional nucleotide analogs as described herein. For example, in various embodiments, from about 1% to about 20%, from about 1% to about 25%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70% , about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100% or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, the functional nucleotide analogs can be present at any position of the nucleic acid molecule, including the 5'-end, the 3'-end and/or one or more internal positions. In some embodiments, a single nucleic acid molecule may contain different sugar modifications, different core base modifications and/or different types of nucleoside bonds (such as backbone structures).

As described herein, 0% to 100% of all nucleotides of one type (e.g., all purine-containing nucleotides of one type, or all pyrimidine-containing nucleotides of one type, or "as a" in all A, G, C, T, or U payload nucleic acid molecules ranging from 0% to 100%) can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1% to about 20%, from about 1% to about 25%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100% or about 95% to about 100% are functional nucleotide analogs as described herein. In any of these embodiments, functional nucleotide analogs can be present in any position of the nucleic acid molecule, including the 5'-end, the 3'-end and/or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different core base modifications and/or different types of nucleoside bonds (such as backbone structures).

Modification of bases

In some embodiments, the functional nucleotide analogs include non-standard nucleobases. In some embodiments, standard nucleobases (e.g., adenine, guanine, uracil, thymine and cytosine) in a nucleotide can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy and/or thio substitutions; one or more fused rings or ring openings, oxidation and/or reduction.

In some embodiments, the non-standard nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracils include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (S ² U), 4-thio-uracil (S ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho ⁵ U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromouracil), 3-methyluracil (m ³ U), 5-methoxyuracil (mo ⁵ U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uracil (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm ⁵ U), and 5-hydroxy-uracil (h ^{5 U} ). U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl uracil (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thiouracil (mcm ⁵ s ² U), 5-aminomethyl-2-thiouracil (nm ⁵ s ² U), 5-methylaminomethyl-2-uracil (mnm ⁵ U), 5-methylaminomethyl-2-thiouracil (mnm ⁵ s ² U), 5-methylaminomethyl-2-selenouracil (mnm ⁵ se ² U), 5-carbamoylmethyl uracil (ncm5U), 5-carboxymethylaminomethyl uracil (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm ⁵ s ² U), 5-propynyl uracil, 1-propynyl-pseudouracil, 5-taurine methyl uracil (τm ⁵ U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouracil (τm ⁵ s ² U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uracil (m ⁵ U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudoneuroside (m ¹ ψ), 1-ethyl-pseudoneuroside (Et ¹ ψ), 5-methyl-2-thio-uracil (m ⁵ s ² U), 1-methyl-4-thio-guduridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-guduridine, 3-methyl-guduridine (m ³ ψ), 2-thio-1-methyl-duduridin, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m ⁵ D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thiouracil, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uracil (m ⁵ U), 5-(isopentenyl)aminomethyl)-2-thiouracil (m ⁵ s ² U), 5,2'-O-dimethyluridine (m ⁵ Um), 2-thio-2'-O-methyluridine (s ² Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm ⁵ Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm ⁵ Um), 3,2'-O-dimethyluridine (m ³ Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm ⁵ Um). Um), 1-thio-uracil, deoxythymidine, 5-(2-carbonylmethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carbamoyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil and 5-3-(1-E-propyleneamino)uracil.

In some embodiments, the non-standard nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-azacytosine, 6-azacytosine, pseudoisocytidine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g. 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytosine, pyrrolopseudoisocytidine, 2-thiocytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine zebularine), 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methylcytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine (k2C), 5,2'-O-dimethylcytidine (m5Cm), N4-acetyl-2'-O-methylcytidine (ac4Cm), N4,2'-O-dimethylcytidine (m4Cm), 5-formyl-2'-O-methylcytidine (fSCm), N4,N4,2'-O-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine and 5-(2-azidoethyl)-cytosine.

In some embodiments, the non-standard nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a substituted adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-nitrogen-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-nitrogen-2,6 -diaminopurine, 1-methyladenine (m1A), 2-methyladenine (m2A), N6-methyladenine (m6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-isopentenyladenine (i6A), 2-methylthio-N6-isopentenyladenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycylaminoformyl -adenine (g6A), N6-threonine methylcarbamoyl-adenine (t6A), N6-methyl-N6-threonine methylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonine methylcarbamoyl-adenine (ms2g6A), N6, N6-dimethyl-adenine (m62A), N6-hydroxy-n-pentylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxy-n-pentylcarbamoyl-adenine (ms2hn6A), N6-acetyl adenine ( ac6A), 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 1,2'-O-dimethyladenosine (m1Am), 2-amino-N6-methylpurine, 1-thioadenine, 8-azidoadenine, N6-(19-amino-pentaoxadodecane)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine and N6-hydroxymethyl-adenine

In some embodiments, the non-standard nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanine include inosine (I), 1-methylinosine (m1I), inosine (imG), methylinosine (mimG), 4-demethylinosine (imG-14), isotyrosine (imG2), wybutosine (yW), peroxotyrosine (o2yW), hydroxytyrosine (OHyW), undermodified hydroxytyrosine (OHyW*), 7-deazaguanine, quercetin ( Q), epoxyquinone (oQ), galactosylquinone (galQ), mannosylquinone, 7-cyano-7-deazaguanine (preQO), 7-aminomethyl-7-deazaguanine (preQ1), paleoalkaloid (G+), 7-deaza8-azaguanine, 6-thioguanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methylguanine Purine, 7-methyl-inosine, 6-methoxy-guanine, 1-methylguanine (m1G), N2-methylguanine (m2G), N2,N2-dimethylguanine (m22G), N2,7-dimethylguanine (m2,7G), N2,N2,7-dimethylguanine (m2,2,7G), 8-oxoguanine, 7-methyl-8-oxoguanine, 1-methyl-6-thioguanine, N2-methyl-6-thioguanine, N2,N2 -dimethyl-6-thioguanine, N2-methyl-2'-O-methyl-guanine (m2Gm), N2,N2-dimethyl-2'-O-methylguanosine (m22Gm), 1-methyl-2'-O-methylguanosine (m1Gm), N2,7-dimethyl-2'-O-methylguanosine (m2,7Gm), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (mIm), 1-thioguanine and O-6-methylguanine.

In some embodiments, the non-standard nucleobase of the functional nucleotide analog can independently be a purine, a pyrimidine, a purine or a pyrimidine analog. For example, in some embodiments, the non-standard nucleobase can be a modified adenine, cytosine, guanine, uracil or hypoxanthine. In other embodiments, non-canonical nucleobases can also include, for example, naturally occurring and synthetic derivatives of bases, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol , 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenosine, 7-deazaadenosine, 3-deazaadenosine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazine, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, or 1,3,5-triazine.

Sugar modification

In some embodiments, the functional nucleotide analogs include non-standard sugar groups. In various embodiments, the non-standard sugar group can be a 5-carbon or 6-carbon sugar (e.g., pentose, ribose, arabinose, xylose, glucose, galactose or its deoxy derivatives) with one or more substituents, and the substituents can be halogen, hydroxyl, thiol, alkyl, alkoxy, alkenyloxy, alkynyloxy, cycloalkyl, aminoalkoxy, alkoxyalkoxy, hydroxyalkoxy, amino, azido groups, aryl, aminoalkyl, aminoalkenyl, aminoalkynyl, etc.

Typically, RNA molecules contain a ribose group, which is a five-membered ring with an oxygen. Exemplary non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or an alkylene group such as methylene or ethylene); addition of double bonds (e.g., replacement of ribose with a cyclopentenyl or cyclohexenyl group); ring closure of ribose (e.g., to form a four-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring with additional carbon atoms or heteroatoms, such as anhydrohexitol, arabitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino (also with a phosphoramidate backbone)); polycyclic forms (e.g., tricyclic and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, in which ribose is replaced by an ethylene glycol unit attached to a phosphodiester linkage), threose nucleic acids (TNA, in which ribose is replaced by an α-L-threofuranofuranosyl-(3'→2')) and peptide nucleic acids (PNA, in which a 2-amino-ethyl-glycine linkage replaces the ribose and phosphodiester backbone).

In some embodiments, the sugar group comprises one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, the nucleic acid molecule may include nucleotides containing, for example, arabinose or L-ribose as sugars. In some embodiments, the nucleic acid molecule includes at least one nucleoside wherein the sugar is L-ribose, 2'-O-methyl ribose, 2'-fluororibose, arabinose, hexitol, LNA or PNA.

Modification of nucleoside bonds

In some embodiments, the payload nucleic acid molecules of the present disclosure may comprise one or more modified nucleoside bonds (eg, phosphate backbones). The phosphate groups of the backbone may be altered by replacing one or more oxygen atoms with different substituents.

In some embodiments, functional nucleotide analogs may include another nucleoside bond to replace the unchanged phosphate moiety. The example of the phosphate group of substitution includes but is not limited to phosphorothioate, phosphite selenate, boric acid phosphate, boric acid phosphate, phosphonate hydrogen, phosphoramidate, phosphorodiamino ester, alkyl or aryl phosphonate and phosphotriester. Two non-connected oxygens of phosphorodithioate are all replaced by sulfur. It is also possible to connect the phosphate bond of change by replacing oxygen with nitrogen (phosphoramidate of bridge), sulfur (phosphorothioate of bridge) and carbon (methylene phosphonate of bridge).

Alternative nucleosides and nucleotides include borane moieties (BH ₃ ), sulfur (thio), methyl, ethyl and/or methoxy groups in place of one or more non-bridging oxygens. As a non-limiting example, two non-bridging oxygens at the same position (such as the α, β or γ position) can be substituted with sulfur (thio) and methoxy groups. The stability of RNA and DNA is enhanced (e.g., against exonucleases and endonucleases) with non-natural thiophosphate backbone linkages by substitution of one or more oxygen atoms at the position of the phosphate moiety (such as α-phosphorothioate). Phosphorothioate DNA and RNA have enhanced nuclease resistance and therefore have a longer half-life in the cellular environment.

Other nucleoside bonds for use in accordance with the present disclosure include nucleoside bonds that do not contain a phosphorus atom.

Other examples of nucleic acid molecules (such as mRNA), compositions, formulations and/or methods related thereto that can be used in conjunction with the present disclosure are further included in WO2002/098443, WO2003/051401, WO2008/052770, WO2009127230, WO2006122828, WO2008/083949, WO2010088927, WO2010/037539, WO2004/0047 43, WO2005/016376, WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592, WO2009/030481, WO2009/095226, WO2011069586, WO2011026641, WO2011/144358, WO2012019780, WO2012013326, WO2 012089338, WO2012113513, WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698, WO2013143699, WO2013143700, WO2013/120626, WO2013120627, WO2013120628, WO2 013120629, WO2013174409/WO2015127917, WO2015024667, WO2015/024665, WO2015/024666, WO2015/024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416, the contents of each of which are incorporated herein in their entirety.

Dosage form

According to the present disclosure, the nanoparticle compositions described herein may include at least one lipid component and one or more other components, such as therapeutic and/or prophylactic agents. The nanoparticle compositions may be designed for one or more specific applications or targets. The elements of the nanoparticle compositions may be selected based on a specific application or target and/or based on the efficacy, toxicity, cost, ease of use, availability or other characteristics of one or more elements. Similarly, a specific formulation of the nanoparticle composition may be selected for a specific application or target based on the efficacy and toxicity of a specific combination of elements.

The lipid component of the nanoparticle composition may include lipids of formula (I) (and its subformulae) as described herein, phospholipids (e.g., unsaturated lipids such as DOPE or DSPC, etc.), PEG lipids, and structural lipids. The elements of the lipid component may be provided in specific ratios.

In one embodiment, provided herein is a nanoparticle composition comprising a cationic or ionizable lipid compound, a therapeutic agent and one or more excipients provided herein. In one embodiment, a cationic or ionizable lipid compound comprises a compound of formula (I) (and its subformula) as described herein, and optionally one or more other ionizable lipid compounds. In one embodiment, one or more excipients are selected from neutral lipids, steroids and polymer-conjugated lipids. In one embodiment, the therapeutic agent is encapsulated in or associated with a lipid nanoparticle.

In one embodiment, the present invention provides a nanoparticle composition (lipid nanoparticle) comprising:

i) 40 to 50 mole percent of a cationic lipid;

ii) neutral lipids;

iii) steroids;

iv) polymer-conjugated lipids; and

v) Therapeutic agents.

As used herein, "molar percentage" refers to the molar percentage of a component relative to the total moles of all lipid components in the LNP (ie, the total moles of cationic lipids, neutral lipids, steroids, and polymer-conjugated lipids).

In one embodiment, lipid nanoparticles account for 41 to 49 mole percent, 41 to 48 mole percent, 42 to 48 mole percent, 43 to 48 mole percent, 44 to 48 mole percent, 45 to 48 mole percent, and the content of cationic lipid is 46-48 mole percent, or 47.2-47.8 mole percent. In one embodiment, lipid nanoparticles account for about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mole percent of cationic lipid.

In one embodiment, the neutral lipid is present at a concentration of 5 to 15 mole percent, 7 to 13 mole percent, or 9 to 11 mole percent. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 mole percent. In one embodiment, the molar ratio of cationic lipid to neutral lipid is about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

In one embodiment, the steroid is present in a concentration range of 39-49 mole percent, 40-46 mole percent, 40-44 mole percent, 40-42 mole percent, 42-44 mole percent or 44-46 mole percent. In one embodiment, the steroid is present at a concentration of 40, 41, 42, 43, 44, 45 or 46 mole percent. In one embodiment, the molar ratio of cationic lipid to steroid is 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the steroid is cholesterol.

In one embodiment, the ratio of therapeutic agent to lipid in the LNP (i.e., N/P, N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) is 2:1 to 2.30:1, such as 3:1 to 22:1. In one embodiment, N/P is 6:1 to 20:1 or 2:1 to 12:1. Exemplary N/P ranges include about 3:1, about 6:1, about 12:1, and about 22:1.

In one embodiment, provided herein is a lipid nanoparticle comprising:

i) cationic lipids with an effective pKa greater than 6.0;

ii) 5 to 15 mole percent of neutral lipids;

iv) 30 to 45 mole percent of a steroid;

v) polymer-conjugated lipids; and

vi) a therapeutic agent or a pharmaceutically acceptable salt or prodrug thereof,

Herein, the mole percentage is determined based on the total moles of lipid present in the lipid nanoparticles.

In one embodiment, the cationic lipid can be any of a variety of lipids with a net positive charge at a selected pH (such as physiological pH). Exemplary cationic lipids are described below. In one embodiment, the pKa of the cationic lipid is greater than 6.25. In one embodiment, the pKa of the cationic lipid is greater than 6.5. In one embodiment, the cationic lipid has a pKa greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65 or greater than 6.7.

In one embodiment, the lipid nanoparticles comprise 40 to 45 mole percent of the cationic lipids. In one embodiment, the lipid nanoparticles comprise 45 to 50 mole percent of the cationic lipids.

In one embodiment, the molar ratio of cationic lipid to neutral lipid is about 2: 1 to about 8: 1. In one embodiment, the neutral lipid accounts for 5 to 10 mole percent of the lipid in the lipid nanoparticle.

Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), or 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG).

In one embodiment, the lipid nanoparticles contain 1 to 10 mol% anionic lipids. In one embodiment, the lipid nanoparticles contain 1 to 5 mol% anionic lipids. The rice grains contain 1 to 9 mol%, 1 to 8 mol%, 1 to 7 mol% or 1 to 6 mol% of anionic lipids. In one embodiment, the molar ratio of anionic lipids to neutral lipids is 1:1 to 1:10.

In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of cationic lipid to cholesterol is about 5:1 to 1:1. In one embodiment, the lipid nanoparticles contain 32 to 40 mol% of the steroid.

In one embodiment, the sum of the molar percentage of neutral lipids and the molar percentage of anionic lipids is 5 to 15 molar percentages. In one embodiment, the sum of the molar percentage of neutral lipids and the molar percentage of anionic lipids is 7 to 12 molar percentages.

In one embodiment, the molar ratio of anionic lipid to neutral lipid is 1:1 to 1: 10. In one embodiment, the sum of the molar percentages of neutral lipid and steroid is 35 to 45 mole percent.

In one embodiment, the lipid nanoparticle comprises:

i) 45-55 mole percent of a cationic lipid;

ii) 5-10 mole percent of neutral lipids;

iii) 1-5 mole percent of anionic lipids; and

iv) 32-40 mole percent of steroid.

In one embodiment, the lipid nanoparticles contain 1.0 to 2.5 mole percent of the polymer-conjugated lipid. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.5 mole percent.

In one embodiment, the neutral lipid is present at a concentration of 5 to 15 mole percent, 7 to 13 mole percent, or 9 to 11 mole percent. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 mole percent. In one embodiment, the molar ratio of cationic lipid to neutral lipid is about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present in a concentration range of 39 to 49 mole percent, 40 to 46 mole percent, 40 to 44 mole percent, 40 to 42 mole percent, 42 to 44 mole percent or 44 to 46 mole percent. In one embodiment, the steroid is present at a concentration of 40, 41, 42, 43, 44, 45 or 46 mole percent. In certain embodiments, the molar ratio of cationic lipid to steroid is 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2.

In one embodiment, the molar ratio of cationic lipid to steroid is from 5:1 to 1:1.

In one embodiment, the lipid nanoparticle contains 1.0 to 2.5 mole percent of polymer-conjugated lipids. In one embodiment, the polymer-conjugated lipids are present at a concentration of about 1.5 mole percent.

In one embodiment, the molar ratio of cationic lipid to polymer-conjugated lipid is about 100:1 to about 20: 1. In one embodiment, the molar ratio of cationic lipid to polymer-conjugated lipid is about 35:1 to about 25:1.

In one embodiment, the average diameter of the lipid nanoparticles is from 50 nm to 100 nm, or from 60 nm to 85 nm.

In one embodiment, the composition comprises cationic lipids, DSPC, cholesterol and PEG-lipids provided herein and mRNA. In one embodiment, the molar ratio of cationic lipids, DSPC, cholesterol and PEG-lipids provided herein is about 50:10:38.5:1.5.

Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and/or prophylactic agents (e.g., RNA) to specific cells, tissues, organs, or systems thereof, etc. in a mammal. The physicochemical properties of nanoparticle compositions can be altered to increase selectivity for specific body targets. For example, particle size can be adjusted based on fenestration size for different organs. The therapeutic and/or prophylactic agents included in the nanoparticle compositions can also be selected based on the desired one or more delivery targets. For example, therapeutic and/or prophylactic agents can be selected for specific indications, conditions, diseases, or disorders and/or for delivery to specific Cells, tissues, organs or systems, etc. (e.g., local or specific delivery). In certain embodiments, nanoparticle compositions may include mRNA encoding an mRNA that can be translated into a polypeptide of interest in a cell. Such compositions may be specifically designed to be delivered to a specific organ. In certain embodiments, compositions may be designed to be specifically delivered to a mammalian liver.

The amount of the therapeutic and/or prophylactic agent in the nanoparticle composition can depend on the size, composition, desired target and/or other properties of the nanoparticle composition and the properties of the therapeutic and/or prophylactic agent. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence and other characteristics of the RNA. The relative amounts of the therapeutic and/or prophylactic agent and other elements (e.g., lipids) in the nanoparticle composition can also be adjusted. In some embodiments, the wt/wt ratio of the lipid component to the therapeutic and/or prophylactic agent in the nanoparticle composition can be about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1 and 60:1. The wt/wt ratio of the lipid component to the therapeutic and/or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the weight/weight ratio is about 20: 1. The amount of the therapeutic and/or prophylactic agent in the nanoparticle composition can be measured by absorption spectroscopy (e.g., UV-visible spectroscopy).

In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N:P ratio is selected. One or more RNAs, lipids, and amounts thereof may be selected so that the N:P ratio is about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio may be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio may be about 5.67:1.

The physical properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different properties than a nanoparticle composition comprising a different structural lipid. Similarly, the properties of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher mole fraction of phospholipids has different properties than a nanoparticle composition comprising a lower mole fraction of phospholipids. The properties can also vary depending on the method and conditions of preparation of the nanoparticle composition.

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (such as a transmission electron microscope or a scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric methods (such as potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. The Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure multiple characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

In various embodiments, the average size of the nanoparticle composition can be between 10 nm and 100 nm. For example, the average size can be between about 40 nm and about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. In some embodiments, the average size of the nanoparticle composition can be between about 50 nm and about 100 nm, about 50 nm and about 90 nm, about 50 nm and about 80 nm, about 50 nm and about 70 nm, about 50 nm and about 60 nm, about 60 nm and about 100 nm, about 60 nm and about 90 nm, about 60 nm and about 80 nm, about 60 nm and about 70 nm, about 70 nm and about 70 nm. In some embodiments, the nanoparticle composition may have an average size of about 70 nm to about 100 nm. In some embodiments, the average size may be about 80 nm. In other embodiments, the average size may be about 100 nm.

The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicate the electromotive force of the composition. For example, the zeta potential can characterize the surface charge of a nanoparticle composition. It is generally desirable to have a relatively low positively or negatively charged nanoparticle composition because more highly charged materials can interact adversely with cells, tissues, and other elements of the human body. In some embodiments, the zeta potential of a nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 ... mV to about +5mV, about -10mV to about 0mV, about -10mV to about -5mV, about -5mV to about +20mV, about -5mV to about +15mV, about -5mV to about +10mV, about -5mV to about +5mV, about -5mV to about 0mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about +5mV to about +15mV, or about +5mV to about +10mV.

The encapsulation efficiency of the therapeutic and/or prophylactic agent describes the amount of the therapeutic and/or prophylactic agent encapsulated or associated with the nanoparticle composition after preparation relative to the initial amount provided. It is desirable that the encapsulation efficiency is high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic and/or prophylactic agent before and after treatment in a solution containing the nanoparticle composition with one or more organic solvents or detergents to decompose the nanoparticle composition. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agents (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and/or prophylactic agent can be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated into a capsule, film, or tablet having a coating. The capsule, film, or tablet of the composition described herein may have any useful size, tensile strength, hardness, or density.

Pharmaceutical composition

According to the present disclosure, nanoparticle compositions can be formulated as part or all of a pharmaceutical composition. A pharmaceutical composition may include one or more nanoparticle compositions. For example, a pharmaceutical composition may include one or more nanoparticle compositions, and one or more different therapeutic agents and/or prophylactic agents. The pharmaceutical composition may further contain one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the preparation and production of pharmaceutical compositions and formulations are described in, for example, Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006, etc. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition unless they are incompatible with one or more components of the nanoparticle composition. Incompatibility. If an excipient or auxiliary ingredient is incompatible with the components of the nanoparticle composition, their combination may result in undesirable biological or deleterious effects.

In some embodiments, one or more excipients or auxiliary ingredients may account for more than 50% of the total mass or volume of the pharmaceutical composition including the nanoparticle composition. For example, typically one or more excipients or auxiliary ingredients may account for 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and/or the International Pharmacopoeia.

According to the one or more nanoparticle compositions in the pharmaceutical composition of the present disclosure, the relative amount of one or more pharmaceutically acceptable excipients and/or any other ingredients may be adjusted depending on their characteristics, size, and other related conditions, and further depending on the administration object and administration route of the composition. For example, the pharmaceutical composition may contain 0.1% to 100% (wt/wt) of one or more nanoparticle compositions.

In certain embodiments, the nanoparticle compositions and/or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and transportation. For example, at a temperature of 4°C or lower, between about -150°C and 0°C or at a temperature of about -80°C to about -20°C, such as about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C. Pharmaceutical compositions in solution form comprising compounds of formula (I) and subformulas thereof are refrigerated for storage or transportation under conditions such as about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C or -80°C. In some embodiments, the present disclosure also relates to methods for improving the stability of nanoparticle compositions and/or pharmaceutical compositions comprising compounds of formula (I) (and subformulas thereof). By storing the nanoparticle compositions and/or pharmaceutical compositions at 4°C or lower, such as between about -150°C and about 0°C or between about -80°C and about -20°C, such as about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C. The nanoparticle compositions and/or pharmaceutical compositions disclosed herein are stable at 4°C or lower (such as between about 4°C and -20°C) for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least one month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In one embodiment, the formulation is stable for at least 4 weeks at about 4°C. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., acetic acid), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pH value of the pharmaceutical composition of the present disclosure is between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8 or 7 and 7.8). The pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, which is suitable for storage or transportation at about -20°C. For example, the pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has a pH of about 7-7.8, which is suitable for storage or transportation at a temperature such as about 4°C or lower. In the context of the present disclosure, "stable" and "stability" refer to the resistance of the nanoparticle composition or pharmaceutical composition disclosed herein to chemical or physical changes (such as degradation, particle size change, aggregation change) under given manufacturing, preparation, transportation, storage and/or use conditions (such as applied stress (shear force, freeze/thaw stress, etc.)).

Nanoparticle compositions and/or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including by delivering therapeutic and/or prophylactic agents to specific cells of the patient or subject. The invention provides a beneficial therapeutic effect by administering to a cell, tissue, organ or system thereof, such as the renal system. Although the description of the nanoparticle composition and the pharmaceutical composition comprising the nanoparticle composition herein is mainly directed to a composition suitable for administration to a human, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. It is well known that the composition is modified to be suitable for administration to a variety of animals, and a veterinary pharmacist of ordinary skill can design and/or perform such modifications only through ordinary experiments. It is contemplated that the subject to whom the composition is administered is, but is not limited to, humans, other primates and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and rats.

Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or later developed in the field of pharmacology. Generally, such preparation methods include combining the active ingredient with an excipient and/or one or more other auxiliary ingredients, and if necessary, the product can also be divided into single or mixed forms and/or packaged into the desired multiple dosage units.

Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and/or sold in bulk as a single unit dose and/or as multiple single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and/or a convenient fraction of the dose, such as half or one-third of the dose.

The pharmaceutical composition can be prepared into various forms suitable for various routes and methods of administration. For example, the pharmaceutical composition can be prepared into liquid dosage forms (such as emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable dosage forms, solid dosage forms (such as capsules, tablets, pills, powders and granules), dosage forms for topical and/or transdermal administration (such as ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and/or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed, peanut, corn, germ, olive oil, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral composition may contain other therapeutic and/or preventive agents, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and/or flavoring agents and other preparations. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent such as Cremophor™, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrin polymers, and/or combinations thereof.

Injectable preparations can be prepared according to known techniques using suitable dispersants, wetting agents and/or suspending agents, such as aqueous or oily suspensions that can be sterile injected. Aseptic injection preparations can be sterile injection solutions, suspensions and/or emulsions in nontoxic parenteral acceptable diluents and/or solvents, such as solutions in 1,3-butanediol. Available acceptable vehicles and solvents include water, American Ringer's solution and isotonic sodium chloride solution. Sterile fixed oils are generally used as solvents or suspension media. For this reason, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injections.

The injectable formulations can be sterilized by filtration through a bacteria-retaining filter and/or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

The present invention discloses methods for delivering therapeutic and/or prophylactic agents to mammalian cells or organs, producing target polypeptides in mammalian cells, and treating diseases or disorders in mammals by contacting mammalian cells with nanoparticle compositions containing therapeutic and/or prophylactic agents and/or administering to the mammal.

Example

The examples in this section are provided by way of example only and are not limiting.

General approach

Conventional preparative HPLC method: HPLC purifications were performed on a Waters 2767 equipped with a diode array detector (DAD) on an Inertsil Pre-C8 OBD column, typically using water with 0.1% TFA as solvent A and acetonitrile as solvent B.

General LCMS Method: LCMS analysis was performed on a Shimadzu (LC-MS2020) system. Chromatography was performed on a SunFire C18, typically using water with 0.1% formic acid as solvent A and acetonitrile with 0.1% formic acid as solvent B.

Example 1: Preparation of Compound 1

Step 1: Preparation of compound 1-2

Compound 1-1 (30.0 g, 0.193 mol, 1.0 eq.) was added to EA (300 mL, 10 V), then cooled to 0-5 °C, TEA (19.5 g, 0.193 mol, 1.0 eq.) was added, Boc ₂ O (42.0 g, 0.193 mol, 1.0 eq)/EA (50 mL) was added dropwise, warmed to room temperature, stirred overnight, water (150 mL) was added, the organic layer was separated, washed with water (100 mL x 2), dried over Na ₂ SO ₄ , and concentrated to give the title compound (42 g, 99% yield) as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.59 (d, J = 6.6 Hz, 1H), 4.38 (s, 1H), 3.91 (ddd, J = 14.8, 10.8, 4.6 Hz, 2H), 3.78 (s, 3H), 3.06 (s, 1H), 1.47 (d, J = 8.1 Hz, 9H).

Step 2: Preparation of compound 1-3

Compound 1-2 (42.0 g, 0.192 mol, 1.0 eq.) was added to acetone (315 mL, 7.5 V), TsOH (1.82 g, 9.6 mmol, 0.05 eq.), compound A (99.7 g, 0.960 mol, 5.0 eq.) was added, heated to 65 ° C, refluxed for 3 h, TEA (1 g) was added, concentrated, EA (200 mL) was added, washed with water (100 mL×2), dried over Na ₂ SO ₄ , filtered and concentrated to give the title compound (49 g, 98.5 yield) as a red oil.

Step 3: Preparation of compound 1-4

Under _N2 atmosphere, compound B (153.2 g, 1.24 mol, 2.0 eq.) was added to anhydrous THF (800 mL, 5.2 V), cooled to -60°C, n-BuLi (491 mL, 2.5 M, 1.23 mol, 1.98 eq.) was added dropwise below -55°C, and then stirred for 40 minutes, compound 1-3 (160.1 g, 0.62 mol, 1.0 eq.)/THF (300 mL) was added dropwise, and then stirred for 10 minutes, quenched with saturated _NH4Cl (500 mL), extracted with EA (1.5 Lx2), washed with saturated sodium chloride (1 L), dried, concentrated and purified by column chromatography (PE:EA=5:1-2:1-DCM:MeOH=20:1-15:1), and concentrated to give the desired product as a yellow oil (113.9 g, yield 81.4%).

Step 4: Preparation of Compound 1-5

Compound 1-4 (10 g, 0.028 mol, 1.0 eq.) was added to CH ₃ CN (100 mL) and water (1.4 mL), C ₁₃ H ₂₇ CHO (6 g, 0.028 mol, 1.0 eq.), K ₂ CO ₃ (7.9 g, 0.056 mol, 2.0 eq.) were added, stirred at room temperature overnight, filtered, washed with PE (100 mL), concentrated, PE (100 mL) and water (100 mL) were added, the organic layer was separated, washed with water (50 mL×2), dried over Na ₂ SO ₄ , and concentrated to give the desired product as a red oil (11 g, 88% yield).

Step 5: Preparation of Compound 1-6

Compound 1-5 (5 g, 0.011 mol, 1.0 eq.) was added to MeOH (100 mL), CeCl ₃ .7H ₂ O (4.86 g, 0.012 mol, 1.1 eq.), and cooled to -18°C. In another round-bottom flask, 0.2% NaOH aqueous solution (2.6 mL) was added. NaBH ₄ (652 mg, 0.017 mol, 1.5 eq.) was added in batches. The solution was then added dropwise to compound 1-5, and the temperature was kept below -15°C. The mixture was stirred for 20 minutes, and water (100 mL) was added. The mixture was extracted with EA (100 mLx3), washed with saturated sodium chloride (50 mLx2), concentrated, and purified by column chromatography (PE:EA=20:1-10:1). The desired product was obtained by concentration as a yellow oil (1 g, 20% yield).

Step 6: Preparation of Compound 1-7

AcCl (356 mg, 4.56 mmol, 2.0 eq.) was added to MeOH (50 mL) at below 10 °C, followed by stirring for 30 minutes, and compound 1-6 (1 g, 2.28 mmol, 1.0 eq.)/MeOH (2 mL) was added, stirred at room temperature for 7 h, concentrated, EA (4 mL) was added, stirred for 10 min, and filtered to give a white solid (460 mg, 54.4% yield).

LCMS: Rt: 4.427 min; MS m/z (ESI): 300.5 [M+H] ⁺ .

Step 7: Preparation of Compound 1-8

Compound 1-7 (1 g, 0.034 mol, 1.0 eq.) was added to DMF (2 mL) and THF (6 mL), stearic acid (950 mg, 0.034 mol, 1.0 eq.), HATU (1.4 g, 0.038 mol, 1.1 eq.) was added, TEA (1.01 g, 0.102 mol, 3.0 eq.) was added dropwise under an ice-water bath, and then stirred at room temperature overnight, water (20 mL) was added, filtered, washed with water, and the solid was added to acetone (5 mL), stirred for 10 minutes, filtered and dried to obtain a white solid (1.76 g, 93% yield).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.25 (dd, J = 50.3, 6.7 Hz, 1H), 5.81 (td, J = 14.7, 7.2 Hz, 1H), 5.57 (dd, J = 15.9, 6.3 Hz, 1H), 4.39 (d, J = 30.9 Hz, 1H), 3.88 (dt, J = 46.7, 10.5 Hz, 2H), 2.50(s, 2H), 2.26(d, J＝6.9Hz, 2H), 2.09(d, J＝6.7Hz, 2H), 1.67(s, 2H), 1.30(s, 50H), 0.92(t, J＝6.3Hz, 6H).

Step 8: Preparation of Compound 1-9

Compound 1-8 (1.76 g, 0.003 mol, 1.0 eq.) was added to DCM (20 mL), EDCI (1.79 g, 0.009 mol, 3.0 eq.), DMAP (418 mg, 0.0033 mol, 1.1 eq.) was added, cooled to 0°C, 6-bromohexanoic acid (604 mg, 0.003 mol, 1.0 eq.)/DCM (1 mL) was added dropwise, stirred at 0-5°C for 2 h, water (20 mL) was added, extracted with DCM (50 mLx3), washed with water, concentrated, purified by column chromatography (PE:EA=15:1-5:1-3:1-2:1), and concentrated to give a white solid (1 g, yield 43.4%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.93–5.70 (m, 2H), 5.52 (dd, J=15.4, 6.5 Hz, 1H), 4.53–4.08 (m, 4H), 3.46 (t, J=6.7 Hz, 2H), 2.39 (t, J=7.4 Hz, 2H), 2.24 (t, J=7.6 Hz, 2H), 2.09 (dd, J=13.9, 6.9 Hz, 2H), 2.02–1.83 (m, 4H), 1.69 (dt, J=16.0, 8.1 Hz, 4H), 1.61–1.22 (m, 50H), 0.93 (t, J=6.8 Hz, 6H).

Step 9: Preparation of Compound 1

Compound 1-9 (200 mg, 0.270 mmol, 1.0 eq.) was added to DMF (2 mL), DIPEA (104.4 mg, 0.809 mmol, 3.0 eq.), NaI (4 mg, 0.027 mmol, 0.1 eq.), compound 1-10 (166 mg, 0.035 mmol, 1.3 eq.) was added, heated to 110 ° C, stirred for 4 h, cooled to room temperature, EA (20 mL) was added, water (10 mL) was added, the organic layer was separated, extracted with EA (20 mL x 2), washed with saturated NaCl (40 mL x 3), dried over Na ₂ SO ₄ , concentrated, purified by column chromatography (DCM: MeOH = 50: 1-30: 1-20: 1-10: 1), concentrated to give a colorless oil (100 mg), and then purified by Pre-HPLC to give a colorless oil (20 mg, yield 6.5%).

LCMS: Rt: 0.093 min; MS m/z (ESI): 1133.9 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.18 (d, J = 7.4 Hz, 1H), 5.83-5.63 (m, 1H), 5.48 (dd, J = 15.4, 6.5 Hz, 1H), 4.43-3.91 (m, 8H), 3.54 (s, 2H), 2.73-1.93 (m, 16H), 1.85-1.01 (m, 91H), 0.88 (t, J = 6.8 Hz, 12H).

The following compounds were prepared in a similar manner to Compound 1 using the corresponding starting materials.

Example 2: Preparation of compound 4.

Step 1: Preparation of compound 4-2

Compound 4-1 (4 g, 0.0215 mmol, 1.0 eq.) was added to DCM (40 mL), EDCI (12.4 g, 0.0645 mmol, 3.0 eq.), DMAP (2.9 g, 0.0236 mmol, 1.1 eq.), and compound C (4.16 g, 0.0215 mmol, 1.0 eq.) were added, stirred overnight, concentrated, PE (100 mL), water (100 mL) were added, the organic layer was separated, washed with 0.5 N HCl (40 mLx1), saturated NaHCO ₃ (40 mLx1), dried over Na ₂ SO ₄ , and concentrated to give a colorless oil (5.2 g, 67% yield).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.10 (t, J = 6.7 Hz, 2H), 3.57 (t, J = 6.7 Hz, 1H), 3.45 (t, J = 6.8 Hz, 1H), 2.36 (t, J = 7.4 Hz, 2H), 2.07–1.77 (m, 2H), 1.77–1.58 (m, 4H), 1.58–1.44 (m, 2H), 1.44–1.18 (m, 18H), 0.92 (t, J = 6.8 Hz, 3H).

Step 2: Preparation of compound 4-3

Compound 4-2 (1.0 g, 2.76 mmol, 1.0 eq.) was added to EtOH (10 mL), followed by compound D (737 mg, 8.29 mmol, 3.0 eq.), stirred at 88°C overnight, concentrated, purified by column chromatography (DCM: MeOH = 50: 1-30: 1-20: 1-10: 1), and concentrated to give an oil (700 mg, 55% yield).

¹ H NMR (400 MHz, DMSO) δ 4.03 (d, J = 6.6 Hz, 2H), 3.42 (t, J = 6.1 Hz, 2H), 2.70 (q, J = 7.2 Hz, 4H), 2.31 (t, J = 7.3 Hz, 2H), 1.52 (ddd, J = 32.5, 14.2, 6.9 Hz, 10H), 1.40–1.16 (m, 22H), 0.88 (t, J = 6.8 Hz, 3H).

LCMS: Rt: 0.205 min; MS m/z (ESI): 372.5 [M+H] ⁺ .

Step 3: Preparation of compound 4

Compound 1-9 (200 mg, 0.270 mmol, 1.0 eq.) was added to DMF (2 mL), DIPEA (105 mg, 0.809 mmol, 3.0 eq.), NaI (5 mg, 0.027 mmol, 0.1 eq.), compound 4-3 (130 mg, 0.351 mmol, 1.3 eq.) was added, heated to 110 ° C, stirred for 4 h, cooled to room temperature, EA (20 mL) was added, water (10 mL) was added, the organic layer was separated, extracted with EA (20 mL x 2), washed with saturated NaCl (40 mL x 3), dried with Na ₂ SO ₄ , concentrated, purified by column chromatography (DCM: MeOH = 50: 1-30: 1-20: 1-10: 1), concentrated to give a colorless oil (107 mg), and then purified by Pre-HPLC to give a colorless oil (26 mg, yield 9.3%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.26 (d, 1H), 5.78 (m, 1H), 5.50 (m, 1H), 4.36 (m, 1H), 4.20 (m, 3H), 4.05 (t, 2H), 3.60 (s, 2H), 2.5 (s, 4H), 2.38 (m, 4H), 2.20 (m, 2H), 2.17 (m, 2H), 1.72–1.59 (m, 15H), 1.30–1.25 (m, 76H), 0.89–0.86 (m, 9H).

LCMS: Rt: 0.567 min; MS m/z (ESI): 1034.0 [M+H] ⁺ .

The following compounds were prepared in a similar manner to Compound 4 using the corresponding starting materials.

Example 3: Preparation of compound 15.

Step 1: Preparation of compound 15-1

Compound 1-7 (2 g, 6.69 mmol, 1.0 eq.) was added to DMF (4 mL) and THF (12 mL), compound E (963 mg, 6.69 mmol, 1.0 eq.), HATU (2.8 g, 7.36 mmol, 1.1 eq.) were added, TEA (2.0 g, 20.07 mmol, 3.0 eq.) was added dropwise under an ice-water bath, and then stirred at room temperature overnight, water (40 mL) was added, extracted with EA (40 mLx3), washed with saturated sodium chloride (40 mLx3), concentrated and purified by column chromatography (PE:EA=5:1-3:1-1:1-EA), and concentrated to give a red solid (2.3 g, yield 82%).

Step 2: Preparation of compound 15-2

Compound 15-2 (2.3 g, 5.41 mmol, 1.0 eq.) was added to DCM (30 mL), EDCI (3.1 g, 16.23 mmol, 3.0 eq.), DMAP (727 mg, 5.95 mmol, 1.1 eq.) was added, cooled to 0°C, compound C (1.0 g, 5.41 mmol, 1.0 eq.)/DCM (2 mL) was added dropwise, stirred at 0-5°C for 2 h, water (20 mL) was added, extracted with DCM (50 mLx3), washed with water, concentrated, purified by column chromatography (PE:EA=15:1-5:1-3:1-2:1), and concentrated to obtain an oil (1.55 g, 48% yield).

Step 3: Preparation of compound 15-3

Compound F (1.26 g, 2.21 mmol, 1.0 eq.) was added to DMF (20 mL), DIPEA (855 mg, 6.63 mmol, 3.0 eq.), NaI (166 mg, 1.11 mmol, 0.1 eq.), and compound 15-2 (1.33 g, 2.21 mmol, 1.0 eq.) were added, heated to 105 ° C, stirred for 4 h, cooled to room temperature, EA (50 mL) was added, water (50 mL) was added, the organic layer was separated, extracted with EA (50 mL x 2), washed with saturated NaCl (40 mL x 3), dried over Na ₂ SO ₄ , concentrated, purified by column chromatography (DCM: MeOH = 50: 1-30: 1-20: 1-10: 1), and concentrated to give an oil (1.24 g).

LCMS: Rt: 0.128 min; MS m/z (ESI): 1093.3 [M+H] ⁺ .

Step 4: Preparation of compound 15-4

Compound 15-3 (1.24 g) was added to DCM (20 mL), TFA (6 mL) was added, and the mixture was stirred at room temperature for 0.5 h, then cooled to 0°C, the pH was adjusted to alkaline with saturated sodium bicarbonate, extracted with DCM (20 mLx3), washed with saturated NaCl (40 mL), dried with Na ₂ SO ₄ , and concentrated to obtain an oil (1.2 g).

LCMS: Rt: 0.165 min; MS m/z (ESI): 992.9 [M+H] ⁺ .

Step 5: Preparation of compound 15-5

Compound 15-4 (1.0 g, 1.0 mmol, 1.0 eq.) was added to EtOH (20 mL), followed by addition of compound G (857 mg, 5 mmol, 5.0 eq.) and DIPEA (260 mg, 2 mmol, 2.0 eq.), stirred at 40° C. overnight, concentrated, purified by column chromatography (DCM: MeOH = 100: 1-80: 1-50: 1-30: 1), and concentrated to give an oil (850 mg, yield 76%).

LCMS: Rt: 0.165 min; MS m/z (ESI): 1116.6 [M+H] ⁺ .

Step 6: Preparation of compound 15

Compound 15-5 (200 mg, 0.18 mmol, 1.0 eq.) was added to EtOH (4 mL), followed by compound H (55 mg, 0.9 mmol, 5.0 eq.), stirred at room temperature for 4 h, concentrated, purified by column chromatography (DCM: MeOH = 50: 1-40: 1-30: 1-20: 1), and concentrated to obtain an oil (188 mg).

LCMS: Rt: 0.165 min; MS m/z (ESI): 1130.2 [M+H] ⁺ .

The following compounds were prepared in a similar manner to Compound 15 using the corresponding starting materials.

Example 4: Preparation of Compound 21.

Step 1: Preparation of compound 21-3

To a solution of 21-1 (10 g, 48.73 mmol) in DCM (200 mL), DIEA (18.89 g, 146.19 mmol), 21-2 (7.13 g, 73.10 mmol) and HATU (37.06 g, 97.46 mmol) were added. The mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was poured into water and extracted with DCM. Washed with brine The combined organic layers were dried over Na2SO4, concentrated and purified by silica gel (EtOAc) column chromatography to give the title compound as a colorless oil (10.0 g, 83% yield). LCMS: Rt: 0.817 min; MS m/z (ESI): 271.1 [M+Na] ⁺ .

Step 2: Preparation of compound 21-4

To a solution of 21-3 (9.0 g, 36.25 mmol) in DCM (400 mL), 1H imidazole (3.70 g, 54.37 mmol) and TBDPSCl (11.96 g, 43.50 mmol) were added. The mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, concentrated and purified by silica gel column chromatography (PE/EtOAc=3/1) to give the title compound as a yellow oil (11.0 g, 62% yield). LCMS: Rt: 1.097 min; MS m/z (ESI): 509.3 [M+Na] ⁺ .

Step 3: Preparation of compound 21-6

To a solution of 21-4 (8.0 g, 16.44 mmol) in THF (80 mL) was added 21-5 (82.19 mL, 82.19 mmol). The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, concentrated and purified by silica gel column chromatography (PE/EtOAc=10/1) to give the title compound (8.0 g, 90% yield) as a yellow oil

Step 4: Preparation of compound 21-7

To a solution of 21-6 (8.0 g, 14.82 mmol) in MeOH (200 mL) was added NaBH4 (1.12 g, 29.64 mmol). The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, concentrated and purified by silica gel column chromatography (PE/EtOAc=4/1) to give the title compound as a colorless oil (6.5 g, 75% yield).

Step 5: Preparation of compound 21-8

To a solution of 21-7 (5.0 g, 9.25 mmol) in DCM (50 mL) was added TFA (10 mL). The mixture was stirred at 35 °C for 10 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the title compound (5.0 g, 97% yield) as a yellow oil.

LCMS: Rt: 0.707 min; MS m/z (ESI): 442.5 [M+H] ⁺ .

Step 6: Preparation of compound 21-10

To a solution of 21-8 (5.0 g, 9.0 mmol) in DCM (150 mL) was added DIEA (3.49 g, 26.99 mmol), 21-9 (1.56 g, 10.8 mmol) and HATU (5.13 g, 13.5 mmol). The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The mixture was poured into water and extracted with DCM. The combined organic layers were washed with brine, Drying over Na2SO4, concentration and purification by silica gel column chromatography (PE/EtOAc=3/1) gave the title compound (4.5 g, 88% yield) as a yellow oil.

Step 7: Preparation of compound 21-11

To a THF (40 mL) solution of 21-10 (4.5 g, 7.92 mmol) was added TBAF (40 mL, 40 mmol). The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, concentrated and purified by silica gel column chromatography (PE/EtOAc=2/1) to obtain the title compound (1.7 g, 65% yield) as a yellow oil.

Step 8: Preparation of compound 21-13

DIEA (1.06 g, 8.19 mmol), EDCI (785 mg, 4.10 mmol) and DMAP (334 mg, 2.73 mmol) were added to a DCM (30 mL) solution of 21-11 (0.9 g, 2.73 mmol) and 21-12 (532 mg, 2.73 mmol). The mixture was stirred at 45 ° C for 10 hours. TLC showed that the reaction was complete. The reaction mixture was concentrated and purified by silica gel column chromatography (PE/EA=3:1) to obtain the title compound (2.0 g, 62% yield) as a white solid.

Step 9: Preparation of Compound 21

To 21-13 (200 mg, 0.39 mmol) and 21-14 (350 mg, 0.79 mmol) in THF (10 mL). The mixture was stirred at 70 ° C for 10 hours. LCMS showed that the reaction was complete. The mixture was concentrated and purified by preparative HPLC to obtain the target compound (51 mg, 15% yield) as a yellow oil.

¹ H NMR (400 MHz, CDCl3) δ: 0.79-0.83 (m, 12H), 1.28-1.59 (m, 58H), 1.90-1.94 (m, 1H), 2.11-2.54 (m, 14H), 3.48-3.50 (m, 3H), 3.96-4.80 (m, 7H), 5.86-5.61 (m, 0.88H), 7.97-8.04 (m, 0.23H).

LCMS: Rt: 0.820 min; MS m/z (ESI): 869.8 [M+H] ⁺ .

Example 5: Preparation of compound 39.

Step 1: Preparation of compound 39-1

A solution of 39-1 (800 mg, 1.24 mmol, 1.0 eq), D (165 mg, 1.85 mmol, 1.5 eq), K2CO3 (513 mg, 3.72 mmol, 3.0 eq), Cs2CO3 (121 mg, 0.37 mmol, 0.3 eq), NaI (18 mg, 0.12 mmol, 0.1 eq) in MeCN (16 ml) was stirred at 80° C. for 16 hours. The mixture was concentrated and purified by column chromatography on silica gel to give 39-1 (360 mg, 39.40% yield) as a yellow oil.

Step 2: Preparation of compound 39

A mixture of 39-1 (280 mg, 0.44 mmol, 1.0 eq) in DMF (3 ml) was added to 39-3 (200 mg, 0.40 mmol, 1.0 eq), NaI (18 mg, 0.12 mmol, 0.3 eq) and DIEA (155 mg, 1.2 mmol, 3.0 eq) and stirred at 105 [UNK] for 3 hours. The reaction mixture was concentrated and purified by PREP-HPLC to give the title 39 as a yellow oil (28 mg, 6.59% yield) MS m/z (ESI): 1063.9 [M+H]+.

¹ H NMR (400 MHz, CDCl3) δ 6.23-5.41 (m, 3H), 4.35-3.95 (m, 9H), 3.71-3.51 (m, 3H), 2.57-1.65 (m, 21H), 1.50-1.35 (m, 21H), 1.30-1.21 (m, 50H), 0.89-0.86 (m, 15H)

The following compounds were prepared in a similar manner to compound 39 using the corresponding starting materials.

Example 6: Preparation of Compound 45.

Step 1: Preparation of compound 45

Pd/C (22 mg) was added to a solution of compound 39 (220 mg, 0.21 mmol, 1.0 eq) in MeOH (2 ml) and reacted at room temperature for 3 h under hydrogen atmosphere. The reaction mixture was concentrated and purified by PREP-HPLC to give the title compound 45 (33 mg, 14.73% yield) as a yellow oil.

MS m/z(ESI):1065.8[M+H]+.

1H NMR (400MHz,CDCl3)δ6.40-6.34(m,1H),4.44-4.01(m,9H),3.66-3.54(m,4H),2.53-2.02(m,17H),1.99-1.59(m,24H),1.49-1.25(m,54H),0.89-0.86(m,15H)

The following compounds were prepared in a similar manner to Compound 51 using the corresponding starting materials.

Example 7: Preparation of compound 46.

Step 1: Preparation of compound 46-1

A solution of 46-1 (1.0 g, 2.17 mmol, 1.0 eq), L (0.5 g, 10.83 mmol, 5.0 eq), NaI (97 mg, 0.65 mmol, 0.3 eq), DIEA (0.56 g, 4.34 mmol, 2.0 eq) in DMF (10 ml) was stirred at 80° C. for 3 hours. The mixture was concentrated and purified by column chromatography on silica gel to give the title compound 46-1 (300 mg, yield 32.50%) as a yellow oil

Step 2: Preparation of compound 46

A solution of 46-1 (230 mg, 0.50 mmol, 1.0 eq), 15-2 (0.3 g, 0.50 mmol, 1.0 eq), NaI (212 mg, 0.15 mmol, 0.3 eq) and DIEA (0.13 g, 1.0 mmol, 2.0 eq) in DMF (4 ml) was stirred at 65° C. for 3 hours. The mixture was quenched with H2O (10 ml) and extracted with DCM (10 mL), then concentrated and purified by PREP-HPLC to give the title compound 46 (100 mg, 6.85% yield) as a yellow oil. MS m/z (ESI): 962.14 [M+H]+.

¹ H NMR (400 MHz, CDCl3) δ 5.80-5.73 (m, 1H), 5.51-5.46 (m, 1H), 4.87-4.84 (m, 1H), 4.42-4.37 (m, 1H), 4.22-4.14 (m, 3H), 2.96 (m, 6H), 2.38-2.23 (m, 5H), 2.04-1.49 (m, 27H), 1.43-1.20 (m, 55H), 1.03-1.01 (m, 3H), 0.90-0.86 (m, 12H)

The following compounds were prepared in a similar manner to Compound 46 using the corresponding starting materials.

Example 8: Preparation of compound A.

Step 1: Synthesis of compound A-2

A mixture of compound A-1 (1.0 g, 4.7 mmol, 1.0 eq) and methylamine in methanol (4 M, 12 ml) was stirred at room temperature overnight. After LCMS showed that the reaction was completed, the reaction mixture was concentrated in vacuo, and the obtained residue was purified by column chromatography (silica gel column, eluent PE: EA = 3: 1 (volume ratio)), and rotary evaporation gave compound 1-2 (390 mg, 34.2% yield). LCMS: Rt: 0.980 min; MS m/z (ESI): 244.3 [M+H] ⁺ .

Step 2: Synthesis of Compound A

Compound A-2 (180.8 mg, 0.74 mmol, 1.0 eq), compound A-3 (620.8 mg, 1.47 mmol, 2.0 eq), K ₂ CO ₃ (306.3 mg, 2.22 mmol, 3.0 eq), Cs ₂ CO ₃ (71.7 mg, 0.22 mmol, 0.3 eq) and NaI (32.9 mg, 0.22 mmol, 0.3 eq) in acetonitrile (15 ml) were stirred at 90°C overnight. LCMS showed that the reaction was complete. The reaction mixture was diluted with EA, washed with water and concentrated. The residue was purified by HPLC to obtain the target product, compound A (46 mg, 10.7% yield).

1H NMR (400MHz,CDCl3)δ:0.86-0.90(m,9H),1.26(s,46H),1.30-1.53(m,4H),1.60-1.67(m,3H),2.24-2.35(m,8H),2.47-2.51(m,1H),3.60-3.63(m,1H),3.97(d,J＝5.6Hz,2H).

LCMS: Rt: 1.050 min; MS m/z (ESI): 582.9 [M+H] ⁺ .

Example 9: Preparation and characterization of lipid nanoparticles

In brief, cationic lipids, DSPC, cholesterol and PEG-lipids provided by the present invention are dissolved in ethanol at a molar ratio of 50:10:38.5:1.5, and mRNA is diluted in 10 to 50 mM citrate buffer, pH = 4. The ethanol lipid solution is mixed with the mRNA aqueous solution at a volume ratio of 1:3 using a microfluidic device at a flow rate of 9-30 mL/min, and the weight ratio of total lipid to mRNA is about 10:1 to 30:1 to prepare LNP. And PBS is used for dialysis instead of ethanol, thereby removing ethanol. Finally, the lipid nanoparticles are filtered through a 0.2 μm sterile filter.

The size of the liposomal nanoparticles was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode. The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK) according to the manufacturer’s instructions.

As reported in the literature, the apparent pKa of LNP formulations is correlated with the delivery efficiency of LNPs to nucleic acids in vivo. The apparent pKa of each formulation was determined using a 2-(p-tolyl)-6-naphthalenesulfonic acid (TNS)-based fluorescence assay. LNP formulations containing cationic lipids/DSPC/cholesterol/DMG-PEG (50/10/38.5/1.5 mol%) were prepared as described above. TNS was made into a 300uM stock solution in distilled water. The LNP formulation was diluted to 0.1mg/ml total lipid in 3mL of a buffer solution containing 50mM sodium citrate, 50mM sodium phosphate, 50mM sodium borate, and 30mM sodium chloride, with a pH of 3 to 9. TNS solution was added to a final concentration of 0.1mg/ml, and after vortex mixing, the fluorescence intensity was measured at room temperature in a Molecular Devices Spectramax iD3 spectrometer using excitation wavelengths of 325nm and 435nm. A sigmoidal best-fit analysis was performed on the fluorescence data, and the pKa values were measured at the pH values that gave half of the maximum fluorescence intensity.

Example 10: Animal Studies

Lipid nanoparticles containing human erythropoietin (hEPO) mRNA encapsulated with the compounds in the table below were administered at a dose of 0.5 mg/kg to 6-8 week old female ICR mice (Xipuer-Bikai, Shanghai) by tail vein injection. Blood samples were taken from mice at specific time points (e.g., 6 hours) after administration. In addition to the above test groups, lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) encapsulating hEPO mRNA were administered at the same dose to mice of similar age and gender as a positive control.

After the last sampling time point, mice were euthanized by overdose of CO _2. Serum was separated from whole blood by centrifugation at 5000 g for 10 min at 4°C, snap frozen and stored at -80°C until analysis. ELSA analysis was performed using a commercially available kit (DEP00, R&D Systems) according to the manufacturer's instructions.

The characteristics of the tested lipid nanoparticles are listed in the table below, including the expression levels over MC3 measured from the test groups.

A: ≥2

B: ≥1 and <2

C: ≥0.1 and <1

D: <0.1.

The compounds of the present invention have the desired size (nm), polydispersity index (PDI), encapsulation efficiency (EE%), expression relative to MC3, and apparent pKa.

Example 11: Preparation of LNP

The compounds were dissolved in ethanol with cholesterol, DSPC, and PEG-DMG at a molar ratio of 50:38.5:10:1.5, and the luciferase mRNA was dissolved in a 50mM citric acid buffered saline solution at pH 4.0. The volume ratio of the two solutions was 1:3. The two phases were quickly mixed using microfluidics and the buffer environment was replaced with PBS at pH 7.4 using dialysis or tangential flow to remove ethanol, and LNPs were prepared respectively. The particle size, PDI, encapsulation efficiency, and apparent pka of each LNP were tested, and the results are shown in the following table

n.dNot determined

The compounds of the present invention have desired size (nm), polydispersity index (PDI), encapsulation efficiency (EE%), and apparent pKa.

Example 12: Animal Experiment

Each prepared LNP was injected into mice via tail vein intravenous injection (dosing concentration 0.25 mpk), and the total fluorescence intensity in the mice was tested after 6 hours (Figure 1 and Table 1). The results showed that the lipid compounds here had good in vivo fluorescence expression, whether relative to compound A or relative to MC3. The organ expression of compound 5 showed that LNP 5 had strong fluorescence intensity not only in the liver (Figure 2), but also in the spleen (Figure 3).

Table 1. Fluorescence expression data of mice intravenously administered with different lipids

Compared with MC3, the compounds of the present invention have higher fluorescence expression in mice after intravenous administration, and will have good applications in the fields of mRNA protein replacement and tumor immunity in the future. The corresponding compounds have good expression in the liver and spleen, and can be used for the application of liver-targeted diseases and the treatment of diseases in the spleen in the future.

Example 13: Intramuscular injection Luc evaluation

Lipid nanoparticles encapsulating luciferase mRNA were administered intramuscularly to 6-8 week old female Balb/c mice (Zhejiang Weitong Lihua Experimental Animal Co., Ltd.) at a dose of 0.05 μg/mouse. The single administration lasted for 6 hours, and the fluorescence expression of the mouse muscles was tested (Table 2)

Table 2. Fluorescence expression data of mouse muscles after administration of different lipids to mouse muscles

Compared with MC3, the compound of the present invention has higher fluorescence expression in the muscle part after intramuscular injection, and will be widely used in the fields of mRNA vaccine, tumor immunity, etc. in the future.

Claims (26)

1. The compound represented by formula (I):
   

   or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

   _L1 is selected from _C1 , _C2 , _C3 , C4, _C5 , _C6 , C7, _C8 , _C9 , _C10 , _C11 or _C12 alkylene, or _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 or _C12 alkenylene or alkynylene, _wherein one or more methylene groups _are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

   _L2 is selected from _C1 , _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 or _C20 alkylene, or _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _{C13 , C14} , _C15 , _C16 , _C17 , _C18 , _C19 or C20 _alkylene . ₂₀ alkenylene or alkynylene, wherein one or more methylene groups are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

   _R1 is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl or heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl or heteroaryl is optionally substituted independently by hydroxy, hydroxy( _C1 - _C6 )alkyl, mercapto, amino, halogen, cyano, aryl, heteroaryl, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or _Rb -X-; optionally, two substituents on the same carbon atom of the alkyl, alkenyl or alkynyl together with the carbon atom form a cycloalkyl; the alkyl is _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , C7 _, C8, _C9 or _C10 alkyl _, the alkenyl or alkynyl is _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 or C10 ₁₀ alkenyl or alkynyl, the cycloalkyl or cycloalkenyl is C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ or C ₁₀ cycloalkyl or cycloalkenyl;

   _R2 is selected from _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 , _C20 , _C21 , _C22 , _C23 , _C24 , _C25 , _C26 , _C27 , _C28 , _C29 or _{C30 alkyl} , alkenyl or alkynyl;

   _R3 is selected from _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 , _C20 , _C21 , _C22 , _C23 , _C24 , _C25 , _C26 , _C27 , _C28 , _C29 or _{C30 alkyl} , alkenyl or alkynyl;

   _R4 is selected from H, _C4 , _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _{C16 , C17, C18, C19, C20, C21, C22, C23 or C24 alkyl , alkenyl or alkynyl , wherein one} or more methylene groups are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

   _R5 is selected from H, _C4 , _C5 , _C6 , _C7 , C8, _C9 , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _{C16 , C17, C18, C19, C20, C21, C22, C23 or C24 alkyl , alkenyl or alkynyl , wherein one} or more methylene groups are optionally independently replaced by -O-, -S-, -S(O)-, -S(O) _2- , -C(O)-, -C(S)-, or -NR _a - instead;

   and _R4 and _R5 are not H at the same time;

   X is selected from O, S or N(R _c );

   _Ra is selected from H, ( _C1 - _C10 ) alkyl (preferably ( _C1 - _C6 ) alkyl), ( _C2 - _C6 ) alkenyl, ( _C2 - _C6 ) alkynyl, ( _C3 - _C10 ) cycloalkyl, hydroxyl or mercapto;

   R _b is selected from H, (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, wherein the (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl or (C ₂ -C ₆ )alkynyl optionally substituted by hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or (C ₃ -C ₁₀ )cycloalkyl; (C 3 -C 10)cycloalkyl, aryl or heteroaryl optionally substituted by (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or (C _{3 -C 10} )cycloalkyl; (C ₄ -C ₁₀ )cycloalkenyl optionally substituted by oxo or -N(R _d ) ₂ , R _d being independently selected from H, or (C ₁ -C ₆ )alkyl, (C ₂ -C _{6 )} alkenyl or (C ₂ -C ₆ )alkynyl optionally substituted by hydroxy; or

   R _c is selected from H, (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, wherein the (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl or (C ₂ -C ₆ )alkynyl is optionally substituted with hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or (C ₃ -C ₁₀ )cycloalkyl; (C 3 -C 10 )cycloalkyl, aryl or heteroaryl optionally substituted with (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, hydroxy, thiol, amino, halogen, cyano, aryl, heteroaryl or (C ₃ -C ₁₀ )cycloalkyl; (C ₄ -C ₁₀ )cycloalkenyl optionally substituted with oxo or _-N (R _{d ) 2} , R _d is independently selected from H, or (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl or (C ₂ -C ₆ )alkynyl optionally substituted with hydroxy; or

   wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, aryl, heteroaryl, phenyl, phenylene, naphthylene or amino group is optionally substituted independently by one or more substituents; preferably, the substituents are selected from (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, (C ₃ -C ₁₀ ) cycloalkyl, (C ₃ -C ₁₀ ) cycloalkenyl, hydroxyl, thiol, amino, halogen, cyano, heterocyclyl, aryl or heteroaryl.
2. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein _L1 is selected from the group consisting of _L1a - _OL1a , _L1a - _SL1a , _L1a -S(O) _-L1a , _L1a -S(O) ₂ - _L1a , _L1a -C(O)-L1a, _L1a -C(S) _-L1a , L1a _{- NRa} - _L1a , _L1a - _OC (O) _-L1a , _L1a -C(O) _-OL1a , L1a _- NRa _-C (O) _-L1a , _L1a -C(O)-NRa _{-L1a ,} L _1a -SC(O)-L _1a or L _1a -OC(S)-L _1a , L _1a at each occurrence is independently selected from _a chemical bond, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ or C ₁₂ alkylene, or C ₂ , C 3 , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ or C ₁₂ alkenylene or alkynylene.
3. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein _L2 is selected from _L2a - _OL2a , _L2a - _SL2a , _L2a -S(O) _-L2a , _L2a -S(O) ₂ - _L2a , L2a-C(O)-L2a, _L2a -C(S) _-L2a , _L2a- NRa- _L2a , _L2a - _OC (O) _-L2a , _L2a - _C (O) _-OL2a , _{L2a -} NRa _-C (O) _-L2a , _L2a -C(O)-NRa _{- L2a} , _L2a -SC(O) _-L2a or _L2a -OC(S) _-L2a , _L2a at each occurrence is independently selected from a chemical bond, _C1 , _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _{C13, C14 , C15} , _C16 , _C17 , _C18 , _C19 or _C20 alkylene, or _C2 , _C3 , C4, _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , _C12 , _{C13 ,} C14, _C15 , _C16 , _C17 , _C18 , _C19 or C20 alkylene _{. 19} or C ₂₀ alkenylene or alkynylene.
4. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ₄ is selected from the group consisting of H, R _4a -OR _4b , R _4a -SR _4b , R _4a -S(O)-R _4b , R _4a -S(O) ₂ -R _4b , R _4a -C(O)-R _4b , R _4a -C(S)-R _4b , R _4a -NR _a -R _4b , R _4a -OC(O)-R _4b , R _4a -C(O)-OR _4b , R _4a -NR _a -C(O)-R _4b , R _4a -C(O)-NR _a -R _4b , R _4a -SC(O)-R _4b or R _4a -OC(S)-R _4b , R _4a is selected from C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , _C10 , _C11 , _C12 , _C13 , _C14 , _C15 , _C16 , _C17 , _C18 , _C19 , _C20 , _C21 , _C22 , _C23 or _C24 alkyl, alkenyl or alkynyl, and _R4b is selected from a chemical bond, _C1 , _C2 , _C3 , C4, _C5 or _C6 alkylene, or _C2 , _C3 , _C4 , _C5 or _C6 alkenylene or _alkynylene .
5. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ₅ is selected from the group consisting of: H, R _5a -OR _5b , R _5a -SR _5b , R _5a -S(O)-R _5b , R _5a -S(O) ₂ -R _5b , R _5a -C(O)-R _5b , R _5a -C(S)-R _5b , R _5a -NR _a -R _5b , R _5a -OC(O)-R _5b , R _5a -C(O)-OR _5b , R _5a -NR _a -C(O)-R _5b , R _5a -C(O)-NR _a -R _5b , _R5a -SC(O) _-R5b or _R5a -OC(S) _-R5b , _R5a is selected from _C4 , _C5 , _C6 , _C7 , C8, _C9 , _C10 , C11, _C12 , _C13 , _C14 , _C15 , _C16 , _{C17 , C18, C19, C20, C21, C22, C23 or C24 alkyl , alkenyl or alkynyl , and R5b is} selected from a chemical bond, _C1 , _C2 , _C3 , _C4 , _C5 or _C6 alkylene, or _C2 , _C3 , _C4 , _C5 or _C6 alkenylene or alkynylene.
6. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the aryl group is phenyl, and/or the heteroaryl group is selected from pyridyl, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyrazinyl, pyrimidinyl, pyridazinyl or triazinyl, and/or the heterocyclic group is selected from (C ₃ -C ₁₀ ) monocyclic cycloalkyl containing one or two heteroatoms selected from oxygen, nitrogen or sulfur.
7. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, wherein

   The alkyl, alkenyl, alkynyl, alkylene, alkenylene or alkynylene group is linear or branched, and the cycloalkyl, cycloalkylene, cycloalkenyl group is monocyclic; preferably, _R4 and _R5 are not H.
8. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from:
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
9. A composition comprising a compound according to any one of claims 1 to 8 and a therapeutic or preventive agent.
10. The composition according to claim 9, further comprising one or more structured lipids.
11. The composition of claim 10, wherein the one or more structured lipids is DSPC.
12. The composition of claim 10 or 11, wherein the molar ratio of the compound to the structural lipid is in the range of about 2:1 to about 8:1.
13. The composition according to any one of claims 9 to 12, further comprising a steroid.
14. The composition of claim 13, wherein the steroid is cholesterol.
15. The composition of claim 13 or 14, wherein the molar ratio of the compound to the steroid is in the range of about 5:1 to about 1:1.
16. The composition according to any one of claims 9 to 15, wherein the composition further comprises one or more polymer-conjugated lipids.
17. The composition according to claim 16, wherein the polymer-conjugated lipid is DMG-PEG2000 or DMPE-PEG2000.
18. The composition of claim 16 or 17, wherein the molar ratio of the compound to the polymer-conjugated lipid is in the range of about 100:1 to about 20:1.
19. The composition according to any one of claims 9 to 18, wherein the therapeutic or prophylactic agent comprises at least one mRNA encoding an antigen or a fragment or epitope thereof.
20. The composition of claim 19, wherein the mRNA is a monocistronic mRNA or a polycistronic mRNA.
21. The composition according to any one of claims 19 to 20, wherein the antigen is a pathogenic antigen.
22. The composition according to any one of claims 19 to 20, wherein the antigen is a tumor-associated antigen.
23. The composition according to any one of claims 19 to 22, wherein the mRNA comprises one or more functional nucleotide analogs.
24. The composition according to claim 23, wherein the functional nucleotide analog is one or more selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.
25. A lipid nanoparticle comprising a compound according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 24.
26. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8, a composition according to any one of claims 9 to 24 or a lipid nanoparticle according to claim 25 and a pharmaceutically acceptable excipient or diluent.