Lipid compound, lipid nanoparticle composition, and use thereof

Abstract

Provided herein are a lipid compound, a lipid nanoparticle composition, and use thereof. First provided herein is a compound represented by formula I or a pharmaceutically acceptable form thereof. The compound represented by formula I is 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 for delivering a therapeutic agent (such as nucleic acid molecules) for therapeutic or prophylactic purposes. Further provided herein is a lipid nanoparticle composition comprising the lipid compound represented by formula I.

Description

Compositions of lipid compounds and lipid nanoparticles and their applications Technical Field

[0001] This disclosure generally relates to lipid compounds that can be used to form lipid nanoparticles for the delivery of therapeutic or preventative agents (e.g., nucleic acids) and related applications. The lipid compounds described herein can be formulated into lipid nanoparticles with other lipids (e.g., phospholipids, steroids, and polymer-bound lipids). This disclosure also relates to the preparation of such lipid compounds and lipid nanoparticles, and their use in the treatment, prevention, or control of certain diseases or conditions. Background Technology

[0002] Gene therapy has the potential to revolutionize precision medicine. Nucleic acid-based gene therapies can be used to treat or prevent many diseases, such as cancer, bacterial infections, viral infections, and genetic disorders. Because nucleic acids are easily degraded in the body, they often need to be protected for efficient delivery to target cells, for example, using viral vectors. Developing safe and efficient delivery platforms is a prerequisite for the clinical success of gene therapy.

[0003] Lipid nanoparticles (LNPs) have been developed for the delivery of non-viral genes. In 2018, the U.S. Food and Drug Administration (FDA) approved LNP-delivered patisirans. It was used to treat hereditary transthyretin amyloidosis. Since then, scientific research using LNP technology to deliver nucleic acid drugs has grown exponentially. In 2020, the FDA approved vaccines against COVID-19 from Moderna and BioNTech & Pfizer, both of which used LNP technology to deliver mRNA encoding the SARS-CoV-2 spike protein to prevent viral infection.

[0004] Liposomes (LNPs) typically comprise ionizable lipids, neutral lipids, steroids, and polymer-bound lipids. Among the components of an LNP, the choice of ionized lipids significantly impacts its performance. Current LNP delivery platforms face several challenges, including low delivery efficiency, low cell permeability, and high degradation sensitivity to certain nucleic acid molecules (e.g., RNA). Therefore, there is a need to develop novel lipid compounds to improve nucleic acid delivery for therapeutic and / or preventative applications. Summary of the Invention

[0005] This disclosure provides a novel compound and composition, as well as a method relating to said compound and composition.

[0006] In one aspect, this disclosure provides a compound of formula I.

[0007] Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0008] G is -OH or -N(A) 1 A 2 A 1 A 2 Each independently is C 1-6 alkyl;

[0009] L 1 C 1-6 Alkylene;

[0010] L 2 C 1-12 Alkylene;

[0011] L 3 For H or C 1-30 alkyl;

[0012] L 4 L 6 Each independently is C 6-30 Alkyl, C 6-30 alkenyl, C 6-30 alkynyl or C 6-30 Alkoxy, wherein the alkyl, alkenyl, alkynyl or alkoxy group has 0 to 3 H atoms, optionally further converted to halogen, hydroxyl, or C atoms. 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted;

[0013] L 5 C 1-12 Alkylene;

[0014] R 1 R 2 Each independently selected The acetylenic group, heteroatom O, N, S, or none; h, i, k are selected from integers from 1 to 10; j is selected from 0 or 1, and the heteroatom is selected from N, O, or S.

[0015] In another aspect, this disclosure provides a compound listed in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof. In yet another aspect, this disclosure provides a composition comprising a compound of this disclosure, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, and one or more lipids selected from neutral lipids, steroids, and polymer-bound lipids.

[0016] In another aspect, this disclosure provides lipid nanoparticles comprising the compounds or compositions of this disclosure.

[0017] In another aspect, this disclosure provides a pharmaceutical composition comprising the compounds, compositions or lipid nanoparticles of this disclosure, and pharmaceutically acceptable excipients or diluents.

[0018] In another aspect, this disclosure also provides the use of the compounds, compositions, lipid nanoparticles or pharmaceutical compositions of this disclosure in the preparation of formulations for in vivo editing of target genes in subjects of need.

[0019] In another aspect, this disclosure also provides a method for delivering therapeutic and / or preventative agents to cells in a subject, the method comprising administering to the subject a composition of this disclosure, a lipid nanoparticle of this disclosure, or a pharmaceutical composition of this disclosure.

[0020] In another aspect, this disclosure also provides a method for organ-specific delivery of therapeutic and / or preventative agents to a subject, the method comprising administering to the subject a composition of this disclosure, a lipid nanoparticle of this disclosure, or a pharmaceutical composition of this disclosure.

[0021] In another aspect, this disclosure also provides a method for generating a target polypeptide in cells within a subject, the method comprising administering to the subject a composition of this disclosure, a lipid nanoparticle of this disclosure, or a pharmaceutical composition of this disclosure.

[0022] In another aspect, this disclosure also provides a method for in vivo editing of a target gene in a subject in need, wherein the method comprises administering lipid nanoparticles comprising at least one lipid and one or more polynucleotides, the one or more polynucleotides comprising or encoding one or more guide molecules, wherein the one or more polynucleotides further encode a Cas protein, wherein the one or more guide molecules and the Cas protein are configured to form a CRISPR-Cas complex capable of targeting and editing the target gene, wherein the at least one lipid is a compound of this disclosure.

[0023] Other features of this disclosure will become apparent to those skilled in the art upon consideration of the detailed description of the following specific embodiments. Attached Figure Description

[0024] Figure 1 shows a schematic diagram of a method for preparing LNP and delivering it to mouse liver cells via tail vein injection to target the PCSK9 gene. Detailed Implementation

[0025] 5.1 General Techniques

[0026] The techniques and procedures described or cited herein include those that are generally well understood by those skilled in the art and / or commonly employed using conventional methods, such as the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003).

[0027] 5.2 Terminology

[0028] Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0029] In this article, the range of values ​​referred to as “value A to value B” is the range that includes the endpoint values ​​A and B.

[0030] In this document, "substantially" or "truly" is used to mean that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0031] In this article, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0032] In this document, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0033] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. The term "pharmaceutically acceptable salt" refers to a salt of the compounds described herein that is substantially non-toxic to organisms. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by the reaction of the compounds described herein with pharmaceutically acceptable inorganic / organic acids or inorganic / organic bases; such salts are also known as acid addition salts or base addition salts. Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide. Common organic bases include (but are not limited to) diethylamine, triethylamine, and ethylaminobutanol.

[0034] Some specific compounds described in this article contain both basic and acidic functional groups, and can therefore be converted into either a base or an acid addition salt.

[0035] The pharmaceutically acceptable salts described herein can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or a mixture of both.

[0036] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key and straight dashed key

[0037] As used herein, the term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that substitution is optional, and unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis.

[0038] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0039] When one of the variables is selected as a bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0040] When one of the variables is selected as missing, it means that it does not exist. For example, in C-R3, when R3 is selected as missing, it means that the structure is actually C.

[0041] When the listed substituents do not specify which atom they are attached to the substituted group, such substituents can be bonded to any of their atoms. For example, a phenyl group can be attached to the substituted group via any carbon atom on the benzene ring.

[0042] Unless otherwise specified, the term "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group, which may be monosubstituted (e.g., -CH₂F) or polysubstituted (e.g., -CF₃), and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), etc.

[0043] Unless otherwise specified, “cycloalkyl” includes any stable cyclic or polycyclic hydrocarbon group in which all carbon atoms are saturated, and which may be monosubstituted or polysubstituted, and may be monovalent, divalent or polyvalent. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, norbornene, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane, etc.

[0044] Unless otherwise specified, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group having at least one carbon-carbon double bond, "C 2-6 "Alkenyl" refers to the above-mentioned hydrocarbon group having 2, 3, 4, 5, or 6 carbon atoms, and "C" represents a carbon group. 2-4 "Alkenyl" refers to the aforementioned hydrocarbon group having 2, 3, or 4 carbon atoms. It should be understood that when the alkenyl group contains more than one carbon-carbon double bond, the double bonds can be separable or conjugated with each other. Example: "C 2-6 "Alkenyl" includes, but is not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, etc.

[0045] Unless otherwise specified, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group having at least one carbon-carbon triple bond. 2-6 "Alkyne group" indicates the above-mentioned hydrocarbon group having 2, 3, 4, 5, or 6 carbon atoms, "C 2-4 "Alkyne group" indicates the above-mentioned hydrocarbon group having 2, 3, or 4 carbon atoms, exemplified by "C". 2-6 "Alynyl" includes, but is not limited to, ethynyl, propynyl, butynyl, penynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, etc.

[0046] Unless otherwise specified, the term "halogen" itself or as part of another substituent means a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.

[0047] Unless otherwise specified, the term "alkoxy" means an alkyl group attached to the remainder of the molecule by an oxygen atom, wherein the alkyl group has the meaning as described herein. Unless otherwise specified, C 1-5 Alkoxy groups include C1, C2, C3, C4, and C5 alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and S-pentoxy. The alkoxy group may optionally be substituted by one or more substituents described herein.

[0048] Unless otherwise specified, the term "amino" means -NH2, -NH (alkyl) or -N (alkyl) (alkyl).

[0049] Unless otherwise specified, the term "aromatic ring" refers to a monocyclic aromatic alkane that is polyunsaturated and may be monosubstituted or polysubstituted.

[0050] Unless otherwise specified, the term "4-6 membered heterocyclic alkyl" refers to a saturated monovalent monocyclic hydrocarbon ring containing 3, 4, or 5 carbon atoms and one or more atoms selected from O, NR, and 24-24-35 ... a heteroatomic groups, wherein R a Represents a hydrogen atom or C 1-6Alkyl; the “4-6 membered heterocyclic alkyl” can be connected to the rest of the molecule by any carbon atom, or, if present, a nitrogen atom.

[0051] Unless otherwise specified, the term "heteroaromatic ring" refers to an aromatic ring containing one to four heteroatoms selected from one or more of N, O, and S.

[0052] Unless otherwise specified, the term "dense aromatic ring" refers to a dense aromatic ring containing two or more aromatic rings.

[0053] Unless otherwise specified, the term "5-membered 6-membered heteroaryl ring" refers to a 5-membered 6-membered fused heteroaryl ring in which at least one of the two fused rings contains one or more heteroatoms (including but not limited to O, S or N), and the entire group is aromatic. Non-limiting examples include benzo5-membered heteroaryl, 6-membered heteroaryl ring and 5-membered heteroaryl ring.

[0054] Unless otherwise specified, the term "6-membered 6-membered heteroaryl ring" refers to a 6-membered 6-membered fused heteroaryl ring in which at least one of the two fused rings contains one or more heteroatoms (including but not limited to O, S or N), and the entire group is aromatic. Non-limiting examples include benzo6-membered heteroaryl and 6-membered heteroaryl rings.

[0055] Unless otherwise specified, the terms "pharmaceutically acceptable salt" or "its pharmaceutically acceptable salt" mean that the compound described herein retains the bioavailability and properties of a free acid or a free base, and that the free acid is obtained by reacting with a non-toxic inorganic or organic base, and the free base is obtained by reacting with a non-toxic inorganic or organic acid.

[0056] Unless otherwise specified, the term "pharmaceutical composition" means a mixture of one or more compounds described herein or pharmaceutically acceptable salts with other chemical components, wherein "other chemical components" means pharmaceutically acceptable carriers, excipients, and / or one or more other therapeutic agents. "Carrier" means a material that does not cause significant irritation to an organism and does not eliminate the biological activity and properties of the administered compound. "Excipient" means an inert substance added to a pharmaceutical composition to facilitate administration of the compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, and disintegrants.

[0057] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to having at least one chiral element (including a chiral center, chiral axis, chiral plane, etc.), thereby enabling the rotation of plane-polarized light. Because the compounds described herein contain asymmetric centers and other chemical structures that may lead to stereoisomerism, in some embodiments, the compounds described herein include stereoisomers of these compounds and mixtures thereof. In some embodiments, the compounds described herein and their salts include asymmetric carbon atoms, and thus can exist as a single stereoisomer, a racemic mixture, or a mixture of enantiomers and diastereomers. Typically, these compounds can be prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched with single stereoisomers (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). The single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or obtained by preparing a mixture of enantiomers followed by separation or resolution, for example, by converting it into a mixture of diastereomers followed by separation or recrystallization, chromatographic treatment, using chiral resolving reagents, or by direct separation of the enantiomers on a chiral chromatographic column. Starting compounds with specific stereochemistry are either commercially available or prepared according to the methods described herein and then resolved using methods well known in the art.

[0058] The term "tautomer" (or "tautomer form") refers to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (or proton transfer tautomers) include (but are not limited to) interconversions via proton transfer, such as keto-enol isomerization, imine-enamine isomerization, amide-imine alcohol isomerization, etc. Unless otherwise stated, the compounds described herein are intended to include all tautomer forms.

[0059] The term "solvent" refers to a substance formed by the combination of a compound described herein or its pharmaceutically acceptable salt with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanol compounds, acetone compounds, etc.

[0060] The term "chelate" refers to a complex with a cyclic structure, obtained through the chelation of two or more ligands with the same metal ion to form a chelate ring.

[0061] The term "non-covalent complex" refers to a complex formed through the interaction of a compound with another molecule, where no covalent bond is formed between the two molecules. Complexation can occur, for example, through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0062] The term "prodrug" refers to a derivative compound that, when administered to a patient, can directly or indirectly provide the compounds described herein. Particularly preferred derivative compounds or prodrugs are those that, when administered to a patient, can improve the bioavailability of the compounds described herein (e.g., facilitate absorption into the bloodstream) or promote the delivery of the parent compound to its site of action (e.g., the lymphatic system). Unless otherwise indicated, the compounds described herein are intended to include all prodrug forms, and various prodrug forms are well known in the art.

[0063] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0064] The terms “contain” and “include” are used in their open, non-restrictive sense.

[0065] The term "alkyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, without unsaturation, and linked to other segments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, and tert-butyl. For example, "C 1-30 "Alkyl" refers to a saturated monovalent straight-chain or branched hydrocarbon group containing 1 to 30 carbon atoms.

[0066] The term "alkylene" refers to a divalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, without saturation, and connected to other segments via two single bonds, including (but not limited to) methylene, 1,1-ethylene, and 1,2-ethylene. For example, "C 1-30 "Alkylene" refers to saturated divalent straight-chain or branched alkyl groups containing 1 to 30 carbon atoms.

[0067] The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon and hydrogen atoms. Cycloalkyl groups can include fused, bridged, or spirocyclic systems. For example, the term "C" as used herein... 3-6"Cycloalkyl" refers to a cycloalkyl group having 3 to 6 carbon atoms. For example, cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or bicyclo[2.2.1]heptyl, etc.

[0068] The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including (but not limited to) cycloalkylene, cycloalkylene, cycloalkylene, cycloalkylene, and cycloalkylene, etc. For example, "C 3-30 "Cycloalkylene" refers to a divalent group obtained by removing hydrogen atoms from a cycloalkyl group containing 3 to 30 carbon atoms.

[0069] The term "branched alkyl" refers to an alkane radical that is attached to a parent molecule and forms at least two branches. For example...

[0070] The term "alkenyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via a single bond. This includes (but is not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups. For example, "C..." 2-30 "Alkenyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0071] The term "alkenyl" refers to a divalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via two single bonds, including (but not limited to) vinylenes. For example, "C 2-30 "Alkenyl" refers to a divalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0072] The term "alkynyl" refers to a monovalent, straight-chain or branched alkane group composed only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other segments by a single bond. This includes (but is not limited to) ethynyl, propynyl, butynyl, and pentyynyl groups. For example, "C..." 2-30 "Alkyne" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

[0073] The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) non-aromatic hydrocarbon group composed only of carbon and hydrogen atoms. Cycloalkenyl groups can include fused, bridged, or spirocyclic systems. Examples include cyclopropenyl and cyclobutenyl.

[0074] The term "cycloene-alkenyl" refers to a divalent group obtained by removing a hydrogen atom from a cycloene-alkenyl group as defined above, including (but not limited to) cycloene-propenyl and cycloene-butenyl groups. For example, "C 3-30 "Biopylene alkenyl" refers to a divalent group obtained by removing hydrogen atoms from a cycloalkenyl group containing 3 to 30 carbon atoms.

[0075] The term "branched alkenyl" refers to an alkene radical that is linked to a parent molecule and forms at least two branches. For example...

[0076] The term "heterocyclic group" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic group whose ring atoms consist of a carbon atom and at least one heteroatom selected from N, O, and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2, or S(=O)(=NR). x ), R x Independently selected from H or C 1-4 Alkyl groups. If the valence requirements are met, a heterocyclic group can be attached to the rest of the molecule via any one ring atom. For example, the term "3-8 membered heterocyclic group" as used herein refers to a heterocyclic group having 3 to 8 ring atoms. Examples of heterocyclic groups include ethylene oxide, aziridine propane, aziridine butane, oxadiazine, tetrahydrofuranyl, dioxadiazopentenyl, pyrrolyl, pyrrolidone, imidazoalkyl, pyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, or trithiaalkyl.

[0077] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C" as used herein... 6-10 "Aryl" refers to an aryl group having 6 to 10 carbon atoms. For example, aryl groups can be phenyl, naphthyl, anthracene, phenanthryl, acenaphthene, azulene, fluorenyl, indene, pyrene, etc.

[0078] The term "heteroaryl" refers to an aromatic group that is a monocyclic or fused polycyclic ring with a conjugated π-electron system, the ring atom consisting of a carbon atom and at least one heteroatom selected from N, O, and S. If the valence requirement is met, the heteroaryl group can be attached to the rest of the molecule through any one of the ring atoms. For example, the term "5-10-membered heteroaryl" as used herein refers to a heteroaryl group having 5 to 10 ring atoms. Examples of heteroaryl groups include thiophene, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl and their benzo[a] derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purine, etc.

[0079] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0080] The term "hydroxyl group" refers to -OH.

[0081] The term "cyano" refers to -CN.

[0082] The term "amino" refers to -NH2.

[0083] The term "nitro" refers to -NO2.

[0084] The term "oxo group" refers to (=O).

[0085] The term "subject" includes both humans and non-human animals. Non-human animals include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cattle, chickens, dogs, mice, rats, goats, rabbits, and pigs. Preferably, the subject is a human. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein.

[0086] The term "nuclease" refers to an enzyme that catalyzes the breaking of phosphodiester bonds between nucleotides in a nucleic acid molecule. In some embodiments, the nuclease is selected from a wide range of nucleases, zinc-finger nucleases (ZFNs), TAL-effector DNA-binding domain-nuclease fusion proteins (TALENs), and RNA-guided nucleases, or variants thereof in which the nuclease activity has been reduced or inhibited.

[0087] In some implementations, the RNA-guided nuclease is a naturally occurring CRISPR-Cas protein or its active variant or fragment. CRISPR-Cas systems are classified into class I or class II systems. Class II systems contain a single-effect nuclease and include types II, V, and VI. Each class is further subdivided into types (I, II, III, IV, V, VI), some of which are further subdivided into subtypes (e.g., II-A, II-B, II-C, VA, VB).

[0088] The terms "type II CRISPR-Cas protein," "type II CRISPR-Cas effector protein," or "Cas9" refer to a CRISPR-Cas effector protein that requires trans-activation RNA (tracrRNA) and contains two nuclease domains (RuvC and HNH) (each responsible for cleaving a single strand of a double-stranded DNA molecule). In other embodiments, the CRISPR-Cas protein is a naturally occurring type V CRISPR-Cas protein or its active variant or fragment.

[0089] As used herein, the terms "Type V CRISPR-Cas protein," "Type V CRISPR-Cas effector protein," or "Cas12" refer to a CRISPR-Cas effector protein that cleaves dsDNA and contains a single RuvC nuclease domain or a split RuvC nuclease domain and lacks an HNH domain. In other embodiments, the CRISPR-Cas protein is a naturally occurring Type VI CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the terms "Type VI CRISPR-Cas protein," "Type VI CRISPR-Cas effector protein," or "Cas13" refer to a CRISPR-Cas effector protein that does not require tracrRNA and contains two HEPN domains that cleave RNA.

[0090] The term "gRNA" refers to a nucleotide sequence that is sufficiently complementary to the target nucleotide sequence to hybridize with it and guide the sequence-specific binding of the associated nuclease to the target nucleotide sequence. For CRISPR-Cas enzymes, the corresponding guide RNA is one or more RNA molecules (usually one or two) that can bind to the Cas enzyme and guide the Cas enzyme to bind to a specific target nucleotide sequence, and in those cases, the Cas enzyme also cleaves the target nucleotide sequence if it has nicking or nuclease activity.

[0091] In this document, references to “one embodiment,” “implementation,” “a specific embodiment,” “a related embodiment,” “an embodiment,” “another embodiment,” or “a further embodiment,” or combinations thereof, refer to a particular element, feature, structure, or characteristic described in connection with that embodiment, which is included in at least one embodiment of this disclosure. Therefore, the foregoing phrases appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this description is intended as a basis for the use of exclusive terms such as “solely,” “only,” etc., or the use of “negative” in relation to the recitation of claim elements.

[0092] 5.3 Lipid compounds

[0093] Unless otherwise specified, the descriptions provided herein apply to all chemical formulas (e.g., chemical formula (I), including their sub-formulas) to the extent applicable.

[0094] In one embodiment, compounds of formula I are provided herein:

[0095] Or its pharmaceutically acceptable salt, prodrug, or stereoisomer, wherein...

[0096] G is -OH or -N(A) 1 A 2 A 1 A 2 Each independently is C 1-6 alkyl;

[0097] L 1 C 1-6 Alkylene;

[0098] L 2 C 1-12 Alkylene;

[0099] L 3 For H or C 1-30 alkyl;

[0100] L 4 L 6 Each independently is C 6-30 Alkyl, C 6-30 alkenyl, C 6-30 alkynyl or C 6-30 Alkoxy, wherein the alkyl, alkenyl, alkynyl or alkoxy group has 0 to 3 H atoms, optionally further converted to halogen, hydroxyl, or C atoms. 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted;

[0101] L 5 C 1-12 Alkylene;

[0102] R 1 R 2 Each independently selected The acetylenic group, heteroatom O, N, S, or none; h, i, k are selected from integers from 1 to 10; j is selected from 0 or 1, and the heteroatom is selected from N, O, or S.

[0103] In one embodiment, this document provides compounds represented by formula Ia, Ib, Ic, Id, Ie, If, or Ig, or pharmaceutically acceptable salts, prodrugs, or stereoisomers thereof.

[0104] Among them, G and L1 L 2 L 3 L 4 L 5 L 6 R 1 and R 2 Same as the definition in Equation I above;

[0105] Each A 3 Each A 4 and A 5 Each independently is hydrogen, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl or C 1-12 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted;

[0106] Each A 6 Each A 7 and A 8 Each independently is hydrogen, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl or C 1-12 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted;

[0107] a and b are each independent integers selected from 0 to 10, for example, selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0108] The condition is Each independently is C 6-30 Alkyl, C 6-30 alkenyl, C 6-30 alkynyl or C 6-30 Alkoxy, wherein the alkyl, alkenyl, alkynyl or alkoxy group has 0 to 3 H atoms, optionally further converted to halogen, hydroxyl, or C atoms. 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted.

[0109] In one implementation, G is -OH or -N(A) 1 A 2 A 1 A 2 Each independently is C 1-4 Alkyl groups; and / or

[0110] L 1 C 2-4 Alkylene; and / or

[0111] L 2 C 1-10 Alkylene; and / or

[0112] L 3 For H or C 1-20 Alkyl groups; and / or

[0113] L 4 L 6 Each independently is C 8-20 Alkyl groups, including straight-chain C 8-20 Alkyl or branched C 8-20 Alkyl groups; and / or

[0114] L 5 C 3-10 Alkylene; and / or

[0115] R 1 R 2 Each independently selected

[0116] In one implementation, G is -OH or -N(A) 1 A 2 A 1 A 2 Each is independently methyl, ethyl, propyl; and / or

[0117] L 1 It is ethylene or propylene; and / or

[0118] L 2 C 1-8 Alkylene; and / or

[0119] L 3 For H or C 1-16 Alkyl groups; and / or

[0120] L 4 L6 Each independently is C 8-18 Alkyl groups, including straight-chain C 8-18 Alkyl or branched C 8-18 Alkyl groups; and / or

[0121] L 5 C 6-10 Alkylene; and / or

[0122] R 1 R 2 Each independently selected

[0123] In one implementation, L 2 It is methylene, ethylene, propylene, butylene, amylene, hexylene, heptaylene, or octylene; and / or

[0124] L 3 H or methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, or hexadecyl; and / or

[0125] L 4 L 6 Each of the following is independently octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, or octadecyl, including straight-chain or branched forms; and / or

[0126] L 5 Hexylene, heptylene, octylene, nonylene, decylene; and / or

[0127] R 1 Selected from and / or

[0128] R 2 Selected from

[0129] In one implementation, L 4 It is octyl, nonyl, decyl, undecyl, or dodecyl, including straight-chain or branched forms; and / or

[0130] L 6 It is nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, or octadecyl, including straight-chain or branched forms.

[0131] In one implementation scheme, each A 3 Each A 4 and A 5 Each independently is hydrogen, C 1-8 Alkyl, C2-8 alkenyl, C 2-8 alkynyl or C 1-8 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-8 alkoxy- or halogen-substituted C 1-8 alkyl or hydroxy substituted C 1-8 Alkyl groups are substituted;

[0132] Each A 6 Each A 7 and A 8 Each independently is hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl or C 1-8 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-8 alkoxy- or halogen-substituted C 1-8 alkyl or hydroxy substituted C 1-8 Alkyl groups are substituted;

[0133] a and b are each an independent integer selected from 1 to 8, for example, selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0134] In one embodiment, the compound is a compound in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0135] Table 1

[0136] It should be understood that any embodiment of the compounds provided herein as shown above, and any particular substituents and / or variables of the compounds provided herein as shown above, may be independently combined with other embodiments and / or substituents and / or variables of the compounds to form embodiments not specifically described above. Furthermore, in the case of listing any particular group or variable of substituents and / or variables, it should be understood that each individual substituent and / or variable may be deleted from a particular embodiment and / or claim, and the remaining list of substituents and / or variables will be considered within the scope of the embodiments provided herein. It should be understood that, in this specification, combinations of various substituents and / or variables are permitted only if such contributions result in stable compounds.

[0137] In some embodiments, the lipid compounds described herein are used to form nanoparticles (e.g., as described in Section 5.4 (Nanoparticle Compositions)).

[0138] In some embodiments, the amount of lipid compounds present in the nanoparticles is about 20 to about 65 mol% of the total lipids in the nanoparticles. In some embodiments, the mol% of lipid compounds in the nanoparticles is about 20 to about 60, about 20 to about 55, about 25 to about 55, about 30 to about 65, about 25 to about 45, about 20, about 25, about 30, about 35, about 40, about 45, about 55, about 60, or about 65.

[0139] 5.4 nanometer lipid particle composition

[0140] In one aspect, this document provides compositions comprising the lipid compounds described herein. In some embodiments, the nanoparticle compositions comprise compounds of formula I and its sub-formulas (e.g., formula Ia, Ib, Ic, Id, Ie, If, or Ig) as described herein (section 5.3), or pharmaceutically acceptable salts, prodrugs, or stereoisomers thereof. For the sake of brevity, when a compound is referred to herein, its pharmaceutically acceptable salts, prodrugs, or stereoisomers are included.

[0141] In some embodiments, such compositions are nanoparticle compositions. In particular embodiments, the nanoparticle compositions herein comprise compounds according to chemical formula I and its sub-formulas (e.g., formula Ia, Ib, Ic, Id, Ie, If, or Ig) (e.g., Section 5.3 (Lipid Compounds)). In some embodiments, the nanoparticle compositions herein also comprise additional lipids. In some embodiments, the nanoparticle compositions herein also comprise one or more charged or ionizable lipids (e.g., Section 5.4.1 Cationic / Ionizable Lipids). In some embodiments, the nanoparticle compositions herein also comprise neutral lipids (e.g., Section 5.4.2 (Neutral Lipids)). In some embodiments, the nanoparticle compositions also comprise polymer-bound lipids (e.g., Section 5.4.3 (Polymer-Bound Lipids)). In some embodiments, the nanoparticles also comprise structural lipids (e.g., Section 5.4.4 (Structural Lipids)). In some embodiments, the nanoparticles also comprise therapeutic payloads (e.g., Section 5.4.5 (Therapeutic Payloads)).

[0142] Nanoparticle compositions that can be used in conjunction with this disclosure include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or cross-linked with each other. The lipid bilayers may contain one or more ligands, proteins, or channels.

[0143] The characteristics of a nanoparticle composition can depend on its components. For example, a nanoparticle composition containing cholesterol as a structural lipid may have different characteristics than a nanoparticle composition containing different structural lipids. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition containing a higher molar fraction of phospholipids may have different characteristics than a nanoparticle composition containing a lower molar fraction of phospholipids. The characteristics can also vary depending on the preparation method and conditions of the nanoparticle composition.

[0144] According to this disclosure, the nanoparticle compositions described herein may comprise at least one lipid component and one or more non-lipid components, such as therapeutic and / or preventative agents. The nanoparticle compositions may be designed for one or more specific applications or targets. The therapeutic and / or preventative agents 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 components. For example, the therapeutic and / or preventative agents may be selected for a specific indication, disease, ailment, or condition and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In some embodiments, the nanoparticle composition may comprise mRNA encoding a polypeptide of interest, which is capable of intracellular translation to produce the polypeptide of interest. Such compositions may be designed for specific delivery to specific organs. In some embodiments, the composition may be designed for specific delivery to mammalian bone marrow cells.

[0145] In some embodiments, this document provides a nanoparticle composition comprising:

[0146] i) Ionizable lipids of formula (I) (and its sub-formulas such as formula Ia, formula Ib, formula Ic, formula Id, formula Ie, formula If, or formula Ig), or

[0147] Its pharmaceutically acceptable salts, stereoisomers, tautomers, prodrugs, solvates, or isotope analogs;

[0148] ii) Neutral lipids;

[0149] iii) Structural lipids; and

[0150] iv) Polymer-bound lipids.

[0151] In some embodiments, this document provides a nanoparticle composition comprising:

[0152] i) Approximately 20 to approximately 65 mol% of ionizable lipids;

[0153] ii) Approximately 5 to approximately 25 mol% of neutral lipids;

[0154] iii) approximately 25 to approximately 55 mol% of structural lipids; and

[0155] iv) Approximately 0.3 to approximately 15 mol% of polymer-bound lipids.

[0156] In some embodiments, this document provides a nanoparticle composition comprising:

[0157] i) about 20 to about 55 mol% of an ionizable lipid of formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0158] ii) Approximately 0 to approximately 13 mol% of anionic lipids;

[0159] iii) Approximately 5 to approximately 25 mol% of neutral lipids;

[0160] iv) approximately 25 to approximately 51.5 mol% of structural lipids; and

[0161] v) Approximately 0.5 to approximately 15 mol% of polymer-bound lipids.

[0162] In some embodiments, this document provides a nanoparticle composition comprising:

[0163] i) about 20 mol% of an ionizable lipid of chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0164] ii) Approximately 20 mol% of anionic lipids;

[0165] iii) Approximately 5 mol% of neutral lipids;

[0166] iv) Approximately 50 mol% of structural lipids; and

[0167] v) Approximately 5 mol% of polymer-bound lipids.

[0168] In some embodiments, this document provides a nanoparticle composition comprising:

[0169] i) about 30 mol% of an ionizable lipid of chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0170] ii) Approximately 5 mol% of anionic lipids;

[0171] iii) Approximately 25 mol% of neutral lipids;

[0172] iv) Approximately 30 mol% of structural lipids; and

[0173] v) Approximately 10 mol% of polymer-bound lipids.

[0174] In some embodiments, this document provides a nanoparticle composition comprising:

[0175] i) about 20 mol% of an ionizable lipid of chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0176] ii) Approximately 5 mol% of anionic lipids;

[0177] iii) Approximately 5 mol% of neutral lipids;

[0178] iv) Approximately 55 mol% of structural lipids; and

[0179] v) Approximately 15 mol% of polymer-bound lipids.

[0180] In some embodiments, this document provides a lipid nanoparticle composition comprising:

[0181] i) about 65 mol% of an ionizable lipid of chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0182] ii) Approximately 9.7 mol% of neutral lipids;

[0183] iii) Approximately 25 mol% of structural lipids; and

[0184] iv) Approximately 0.3 mol% of polymer-bound lipids.

[0185] In some embodiments, this document provides a nanoparticle composition comprising:

[0186] i) about 50 mol% of an ionizable lipid of chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0187] ii) Approximately 10 mol% of neutral lipids;

[0188] iii) Approximately 38.5 mol% of structural lipids; and

[0189] iv) Approximately 1.5 mol% of polymer-bound lipids.

[0190] As used in this article, “molar%” refers to the molar percentage of a component relative to the total number of moles of all lipid components in the LNP.

[0191] In some embodiments, the lipid nanoparticles further comprise one or more therapeutic payloads (e.g., Section 5.4.5 (Therapeutic Payload)). In some embodiments, the therapeutic payload comprises one or more nucleic acids. In some embodiments, the therapeutic payload comprises one or more RNAs. In some embodiments, the therapeutic payload comprises mRNA. In some embodiments, the therapeutic payload comprises sgRNA. In some embodiments, the therapeutic payload comprises both mRNA and sgRNA.

[0192] In some embodiments, this document provides a nanoparticle comprising:

[0193] i) an ionizable lipid of chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If or Ig), or a pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate or isotope analog thereof.

[0194] ii) A neutral lipid;

[0195] iii) A structural lipid;

[0196] v) A polymer-bound lipid; and

[0197] iv) One or more therapeutic payloads.

[0198] In some embodiments, the molar ratio of therapeutic payload to lipids in the LNP (i.e., the N / P ratio, where N represents the number of moles of ionizable lipids and P represents the number of moles of phosphate present as part of the nucleic acid backbone) ranges from about 3:1 to about 40:1. In some embodiments, the N / P ratio is about 5:1 to about 30:1, about 5:1 to about 20:1, about 5:1 to about 10:1, about 30:1, about 20:1, about 15:1, about 10:1, about 5:1, or about 3:1.

[0199] 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 preventative agents (e.g., RNA) to specific cells, tissues, organs, or systems or combinations thereof in a mammal. The physicochemical properties of the nanoparticle composition can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the pore size of different organs. The therapeutic and / or preventative agents contained in the nanoparticle composition can also be selected based on the desired delivery target. For example, therapeutic and / or preventative agents can be selected for a specific indication, condition, disease, or disorder and / or for delivery to specific cells, tissues, organs, or systems or combinations thereof (e.g., local or specific delivery). In some embodiments, the nanoparticle composition may contain mRNA encoding a polypeptide of interest, which is capable of being translated within the cell to produce the polypeptide of interest.

[0200] The physical properties of nanoparticle compositions can depend on their components. For example, a nanoparticle composition containing cholesterol as a structural lipid can have different properties compared to a nanoparticle composition containing different structural lipids. Similarly, the properties of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition containing a higher molar fraction of phospholipids can have different properties compared to a nanoparticle composition containing a lower molar fraction of phospholipids. Properties can also vary depending on the preparation method and conditions of the nanoparticle composition.

[0201] 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 preventative agents (such as RNA) to specific cells, tissues, organs, or systems or combinations thereof in a mammal. The physicochemical properties of the nanoparticle composition can be modified to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the pore size of different organs. The therapeutic and / or preventative agents contained in the nanoparticle composition can also be selected based on the desired delivery target. For example, therapeutic and / or preventative agents can be selected for a specific indication, condition, disease, or disorder and / or for delivery to specific cells, tissues, organs, or systems or combinations thereof (e.g., local or specific delivery). In some embodiments, the nanoparticle composition may contain mRNA encoding a polypeptide of interest, which is capable of being translated within the cell to produce the polypeptide of interest.

[0202] In some embodiments, the specific cells are or include spleen cells (e.g., spleen B cells, spleen T cells, spleen monocytes), hepatocytes (e.g., liver cells), bone marrow cells (e.g., bone marrow monocytes), immune cells, kidney cells, muscle cells, heart cells, lung cells, or central nervous system cells. In some embodiments, the cells are bone marrow cells.

[0203] In some embodiments, the organ is the lung, spleen, bone marrow, or lymph node. In some embodiments, the organ is bone marrow.

[0204] The physical properties of nanoparticle compositions can depend on their components. For example, a nanoparticle composition containing cholesterol as a structural lipid can have different properties compared to a nanoparticle composition containing different structural lipids. Similarly, the properties of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition containing a higher molar fraction of phospholipids can have different properties compared to a nanoparticle composition containing a lower molar fraction of phospholipids. Properties can also vary depending on the preparation method and conditions of the nanoparticle composition.

[0205] Nanoparticle compositions can be characterized using a variety of methods. For example, the morphology and particle size distribution of the nanoparticle composition can be examined using microscopy (e.g., transmission electron microscopy or scanning electron microscopy). The zeta potential can be measured using dynamic light scattering or potentiometry (e.g., potentiometric titration). Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) can also be used to measure various properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0206] Particle size: The average particle size of the nm particle composition can be from about 30 nm to about 500 nm. In some embodiments, the average particle size of the nm particle composition can be from about 30 nm to about 450 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 50 nm to about 130 nm, from about 50 nm to about 120 nm, from about 50 nm to about 110 nm, from about 60 nm to about 110 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 70 nm to about 120 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 30 nm, from about 40 nm, from about 50 nm, from about 60 nm, from about 70 nm, from about 80 nm, from about 90 nm, from about 100 nm, from about 110 nm, from about 120 nm, from about 150 nm, from about 250 nm, from about 350 nm, or from about 500 nm. In some implementations, the particle size of lipid particles is measured by dynamic light scattering (DLS).

[0207] Polydispersity Index (PDI): The nanoparticle composition can be relatively homogeneous. PDI can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A smaller PDI (e.g., less than 0.3) generally indicates a narrow particle size distribution. The PDI of the nanoparticle composition can be from about 0 to about 0.25, from about 0.05 to about 0.20, for example 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 PDI of the nanoparticle composition can be from about 0.10 to about 0.20.

[0208] Encapsulation efficiency (EE): Encapsulation efficiency of the therapeutic and / or preventative agents describes the amount of therapeutic and / or preventative agents encapsulated or otherwise associated with the nanoparticle composition after preparation, relative to the initial amount provided. Encapsulation efficiency is expected to be high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amounts of therapeutic and / or preventative agents in a solution containing the nanoparticle composition before and after the nanoparticle composition is disrupted using one or more organic solvents or detergents. Encapsulation efficiency of RNA can be measured using a fluorescence assay (e.g., the RiboGreen RNA assay). For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or preventative agents 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 75%. In some embodiments, the encapsulation efficiency can be at least 90%.

[0209] Apparent pKa: Apparent pKa is the pH value at which the number of ionized (e.g., protonated) and deionized groups in a system are equal. The surface charge and ionic interactions of the assembled components in nanoparticles can be estimated based on the apparent pKa. The apparent pKa of a nanoparticle is the result of the average ratio of all ionized to deionized groups in the nanoparticle. Without theoretical constraints, nanoparticles with optimal pKa carry low surface charges at physiological pH, thereby minimizing nonspecific binding and toxicity in vivo. Optimal apparent pKa of nanoparticles can facilitate endosome escape and release of therapeutic agents (e.g., RNA) into the cytoplasm. The apparent pKa of nanoparticles can be measured by acid-base titration and 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescence methods, as described by Patel P. et al. in “The Importance of Apparent pKa in the Development of Nanoparticles Encapsulating siRNA and mRNA” (Trends Pharmacol Sci. 2021, 42(6):448-460), which is incorporated herein by reference. In some embodiments, the apparent pKa of the nanoparticles described herein is about 4 to about 8, about 5 to about 7, or about 5.5 to about 6.5.

[0210] Zeta potential: The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions with relatively low surface charges (positive or negative) are generally desirable because substances with higher charges may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -30 mV to about +30 mV, about -20 mV to about +20 mV, or about -10 mV to about +10 mV. In some embodiments, the zeta potential is measured by DLS.

[0211] As described herein, in some embodiments, the nanoparticle compositions provided herein, in addition to comprising a lipid compound according to chemical formula (I) (and its sub-formulas such as formula Ia, Ib, Ic, Id, Ie, If, or Ig) as described herein, also comprise one or more additional lipid or non-lipid components as described below.

[0212] In some embodiments, the additional lipids are present in the nanoparticle composition in an amount of about 0 to about 20 mol% of the total lipids in the nanoparticles. In some embodiments, the mol% of lipids is about 0.001 to about 20, about 0.01 to about 20, about 0.1 to about 20, about 1 to about 20, about 0 to about 13, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 10, about 15, or about 20. In some embodiments, the molar ratio of the additional lipids to the ionizable lipids of formula (I) (and its sub-formulas such as formula Ia, formula Ib, formula Ic, formula Id, formula Ie, formula If, or formula Ig) is about 0 to about 10:1, about 0.01 to about 10:1, about 10:1 to about 1:10, about 1:1, about 2:1, about 5:1, about 7.5:1, about 10:1, about 1:2, about 1:1, about 1:7.5, or about 1:10.

[0213] 5.4.1 Cationic / Ionizable Lipids

[0214] As described herein, in some embodiments, the nanoparticle compositions provided herein, in addition to comprising lipids according to formula I (and its sub-formulas, such as formula Ia, Ib, Ic, Id, Ie, If, or Ig) as described herein, also comprise one or more charged or ionizable lipids. Without being bound by theory, it is anticipated that certain charged or zwitterionic lipid components of the nanoparticle compositions will resemble lipid components in cell membranes, thereby improving cellular uptake of the nanoparticles. Exemplary charged or ionizable lipids that may form part of the nanoparticle compositions herein include 1,2-diolenoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA), DLin-MC2-MPZ, 2,2-diolenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and 1,2-dioleoyl-3-trimethylammonium-propane (DDMA). One or more of OTAP, 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecane-2-ol C12-200, 3β[N-N'N'-dimethylaminoethane)-carbamoyl]cholesterol DC-Chol, and N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylamine chloride DOTMA. Additional exemplary charged or ionizable lipids that may form part of the nanoparticle compositions of this disclosure include lipids (e.g., lipid 5) 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, all of which are incorporated herein by reference.

[0215] 5.4.2 Neutral lipids

[0216] In some embodiments, the lipid component of the nanoparticle composition may include one or more neutral lipids, such as neutral phospholipids. Without being theoretically limited, it is conceivable that phospholipids can assemble into one or more lipid bilayer structures.

[0217] Exemplary phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether). PC), 1-oleoyl-2-cholesterolylhemisuccino-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso) PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate choline, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate racemic-(1-glycerol) sodium salt (DOPG) and sphingomyelin. In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0218] Other exemplary neutral lipids include, for example, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). In one embodiment, the neutral lipids are selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0219] In one embodiment, the neutral lipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG). Additionally, extra phospholipids that may form part of the nanoparticle compositions herein include those described in WO2017 / 112865, the entire contents of which are incorporated herein by reference.

[0220] In some embodiments, the amount of neutral lipids present in the nanoparticles is about 5 to about 25 mol% of the total lipids in the nanoparticles. In some embodiments, the mol% of neutral lipids is about 5 to about 20, about 5 to about 15, about 7.5 to about 15, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, or about 25.

[0221] 5.4.3 Polymer-bound lipids

[0222] In some embodiments, the lipid component of the nanoparticle composition may include one or more polymer-bound lipids. A polymer-bound lipid is a molecule that simultaneously comprises a lipid moiety and a polymer moiety. An example of a polymer-bound lipid is a polyethylene glycol-modified lipid. A polyethylene glycol-modified lipid refers to a molecule that simultaneously comprises a lipid moiety and a polyethylene glycol moiety. Without being theoretically limited, it is conceivable that the polymer-bound lipid component in the nanoparticle composition can improve colloidal stability and / or reduce protein uptake by the nanoparticles.

[0223] In some embodiments, the polymer-bound lipid is a polyethylene glycol-modified lipid. Exemplary lipids that may be used include, but are not limited to, polyethylene glycol-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidic acid, polyethylene glycol-modified ceramide, polyethylene glycol-modified dialkylamine, polyethylene glycol-modified diacylglycerol, polyethylene glycol-modified dialkyloxypropylcarbamate, polyethylene glycol-modified sphingolipid, polyethylene glycol-modified diacrylamide, or combinations thereof. In some embodiments, the polyethylene glycol portion of the polyethylene glycol-modified lipid has a molecular weight of about 500 Da to about 10,000 Da, about 1,000 Da to about 5,000 Da, about 1,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, or about 5,000 Da. In some embodiments, the polyethylene glycol portion of the polyethylene glycol-modified lipid has a molecular weight of about 2,000 Da (i.e., PEG2000 lipid).

[0224] In some embodiments, the PEGylated lipids are 1,2-dimyristoyl-sn-glycerol methoxy-polyethylene glycol PEG-DMG, dimyristoylglycerol-polyethylene glycol PEG-c-DMG, polyethylene glycol-dimyristoylglycerol PEG-C14, PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEGylated phosphatidylethanolamine PEG-PE, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and Tween-20. Tween-80, 1,2-dipalmityl-sn-glycerol-methoxy polyethylene glycol PEG-DPG, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol PEG-c-DOMG, and N-acetylgalactosamine ((R)-2,3-bis(octadecanoyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate))GalNAc-PEG-DSG, or combinations thereof.

[0225] In some embodiments, the amount of polymer-bound lipids present in the nanoparticles is from about 0.1 to about 15 mol% of the total lipids in the nanoparticles. In some embodiments, the mol% of polymer-bound lipids is from about 0.3 to about 15, from about 0.5 to about 15, from about 0.3 to about 10, from about 0.5 to about 10, from about 0.5 to about 5, from about 0.1, from about 0.2, from about 0.3, from about 0.5, from about 1, from about 2, from about 3, from about 4, from about 5, from about 10, or from about 15.

[0226] In some embodiments, the amount of polymer-bound lipids present in the nanoparticles is from about 0.1 to about 15 mol% of the total lipids in the nanoparticles. In some embodiments, the mol% of polymer-bound lipids is from about 0.3 to about 15, from about 0.5 to about 15, from about 0.3 to about 10, from about 0.5 to about 10, from about 0.5 to about 5, from about 0.1, from about 0.2, from about 0.3, from about 0.5, from about 1, from about 2, from about 3, from about 4, from about 5, from about 10, or from about 15.

[0227] 5.4.4 Structural lipids

[0228] In some embodiments, the lipid component of the nanoparticle composition may include one or more structural lipids. In some embodiments, the structural lipids are steroids or steroid analogs. Without being theoretically limited, it is conceivable that structural lipids can stabilize the amphiphilic structure of the nanoparticles, such as, but not limited to, the lipid bilayer structure of the nanoparticles. Exemplary structural lipids that may be used include, but are not limited to, cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroids, lycopene, ursolic acid, α-tocopherol, coprosterol, or corticosteroids, or combinations thereof.

[0229] In some embodiments, the structural lipids are present in the nanoparticles in an amount of about 25 to about 55 mol% of the total lipids in the nanoparticles. In some embodiments, the mol% of the structural lipids is about 25 to about 51.5, about 25 to about 45, about 25 to about 40, about 30 to about 55, about 25, about 30, about 35, about 40, about 45, about 50, about 51.5, or about 55.

[0230] 5.4.5 Therapeutic payload

[0231] According to this disclosure, the nanoparticle compositions described herein may also contain one or more therapeutic and / or preventative agents. In this disclosure, these therapeutic and / or preventative agents are sometimes referred to as "therapeutic payloads" or "payloads." In some embodiments, nanoparticles can be used as delivery carriers to administer therapeutic payloads in vivo or in vitro.

[0232] In some embodiments, the nanoparticle composition comprises, as a therapeutic payload, a small molecule compound, an antitumor agent, an anti-infective agent, a local anesthetic, a β-adrenergic blocker, an antihypertensive agent, an antidepressant, an anticonvulsant, an antihistamine, an antibiotic / antibacterial agent, an antifungal agent, an antiparasitic agent, a hormone, a hormone antagonist, an immunomodulator, a neurotransmitter antagonist, an antiglaucoma agent, a vitamin, an anesthetic, and an imaging agent.

[0233] In some implementations, the therapeutic payload is a nucleic acid. In some implementations, the therapeutic payload is RNA, DNA, antisense nucleic acid, adaptor, nuclease, immunostimulatory nucleic acid or peptide nucleic acid (PNA), or a combination thereof.

[0234] The amount of therapeutic and / or preventative agents in the nanoparticle compositions described herein can depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the nature of the therapeutic and / or preventative agents. For example, the amount of useful RNA in the nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or preventative agents with other elements (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight / weight ratio of lipid components to therapeutic and / or preventative agents in the nanoparticle composition can be from about 2:1 to about 200:1, such as 2:1, 3:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 80:1, 90:1, 100:1, or 200:1.

[0235] In some embodiments, the nanoparticle compositions herein comprise one or more nucleic acid molecules (e.g., DNA or RNA molecules) as a therapeutic payload. Exemplary forms of nucleic acid molecules that may be included as a therapeutic payload in the nanoparticle compositions include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger RNA (mRNA), hybrids thereof, RNAi inducers, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. In some embodiments, the therapeutic payload comprises RNA. RNA molecules that may be included as a therapeutic payload in the nanoparticle compositions include, but are not limited to, short RNA, agomir, antagomir, antisense RNA, 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.

[0236] In some embodiments, the therapeutic payload is RNA. In some embodiments, the therapeutic payload comprises mRNA, rRNA, circular (cirRNA), siRNA, saRNA, tRNA, snRNA, antagomir, miRNA, microRNA inhibitor, microRNA activator, shRNA, or combinations thereof.

[0237] In some embodiments, the therapeutic payload is mRNA. In some embodiments, the therapeutic payload is rRNA. In some embodiments, the therapeutic payload is cirRNA. In some embodiments, the therapeutic payload is siRNA. In some embodiments, the therapeutic payload is saRNA. In some embodiments, the therapeutic payload is tRNA. In some embodiments, the therapeutic payload RNA is snRNA. In some embodiments, the therapeutic payload is antagomir. In some embodiments, the therapeutic payload is miRNA. In some embodiments, the therapeutic payload is a microRNA inhibitor. In some embodiments, the therapeutic payload is a microRNA activator. In some embodiments, the therapeutic payload is shRNA.

[0238] In some embodiments, the therapeutic payload comprises an RNA-guided nuclease system, or one or more polynucleotides encoding such a system or components thereof. In some embodiments, the RNA-guided nuclease system is a CRISPR-Cas system.

[0239] In some embodiments, the therapeutic payload comprises a polynucleotide (e.g., mRNA or circular RNA) encoding a Cas protein. In some embodiments, the therapeutic payload comprises a guide molecule or a polynucleotide (e.g., mRNA or circular RNA) encoding a guide molecule. In some embodiments, the therapeutic payload comprises a polynucleotide encoding a Cas protein and also comprises a nucleic acid-based guide molecule, wherein the Cas protein and the guide molecule are configured to form an activated CRISPR-Cas complex capable of targeting and editing target nucleic acids.

[0240] In some implementations, the guide molecule is selected from sgRNA, crRNA, gRNA, and tracrRNA (tcRNA).

[0241] In some embodiments, the Cas protein is a naturally occurring protein. In some embodiments, the Cas protein is a non-naturally occurring protein. In some embodiments, the Cas protein is selected from Cas9 protein, Cas12 protein, Cas13 protein, IscB protein, TnpB protein, IsrB protein, or functional derivatives thereof.

[0242] In some embodiments, the therapeutic payload comprises a Cas9 effector protein and sgRNA. In some embodiments, the therapeutic payload comprises one or more polynucleotides encoding the Cas9 effector protein and sgRNA. In some embodiments, the therapeutic payload comprises sgRNA and a polynucleotide encoding the Cas9 effector protein. In any of the embodiments described in this paragraph, the encoding polynucleotide is mRNA. In any of the embodiments described in this paragraph, the encoding polynucleotide is circular RNA.

[0243] In some embodiments, the therapeutic payload comprises a Cas12 effector protein and crRNA. In some embodiments, the therapeutic payload comprises one or more polynucleotides encoding the Cas12 effector protein and crRNA. In some embodiments, the therapeutic payload comprises crRNA and a polynucleotide encoding the Cas12 effector protein. In any embodiment described in this paragraph, the encoding polynucleotide is mRNA. In any embodiment described in this paragraph, the encoding polynucleotide is circular RNA.

[0244] In some embodiments, the therapeutic payload comprises a Cas13 effector protein and crRNA. In some embodiments, the therapeutic payload comprises one or more polynucleotides encoding the Cas13 effector protein and crRNA. In some embodiments, the therapeutic payload comprises crRNA and a polynucleotide encoding the Cas13 effector protein. In any of the embodiments described in this paragraph, the encoding polynucleotide is mRNA.

[0245] In some embodiments, the therapeutic payload comprises an IscB effector protein and ωRNA. In some embodiments, the therapeutic payload comprises one or more polynucleotides encoding an IscB effector protein and ωRNA. In some embodiments, the therapeutic payload comprises ωRNA and a polynucleotide encoding an IscB effector protein. In any of the embodiments described in this paragraph, the encoding polynucleotide is mRNA.

[0246] In some embodiments, the Cas protein is also associated with one or more functional domains selected from deaminase (e.g., adenosine deaminase or cytidine deaminase) catalytic domains, DNA methylation catalytic domains, DNA demethylation catalytic domains, histone residue modification domains, nuclease catalytic domains, fluorescent proteins, transcriptional modification factors (e.g., transcription activation catalytic domains, transcription repression catalytic domains), nuclear localization signals (NLS), nuclear output signals (NES), light-gated factors, chemically induced factors, or chromatin visualization factors; preferably, the functional domains are selected from adenosine deaminase catalytic domains or cytidine deaminase catalytic domains.

[0247] In some implementations, the adenine base editor is ABE8e (Addgene, plasmid #138489). ABE8e is described in Richter et al., “Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity,” Nature Biotechnology, 2020, 38(7):883-891.

[0248] In some embodiments, the therapeutic payload includes and / or encodes a CRISPR-Cas system capable of targeting and editing disease-related target genes. In some embodiments, the target genes are associated with metabolic diseases, genetic diseases, cancer, cardiovascular diseases, or infectious diseases. In some embodiments, the therapeutic payload includes and / or encodes a CRISPR-Cas system capable of targeting and editing target genes associated with familial hypercholesterolemia (FH), transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1), hereditary angioedema (HAE), or hepatitis B.

[0249] In some embodiments, the nanoparticle compositions described herein comprise a therapeutic payload comprising both mRNA and sgRNA. In some embodiments, the mRNA encodes a Cas protein. In some embodiments, Cas is ABE8e or a functional derivative thereof. In some embodiments, the sgRNA targets the PCSK9 gene in a subject.

[0250] Chemical modifications can be made to the phosphate backbone, sugars, and / or bases of payload polynucleotide molecules. Backbone modifications such as phosphate thioates alter the charge on the phosphate backbone and contribute to oligonucleotide delivery and nuclease resistance (e.g., see Eckstein, “Phosphorothioates, essential components of therapeutic oligonucleotides”, Nucl. Acid Ther., 2014, Vol. 24, pp. 374-387); sugar modifications, such as 2'-O-methyl (2'-OMe), 2'-F, and locked nucleic acids (LNA), enhance base pairing and nuclease resistance (e.g., see Allen et al., “Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA”, J. Med. Chem., 2005, Vol. 48.4, pp. 901-904). Chemically modified bases, such as 2-thiouridine or N6-methyladenosine, can allow for stronger or weaker base pairings (see, for example, Bramsen et al., “Development of therapeutic-grade small interfering RNAs by chemical engineering”, Front. Genet., August 20, 2012; 3:154). Furthermore, RNA readily conjugates at its 5' and 3' ends to a variety of functional motifs, including fluorescent dyes, polyethylene glycol, or proteins.

[0251] In some embodiments, the sgRNA includes one or more phosphate thioester modifications. In some embodiments, the sgRNA includes one or more locked nucleic acids for the purpose of enhancing base pairing and / or increasing nuclease resistance.

[0252] A summary of these chemical modifications can be found, for example, in Kelley et al., “Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing,” J. Biotechnol. 233:74-83, 2016; WO 2016205764; and U.S. Patent No. 8,795,965B2; the full text of each reference is incorporated herein by reference.

[0253] Chemically synthesized payload polynucleotide molecules can be modified in various ways. For example, modifying oligonucleotides with 2'-OMe to improve nuclease resistance can alter the binding energy of Watson-Crick base pairing. Furthermore, 2'-OMe modification can affect the interaction of oligonucleotides with transfection reagents, proteins, or any other molecules in the cell. The effects of these modifications can be determined through empirical testing.

[0254] In some embodiments, the payload polynucleotide molecule includes one or more phosphate thioester modifications. In some embodiments, the payload polynucleotide molecule includes one or more locked nucleic acids to enhance base pairing and / or increase resistance to nucleases.

[0255] The sequence and length of the CRISPR guide molecules described herein can be optimized. In some embodiments, the optimized length of the guide molecule can be determined by identifying the processing form of the guide molecule or by empirical length studies of the guide molecule.

[0256] The guide molecule may also include one or more aptamer sequences. An aptamer is an oligonucleotide or peptide molecule capable of binding to a specific target molecule. The aptamer may be specific to a gene effector, gene activator, or gene repressor. In some embodiments, the aptamer may be specific to a protein that is in turn specific to and recruits / binds to a specific gene effector, gene activator, or gene repressor. The effector, activator, or repressor may be present in the form of a fusion protein. In some embodiments, the guide molecule has two or more aptamer sequences specific to the same adaptor protein. In some embodiments, two or more aptamer sequences are specific to different adaptor proteins. Adaptor proteins may include, for example, MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. Therefore, in some embodiments, the aptamer is selected from binding proteins that specifically bind to any of the adaptor proteins described herein. In some embodiments, the aptamer sequence is an MS2 loop. Detailed descriptions of aptamers can be found, for example, Nowak et al., “Guide RNA engineering for versatile Cas9 functionality,” Nucl. Acid. Res., 16 Nov 2016; 44(20):9555-9564; and WO 2016205764, which are incorporated herein by reference in their entirety.

[0257] This disclosure considers all possible variants of nucleic acids, such as cDNA, which can be prepared through combinatorial selection based on possible codon selections. These combinations are made according to the standard triplet genetic code applied to encoding polynucleotides of naturally occurring variants, and all such variants should be considered as specifically disclosed. For example, this document discloses payload polynucleotide sequences encoding functional derivatives of Cas proteins, which have been codon-optimized for expression in bacteria (e.g., *E. coli*) and human cells. For example, codon-optimized sequences for human cells can be generated by replacing codons that occur more frequently in human cells with codons that occur less frequently in human cells. The frequency of codon occurrence can be determined by calculations known in the art. Examples of calculations of these codon frequencies in various host cells (e.g., *E. coli*, yeast, insects, *C. elegans*, *Drosophila melanogaster*, humans, mice, rats, pigs, *Pichia pastoris*, *Arabidopsis thaliana*, maize, and tobacco) have been provided by, for example, *E. coli*, yeast, insects, *Caenorhabditis elegans*, *Drosophila melanogaster*, humans, mice, rats, pigs, *Pichia pastoris*, *Arabidopsis thaliana*, maize, and tobacco) The source of the codon frequency table tool is published or provided.

[0258] 5.5 Drug Combinations

[0259] According to this disclosure, the nanoparticle compositions herein can be formulated, in whole or in part, into pharmaceutical compositions. A pharmaceutical composition may include one or more nanoparticle compositions. For example, a pharmaceutical composition may include one or more nanoparticle compositions containing one or more different therapeutic and / or preventative agents. A pharmaceutical composition may further include one or more pharmaceutically acceptable excipients or adjuvants, as described herein. General guidance on the formulation and preparation of pharmaceutical compositions and agents can be found, for example, in Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and adjuvants may be used in any pharmaceutical composition unless any conventional excipient or adjuvant is incompatible with one or more components of the nanoparticle composition. An excipient or adjuvant is incompatible with a component of the nanoparticle composition if its combination with any component would result in any adverse biological effect or other harmful effect.

[0260] In some embodiments, one or more excipients or adjuvants may constitute more than 50% of the total mass or total volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical formulation. 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 conforms to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0261] According to this disclosure, the relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any other ingredients in the pharmaceutical compositions herein will vary depending on the identity, size, and / or condition of the subject being treated, and also on the route of administration of the composition. For example, the pharmaceutical composition may comprise 0.1% to 100% (by weight) of one or more nanoparticle compositions.

[0262] Nanoparticle compositions may optionally include one or more coatings. For example, nanoparticle compositions may be formulated into coated capsules, films, or tablets. Capsules, films, or tablets containing the compositions described herein may have any useful dimensions, tensile strength, hardness, or density.

[0263] In some embodiments, the nanoparticle compositions and / or pharmaceutical compositions of this disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures of 4°C or lower, such as from about -150°C to about 0°C or at temperatures between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, or -90°C). For example, pharmaceutical compositions comprising any chemical formula (I) and its sub-formulas (e.g., formula Ia, formula Ib, formula Ic, formula Id, formula Ie, formula If, or formula Ig) are stored at, for example, about -20°C, Solutions refrigerated at -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C for storage and / or transport. In some embodiments, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable at temperatures, for example, 4°C or lower (e.g., between about 4°C and -80°C) for 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 1 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 16 months, at least 20 months, or at least 24 months. In some embodiments, the pH of the pharmaceutical compositions disclosed herein is between about 6 and 8.

[0264] In some embodiments, the pharmaceutical composition is formulated for administration by means of: oral, intrafacial, intra-articular, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravesical, intravitreal, liposome, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, topical, buccal, percutaneous, intravaginal, in cream, in lipid composition, via catheter, via irrigation, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intra-arterial injection. In some embodiments, the excipient is a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical composition is formulated in unit doses.

[0265] Pharmaceutical compositions comprising one or more nanoparticles can be prepared by any method known or subsequently developed in the field of pharmacology. Generally, such preparation methods involve combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dispensing, shaping, and / or packaging the product into the desired single- or multi-dose units.

[0266] According to this disclosure, the pharmaceutical compositions herein may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, 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 active ingredient is generally equal to the dose of active ingredient to be given to a subject and / or a convenient fraction of that dose, such as half or one-third of the dose.

[0267] Pharmaceutical compositions can be formulated into various forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions can be formulated into liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), topical and / or transdermal dosage forms (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and patches), suspensions, powders, and other forms.

[0268] 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, liquid dosage forms may 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-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol fatty acid esters, and mixtures thereof. Besides inert diluents, oral compositions may include other therapeutic and / or preventative agents, other agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or aromatizers. In some embodiments for parenteral administration, the composition is mixed with solubilizers such as Cremophor™, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0269] For example, sterile aqueous or oily suspensions for injection can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic, parenteral-acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable carriers and solvents that can be used include water, Ringer's solution, United States Pharmacopeia, and isotonic sodium chloride solution. Sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectable formulations.

[0270] Injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0271] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified. As used herein, "appropriate amount" refers to a relatively wide range of adjustable solvent or reagent amounts with minimal impact on the synthesis results, and is not specifically limited. In the following examples, all solvents and reagents used are analytically pure or chemically pure; all solvents were redistilled before use; and all anhydrous solvents were treated according to standard or literature methods.

[0272] Example 1: Preparation of Compound 1

[0273] 8-(5-hexadecyl-1-hydroxy-7-oxoylide-3-aza-6-oxahexadecane-3-yl)octanoic acid heptadecan-9-yl ester

[0274] Step 1: Synthesis of compounds 1-3

[0275] Compound 1-1 (2.0 g, 8.96 mmol, 1.0 eq), EDCI (2.58 g, 13.44 mmol, 1.5 eq), DMAP (0.55 g, 4.48 mmol, 0.5 eq), and diisopropylethylamine (2.32 g, 17.92 mmol, 2.0 eq) were added sequentially to 30 mL of dichloromethane. After stirring the reaction solution at 25°C for 0.5 hours, compound 1-2 (2.30 g, 8.96 mmol, 1.0 eq) was added, and the reaction was continued at 25°C for 15.5 hours. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography to give compound 1-3 (2.80 g, 67.7% yield).

[0276] Step 2: Synthesis of compounds 1-5

[0277] Compounds 1-3 (2.80 g, 6.07 mmol, 1.0 eq), 1-4 (1.06 g, 6.07 mmol, 1.0 eq), potassium carbonate (1.26 g, 9.11 mmol, 1.5 eq), and potassium iodide (0.50 g, 3.04 mmol, 0.5 eq) were added sequentially to 20 mL of acetonitrile. The reaction solution was stirred at 80 °C for 16 hours. The solution was concentrated under reduced pressure, diluted with 50 mL of water, and extracted three times with 20 mL of water each time. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography to give compound 1-5 (1.85 g, 55.0% yield).

[0278] Step 3: Synthesis of compounds 1-7

[0279] Compounds 1-5 (1.85 g, 3.33 mmol, 1.0 eq) and 1-6 (4.47 g, 16.65 mmol, 5.0 eq) were added to 20 mL of isopropanol. The reaction solution was stirred at 80 °C for 16 h, concentrated under reduced pressure, and column chromatography was performed to give compound 1-7 (1.70 g, yield 62.0%).

[0280] Step 4: Synthesis of compounds 1-9

[0281] Compounds 1-8 (0.53 g, 3.09 mmol, 1.5 eq), EDCI (0.59 g, 3.09 mmol, 1.5 eq), DMAP (0.13 g, 1.03 mmol, 0.5 eq), and diisopropylethylamine (0.53 g, 4.12 mmol, 2.0 eq) were added sequentially to 20 mL of dichloromethane. After stirring the reaction solution at 25 °C for 0.5 hours, compound 1-7 (1.70 g, 2.06 mmol, 1.0 eq) was added. The reaction solution was stirred for another 15.5 hours, then concentrated under reduced pressure, and column chromatography was used to obtain compound 1-9 (1.32 g, 65.4% yield).

[0282] Step 5: Synthesis of Compound 1

[0283] Compounds 1-9 (1.32 g, 1.35 mmol, 1.0 eq) and TBAF (5.4 mL, 5.40 mmol, 4.0 eq, 1.0 M tetrahydrofuran solution) were added sequentially to 20 mL of tetrahydrofuran. The reaction solution was stirred at 25 °C for 2 hours, concentrated under reduced pressure, diluted with 20 mL of water, and extracted three times with 20 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was used to give compound 1 (0.96 g, 82.3% yield). MS: m / z [M+H] + =864.8.

[0284] Example 2 Preparation of compounds 2-3

[0285] Compounds 2-3 were prepared according to the preparation method of compound 1 in Example 1, by successively replacing intermediates 1-2 with undecane-5-ol and nonane.

[0286] Example 3 Preparation of Compound 4

[0287] Compound 4 was prepared according to the preparation method of Compound 1 in Example 1, except that intermediates 1-4 were replaced with compound 3-{[dimethyl(2-methylprop-2-yl)methoxy}prop-1-amine.

[0288] Example 4 Preparation of Compound 5

[0289] 8-(5-hexadecyl-1-hydroxy-7-oxoylide-3-aza-6,8-dioxahexadec-3-yl)octanoic acid heptadecan-9-yl ester

[0290] Step 1: Compounds 1-7 (0.5 g, 0.61 mmol, 1.0 eq), 5-1 (0.22 g, 0.73 mmol, 1.2 eq), DMAP (37.0 mg, 0.30 mmol, 0.5 eq), and pyridine (0.14 g, 1.83 mmol, 3.0 eq) were added sequentially to 5 mL of acetonitrile. The reaction solution was stirred at 25 °C for 16 hours, concentrated under reduced pressure, diluted with 20 mL of water, and extracted three times with 10 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give compound 5-2 (0.38 g, yield 63.9%).

[0291] Step 2: Compound 5-2 (0.38 g, 0.39 mmol, 1.0 eq) and TABF (1.56 mL, 1.56 mmol, 4.0 eq) were added to 5 mL of tetrahydrofuran. The reaction solution was stirred at 25 °C for 3 hours, concentrated under reduced pressure, diluted with 10 mL of water, and extracted three times with 10 mL of ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography to give compound 5 (0.16 g, yield 47.7%). MS: m / z [M+H] + =866.8.

[0292] Example 5 Preparation of Compound 6

[0293] Compound 6 was prepared according to the preparation method of Compound 1 in Example 1, by replacing intermediates 1-6 and 1-8 with 2-octyloxetine and tridecanoic acid, respectively.

[0294] Example 6 Preparation of Compound 7

[0295] Compound 7 was prepared according to the preparation method of Compound 1 in Example, by replacing intermediates 1-6 and 1-8 with 2-butyloxacyclopropane and tridecanoic acid, respectively.

[0296] Example 7 Preparation of Compound 8

[0297] Tridecanoic acid-19-(2-hydroxyethyl)-9-octyl-11-oxoylide-19-aza-10-oxaenoic acid-21-yl ester

[0298] Step 1: Synthesis of Compound 8-3

[0299] Compounds 8-1 (1.00 g, 4.48 mmol, 1.0 eq), 8-2 (1.15 g, 4.48 mmol, 1.0 eq), EDCI (1.03 g, 5.38 mmol, 1.2 eq), DMAP (0.27 g, 2.24 mmol, 0.5 eq), and diisopropylethylamine (0.87 g, 6.72 mmol, 1.5 eq) were added sequentially to 10 mL of dichloromethane. The reaction solution was stirred at 25 °C for 16 hours, extracted three times with 10 mL of dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography to give compound 8-3 (1.65 g, 79.8% yield).

[0300] Step 2: Synthesis of Compound 8-5

[0301] Compounds 8-3 (1.65 g, 3.57 mmol, 1.0 eq), 8-4 (0.22 g, 3.57 mmol, 1.0 eq), potassium carbonate (0.74 g, 5.35 mmol, 1.5 eq), and potassium iodide (0.30 g, 1.78 mmol, 0.5 eq) were added to 20 mL of acetonitrile. The reaction solution was stirred at 80 °C for 16 hours. The solution was concentrated under reduced pressure, diluted with 20 mL of water, and extracted three times with 15 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography to give compound 8-5 (0.83 g, 52.6% yield).

[0302] Step 3: Synthesis of Compound 8

[0303] Compounds 8-5 (0.83 g, 1.88 mmol, 1.0 eq), 8-6 (0.91 g, 2.82 mmol, 1.5 eq), potassium carbonate (0.52 g, 3.76 mmol, 2.0 eq), and potassium iodide (0.16 g, 0.94 mmol, 0.5 eq) were added sequentially to 10 mL of acetonitrile. The reaction solution was stirred at 80 °C for 16 h, concentrated under reduced pressure, diluted with 20 mL of water, and extracted three times with 10 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography to give compound 8 (250.0 mg, yield 19.51%). MS: m / z [M+H] + =682.6.

[0304] Example 8 Preparation of Compound 9

[0305] 2-Butyloctanoic acid-7-butyl-19-(2-hydroxyethyl)-8-oxoylide-19-aza-9-oxaoctacosane-28-yl ester

[0306] Step 1: Synthesis of intermediate 9-3

[0307] Compounds 9-1 (1.00 g, 4.48 mmol, 1.0 eq), 9-2 (1.35 g, 6.72 mmol, 1.5 eq), EDCI (1.29 g, 6.72 mmol, 1.5 eq), DMAP (0.27 g, 2.24 mmol, 0.5 eq), and diisopropylethylamine (1.16 g, 8.96 mmol, 2.0 eq) were added sequentially to 10 mL of dichloromethane. The reaction solution was stirred at 25 °C for 16 h, extracted three times with 10 mL of dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and column chromatography was performed to give compound 9-3 (1.16 g, yield 63.8%).

[0308] Step 2: Synthesis of Compound 9

[0309] Compounds 9-3 (1.13 g, 2.78 mmol, 2.4 eq), 9-4 (71.0 mg, 1.16 mmol, 1.0 eq), potassium carbonate (0.48 g, 3.48 mmol, 3.0 eq), and potassium iodide (19.0 mg, 1.16 mmol, 1.0 eq) were added sequentially to 10 mL of acetonitrile. The reaction mixture was stirred at 80 °C for 16 h, concentrated under reduced pressure, diluted with 20 mL of water, and extracted three times with 10 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography to give compound 9 (0.32 g, yield 38.8%). MS: m / z [M+H] + =710.7.

[0310] Example 9 Preparation of Compound 10

[0311] Compound 10 was prepared according to the preparation method of compound 9 in Example 8, except that intermediate 9-4 was replaced with 3-aminoprop-1-ol.

[0312] Example 10 Preparation of compounds 11-15

[0313] Compounds 11-15 were synthesized according to the synthesis method of compounds 1-5 in Examples 1-4, by replacing the intermediate 2-aminoethyl-1-ol with 2-(diethylamino)ethyl-1-amine, or by replacing the intermediate 3-aminoprop-1-ol with 3-(diethylamino)prop-1-amine.

[0314] Example 11: Preparation, characterization, and in vivo editing evaluation of lipid nanoparticles

[0315] 1. Animal Experiment Design

[0316] mRNA and sgRNA delivery experiments of the PCSK9-targeting base editor ABE8e

[0317] Cholesterol in the blood is mainly synthesized by the liver, which is also the main organ for breaking down excess cholesterol. On the surface of the liver, there is a low-density lipoprotein (LDL) receptor (LDLR). The LDL receptor binds to cholesterol circulating back to the liver, breaking it down into bile acids, which are then excreted through the intestines. PCSK9 is a liver-synthesized protease that binds to the LDL receptor, promoting its entry into hepatocytes and leading to its degradation by lysosomes, thus reducing the number of LDL receptors. Therefore, inhibiting the activity of PCSK9 increases the number of LDLR receptors, thereby enhancing the uptake and breakdown of cholesterol. Basic and clinical studies have shown that the PCSK9 gene is an effective target for treating hyperlipidemia and atherosclerosis. Figure 1 illustrates the changes in the number of LDL receptors and the resulting alterations in cholesterol metabolism before and after editing a specific site in the PCSK9 gene.

[0318] The strategy for PCSK9 gene editing delivery in mouse hepatocytes is shown in Figure 1. The main process is as follows: The prepared lipid nanoparticles are used to target and deliver the mRNA and sgRNA encoding ABE8e to mouse hepatocytes via intravenous injection. Under the action of ABE8e and sgRNA, mutations are introduced into the PCSK9 gene, and the A to G mutation is achieved at a specific site. The editing efficiency is calculated by sequencing.

[0319] The specific experimental design is as follows:

[0320] 1.1 Selection of suitable mutation sites and editing design

[0321] The single-base editor ABE8e achieves precise A-to-G base substitution without requiring a donor template or causing DSB (Dissociative Identity Deficit Hyperbacterial Syndrome). Based on this, the first exon of the PCSK9 gene was selected as the screening mutation site. The mRNA and sgRNA encoding the single-base editing tool ABE8e were co-delivered to the animal via lipid nanoparticles. The mRNA encoding ABE8e was translated into protein in the cytoplasm, formed a complex with sgRNA, and entered the nucleus. Guided by sgRNA, the base editor ABE8e targeted the splicing donor site in the first exon of the PCSK9 gene, deaminated adenine (A) on the template strand of the first exon to convert it to inosine (I). I is read and replicated at the DNA level as G, ultimately achieving the A-to-G substitution, thereby disrupting the splicing donor site and causing premature termination of the PCSK9 gene reading frame.

[0322] 1.2 Preparation of mRNA and sgRNA of base editor ABE8e

[0323] The sequences of the first exon and the first intron of the mouse PCSK9 gene (NCBI Gene ID: 100102) were selected as the target region to determine the target sequence PCSK9-sgRNA for single-base editing of the PCSK9 gene.

[0324] By analyzing the sequence across the first exon and intron of the PCSK9 gene, a target region sgRNA was designed: PCSK9-sgRNA (synthesized by Nanjing Genscript Biotech Co., Ltd.). The PCSK9-sgRNA sequence is as follows:

[0325] PCSK9-sgRNA: 5'-CCCATACCTTGGAGCAACGG-3' (SEQ ID NO: 1);

[0326] sgRNAs were designed and oligonucleotides were synthesized based on the target sequence. The sgRNA sequences used are shown in SEQ ID NO:1. A CACC sequence was added to the 5' end of the upstream sequence of each sgRNA, and an AAAC sequence was added to the 5' end of the downstream sequence. After synthesis, the upstream and downstream sequences were annealed using a preset program (95℃, 5 min; 95℃-85℃ at -2℃ / s; 85℃-25℃ at -0.1℃ / s; held at 4℃). The annealed products were then ligated into the lenti U6-sgRNA / EF1a-mCherry vector (Addgene, Plasmid, #114199) linearized with BbsI (NEB, R3539S).

[0327] The system used in the construction of the sgRNA plasmid is as follows:

[0328] The linearization system of lenti U6-sgRNA / EF1a-mCherry vector is as follows: 3 μg vector; 6 μL buffer (NEB: R0539L); 2 μL BbsI; ddH2O to make up to 60 μL, digested overnight at 37℃.

[0329] The ligation system for the annealed sgRNA product and the linearized vector was as follows: 1 μL of T4 ligase buffer (NEB: M0202L), 20 ng of linearized vector, 5 μL of annealed oligo fragment (10 μM), 0.5 μL of T4 ligase (NEB: M0202L), and ddH2O to a final volume of 10 μL. The mixture was incubated overnight at 16°C.

[0330] The ligation vector was transformed into *E. coli* DH5α competent cells (Weidi Bio, DL1001). The specific procedure is as follows: DH5α competent cells were removed from -80℃ and immediately placed on ice. After 5 minutes, once the bacterial block had thawed, the ligation product was added, and the mixture was gently stirred by tapping the bottom of the centrifuge tube. The cells were then incubated on ice for 25 minutes. A heat shock was performed at 42℃ for 45 seconds, followed by immediate return to ice and incubation for 2 minutes. 700 μl of antibiotic-free sterile LB medium was added to the centrifuge tube, mixed, and incubated at 37℃, 200 rpm for 60 minutes. The cells were harvested by centrifugation at 5000 rpm for one minute. Approximately 100 μl of the supernatant was collected, gently resuspended by pipetting, and spread onto LB medium containing Amp antibiotics. The plates were inverted and incubated overnight at 37℃. Single colonies were picked, confirmed by sequencing, and positive clones were shaken and plasmids (TIANGEN: DP120-01) were extracted and their concentration determined. The plasmids were then stored at -20℃ for later use.

[0331] The base editor ABE8e used in this experiment is the highly efficient base editor ABE8e evolved by David R. Liu's team (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol. 2020 Jul; 38(7):883-891. doi:10.1038 / s41587-020-0453-z. Epub 2020 Mar 16. Erratumin: Nat Biotechnol. 2020 May 20; PMID:32433547; PMCID: PMC7357821). The ABE8e plasmid (Addgene, Plasmid #138489) was purchased from Addgene and the ABE8e mRNA was expressed and purified in the laboratory for later use.

[0332] 2. Lipid nanoparticles were prepared by ionizable lipids (the compounds provided in this paper or MC3) / DSPC / cholesterol / PEG-lipids in a molar ratio of 50:10:38.5:1.5.

[0333] 2.1 Dilinylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) and compounds 1-15 provided herein were dissolved in anhydrous ethanol in the above molar ratio with DSPC, cholesterol, and PEG-DMG, respectively.

[0334] 2.2 Ethanol solutions of different lipid carriers were mixed with mRNA buffer at a 1:3 (volume / volume) ratio (total lipid to mRNA mass ratio of 40:1, sgRNA:ABE8e mRNA (w / w) ratio of 1:1), and the mixture was flowed at 12 ml / min through a microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) to obtain nucleic acid lipid nanoparticles 1-15. The obtained nucleic acid lipid nanoparticles were immediately diluted 40-fold in 1×DPBS buffer. The diluted nucleic acid lipid nanoparticle solution was concentrated to the target volume using an ultracentrifuge tube. The diluted solution was then used for DLS particle size measurement and encapsulation efficiency detection.

[0335] 2.3 The particle size and polydispersity index of lipid nanoparticles were 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 (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2.

[0336] Table 2 Characterization of some lipid nanoparticles

[0337] 3. In vivo editing experiment evaluation

[0338] 3.1 Lipid nanoparticles containing compounds described herein (see Table 2) encapsulating the mRNA and sgRNA of the base editor ABE8e were administered systemically via tail vein injection to 6-7 week old female C57BL / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.). Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) encapsulating the mRNA and sgRNA of the base editor ABE8e were administered similarly to age- and sex-matched mice as positive controls. Additionally, PBS buffer was administered via tail vein injection to age- and sex-matched mice as negative controls.

[0339] 3.2 Editing Efficiency Test

[0340] One week after administration to mice, the editing efficiency was tested. The mice were sacrificed, liver tissue was collected, the genome was extracted after lysis, and efficiency was analyzed by deep sequencing.

[0341] The deep sequencing steps are as follows:

[0342] (1) Design primers based on the location of the target gene, see Table 3 for details.

[0343] Table 3 Primer design targeting the PCSK9 gene

[0344] (2) Editing efficiency test.

[0345] The PCR program was as follows: 94℃, 2 min; 98℃, 10 s; 60℃, 30 s; 68℃, 20 s; 34 cycles; 68℃, 5 min. After PCR, gel electrophoresis was used to verify the amplification of the product by selecting a suitable and single band. The obtained PCR product was then sent to Nanjing GenScript Biotech Co., Ltd. for sequencing.

[0346] (3) The deep sequencing results were analyzed using the Crispresso software to perform specific site analysis and calculate the editing efficiency. The calculation results are shown in Table 4.

[0347] Table 4. Evaluation of In-Vivo Editing Efficiency

[0348] As shown in Table 4, the LNP containing the lipids described herein can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles disclosed herein have a better particle size distribution, higher encapsulation efficiency, and significantly better delivery effect than the comparative lipid nanoparticles, which can meet the needs of in vivo delivery.

[0349] The above embodiments are intended to be illustrative only, and those skilled in the art will recognize, or be able to determine, other equivalents of a particular compound, material, and procedure through experiments not exceeding conventional methods. All such equivalents are considered to be within the scope of this invention and are covered by the appended claims.

Claims

1. A compound represented by Formula I: Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein: G is -OH or -N(A) 1 A 2 A 1 A 2 Each independently is C 1-6 alkyl; L 1 C 1-6 Alkylene; L 2 C 1-12 Alkylene; L 3 For H or C 1-30 alkyl; L 4 L 6 Each independently is C 6-30 Alkyl, C 6-30 alkenyl, C 6-30 alkynyl or C 6-30 Alkoxy, wherein the alkyl, alkenyl, alkynyl or alkoxy group has 0 to 3 H atoms, optionally further converted to halogen, hydroxyl, or C atoms. 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted; L 5 C 1-12 Alkylene; R 1 R 2 Each independently selected The acetylenic group, heteroatom O, N, S, or none; h, i, k are selected from integers from 1 to 10; j is selected from 0 or 1, and the heteroatom is selected from N, O, or S.

2. The compound according to claim 1, wherein, The compound is a compound represented by formula Ia, Ib, Ic, Id, Ie, If, or Ig: Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein the G, L 1 L 2 L 3 L 4 L 5 L 6 R 1 and R 2 As defined in claim 1; Each A 3 Each A 4 and A 5 Each independently is hydrogen, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl or C 1-12 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted; Each A 6 Each A 7 and A 8 Each independently is hydrogen, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl or C 1-12 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted; a and b are each independent integers selected from 0 to 10, for example, selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The condition is Each independently is C 6-30 Alkyl, C 6-30 alkenyl, C 6-30 alkynyl or C 6-30 Alkoxy, wherein the alkyl, alkenyl, alkynyl or alkoxy group has 0 to 3 H atoms, optionally further converted to halogen, hydroxyl, or C atoms. 1-12 Alkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 1-12 alkoxy- or halogen-substituted C 1-12 alkyl or hydroxy substituted C 1-12 Alkyl groups are substituted.

3. The compound according to claim 1 or 2, wherein, G is -OH or -N(A) 1 A 2 A 1 A 2 Each independently is C 1-4 Alkyl groups; and / or L 1 C 2-4 Alkylene; and / or L 2 C 1-10 Alkylene; and / or L 3 For H or C 1-20 Alkyl groups; and / or L 4 L 6 Each independently is C 8-20 Alkyl groups, including straight-chain C 8-20 Alkyl or branched C 8-20 Alkyl groups; and / or L 5 C 3-10 Alkylene; and / or R 1 R 2 Each independently selected 4. The compound according to claim 3, wherein, G is -OH or -N(A) 1 A 2 A 1 A 2 Each is independently methyl, ethyl, propyl; and / or L 1 It is ethylene or propylene; and / or L 2 C 1-8 Alkylene; and / or L 3 For H or C 1-16 Alkyl groups; and / or L 4 L 6 Each independently is C 8-18 Alkyl groups, including straight-chain C 8-18 Alkyl or branched C 8-18 Alkyl groups; and / or L 5 C 6-10 Alkylene; and / or R 1 R 2 Each independently selected 5. The compound according to claim 4, wherein, L 2 It is methylene, ethylene, propylene, butylene, amylene, hexylene, heptaylene, or octylene; and / or L 3 H or methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, or hexadecyl; and / or L 4 L 6 Each of the following is independently octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, or octadecyl, including straight-chain or branched forms; and / or L 5 Hexylene, heptylene, octylene, nonylene, decylene; and / or R 1 Selected from and / or R 2 Selected from 6. The compound according to claim 5, wherein, L 4 It is octyl, nonyl, decyl, undecyl, or dodecyl, including straight-chain or branched forms; and / or L 6 It is nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, or octadecyl, including straight-chain or branched forms.

7. The compound according to claim 2, wherein, Each A 3 Each A 4 and A 5 Each independently is hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl or C 1-8 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-8 alkoxy- or halogen-substituted C 1-8 alkyl or hydroxy substituted C 1-8 Alkyl groups are substituted; Each A 6 Each A 7 and A 8 Each independently is hydrogen, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl or C 1-8 Alkoxy; the alkyl, alkenyl, alkynyl or alkoxy group may optionally be further converted by halogen, hydroxyl, C 1-8 Alkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-8 alkoxy- or halogen-substituted C 1-8 alkyl or hydroxy substituted C 1-8 Alkyl groups are substituted; a and b are each an independent integer selected from 1 to 8, for example, selected from 1, 2, 3, 4, 5, 6, 7 or 8.

8. The compound according to claim 3, wherein, The compound is selected from one or more of compounds 1 to 15 in Table 1.

9. The compound according to claim 3, wherein, The pharmaceutically acceptable salt, prodrug, or stereoisomer is selected from salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs.

10. A compound listed in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

11. A composition comprising the compound of any one of claims 1-10 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, and one or more lipids selected from neutral lipids, steroids, and polymer-bound lipids.

12. The composition according to claim 11, comprising, in mol%... i) 20-65 mol% of the compound of any one of claims 1-10 or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof; ii) 5-25 mol% of neutral lipids; iii) 25-55 mol% steroids; and iv) 0.3-15% of polymer-bound lipids; Optionally, the neutral lipid is selected from 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2- Di-undecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16Lyso) PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME16.0PE), 1,2-distearateoyl-sn-glycerol- The group consisting of 3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-disarachidanoyl-sn-glycerol-3-phosphoethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphoethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol)sodium salt (DOPG), and sphingomyelin; Optionally, the steroid is selected from the group consisting of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and brassosterol; Optionally, the polymer-bound lipid is selected from the group consisting of PEG-DMG, PEG-c-DMG, PEG-C14, PEG-C18, ALC0159, PEG-c-DMA, PEG-DSPE, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG, and GalNAc-PEG-DSG.

13. The composition according to claim 11 or 12, further comprising one or more therapeutic or preventative agents; Preferably, the treatment or preventive agent comprises one or more polynucleotides; Preferably, the one or more polynucleotides are DNA or RNA; Preferably, the DNA is in a vector, or the RNA is mRNA or circular RNA; Preferably, the one or more polynucleotides encode a polypeptide and an RNA molecule; Preferably, the therapeutic or preventative agent comprises one or more RNA-based guide molecules and an mRNA molecule encoding a Cas protein; Preferably, one or more guide molecules and the Cas protein are configured to form a CRISPR-Cas complex capable of targeting and editing target nucleic acids; Preferably, the polynucleotide comprises a modified nucleotide; Preferably, one or more RNA-based guide molecules contain modified nucleotides.

14. A lipid nanoparticle comprising a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, or a composition of any one of claims 11 to 13.

15. A pharmaceutical composition comprising a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, a composition of any one of claims 11 to 13 or the lipid nanoparticles of claim 14, and a pharmaceutically acceptable excipient or diluent.

16. The use of the compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, the composition of any one of claims 11 to 13, the lipid nanoparticle of claim 14, or the pharmaceutical composition of claim 15 in the preparation of a formulation for editing a target gene in vivo in a subject in need.

17. A method of delivering a therapeutic and / or preventive agent to cells in a subject, the method comprising administering to the subject a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, a composition of any one of claims 11 to 13, a lipid nanoparticle of claim 14, or a pharmaceutical composition of claim 15.

18. A method for organ-specific delivery of a therapeutic agent and / or a preventive agent to a subject, the method comprising administering to the subject a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, a composition of any one of claims 11 to 13, a lipid nanoparticle of claim 14, or a pharmaceutical composition of claim 15.

19. A method for generating a target polypeptide in cells of a subject, the method comprising administering to the subject a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, a composition of any one of claims 11 to 13, a lipid nanoparticle of claim 14, or a pharmaceutical composition of claim 15.

20. A method for in vivo editing of a target gene in a subject in need, wherein the method comprises administering lipid nanoparticles comprising at least one lipid and one or more polynucleotides, wherein the one or more polynucleotides comprise or encode one or more guide molecules, wherein the one or more polynucleotides further encode a Cas protein, wherein the one or more guide molecules and the Cas protein are configured to form a CRISPR-Cas complex capable of targeting and editing the target gene, wherein the at least one lipid is a compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

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