Ionizable lipids suitable for lipid nanoparticles

Abstract

The present disclosure provides nitrogen-containing siloxane ionizable lipid compounds and lipid nanoparticles comprising the same. The present disclosure also relates to lipid nanoparticles comprising the provided ionizable lipid compounds, as well as a phospholipid, e.g., a phospholipid comprising at least one unsaturated tail and a head group having a positively charged nitrogen. The provided materials are particularly beneficial in applications involving nucleic acid delivery. The present disclosure also provides pharmaceutical compositions and methods comprising the provided ionizable lipids and / or lipid nanoparticles.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 613,515, filed December 21, 2023, and U.S. Provisional Application No. 63 / 566,701, filed March 18, 2024, the entire contents of which are incorporated herein by reference for all purposes. Background Technology

[0002] Lipid nanoparticles (LNPs) have become a key technology in drug delivery, particularly for the encapsulation and systemic delivery of nucleic acids such as mRNA and siRNA. Their importance lies in their ability to protect these therapeutic agents from degradation, enhance cellular uptake, and control the release of the encapsulating agent. However, despite the success of LNPs, there is still a pressing need for improvement. Current LNPs may have several limitations, including poor biodistribution and / or clearance, induction of immune responses, potential off-target effects, and low expression efficiency of the delivered payload (such as mRNA). Therefore, developing next-generation LNPs with, for example, enhanced stability, targeted delivery, and minimized side effects is crucial to fully realizing the potential of LNPs in nucleic acid therapy. This disclosure addresses these needs and provides related and other advantages. Summary of the Invention

[0003] This summary provides a high-level overview of various aspects of this disclosure and introduces some concepts described and illustrated in this document and the accompanying drawings. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to define the scope of the claimed subject matter. The embodiments covered by this disclosure are defined by the claims rather than by this summary. The subject matter should be understood by referring to appropriate portions of the specification throughout, any or all of the accompanying drawings, and each claim. Some exemplary embodiments of this disclosure are discussed below.

[0004] On the one hand, this disclosure provides compounds having the following formula or a pharmaceutically acceptable salt thereof: (Ia). R of equation (Ia) 1 and R 2 Each is independently hydrogen or C 1-4 Alkyl group. Or, R 1 and R 2 They combine with the nitrogen atoms to form 3- to 8-membered heterocyclic groups. R in formula (Ia) 3 R 4 and R 5 Each is independently a cis-C with only one unsaturated bond. 5-20 alkenyl or C 5-20 Alkyne group. The subscript n in formula (Ia) is an integer from 4 to 8.

[0005] On the other hand, this disclosure provides lipid nanoparticles having a pKa of 5.8 to 6.9, i.e., a pKa of not less than 5.8 and not greater than 6.9. The lipid nanoparticles comprise ionizable lipids or pharmaceutically acceptable salts thereof. The ionizable lipids have the following formula: (I). R in equation (I) 1 and R 2 Each is independently hydrogen, C 1-4 Alkyl or 2- to 4-membered heteroalkyl, wherein the alkyl and heteroalkyl optionally have one or more substituents, and wherein each of the substituents is independently a hydroxyl, C 1-6 Hydroxyalkyl or fluorine. Or, R 1 and R 2 They combine with the nitrogen atoms to form 3- to 8-membered heterocyclic groups optionally having one or more substituents, wherein each substituent is independently a hydroxyl group, C10-20-30-30-30-4 ... 1-6 Hydroxyalkyl or fluorine. R of formula (I) 3 R 4 and R 5 Each is independently a C that optionally has one or more substituents. 5-20 Alkyl, C 5-20 alkenyl, C 5-20 alkynyl or C 5-12 Cycloalkyl, wherein each of the substituents is independently C10. 1-6 Alkyl or C 2-6 Alkenyl. R of formula (I) 6 and R 7 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group, condition is R 6 and R 7 No more than one of them is hydrogen. Or, R 6 and R 7 They combine with the carbon atoms to form cyclopropyl or cyclobutyl groups. R in formula (I) 8 and R 9 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group, condition is R 8 and R 9 No more than one of them is hydrogen. Or, R 8 and R 9 They combine with the carbon atoms to form cyclopropyl or cyclobutyl groups. R in formula (I) 10 and R 11 Each is independently hydrogen, C 1-20Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group, condition is R 10 and R 11 No more than one of them is hydrogen. Or, R 10 and R 11 They combine with the carbon atoms they are attached to to form cyclopropyl or cyclobutyl groups. 12 and R 13 Each is independently hydrogen or optionally has one or more substituents. 1-6 Alkyl group, wherein each of the substituents is independently a hydroxyl group, C10, or C20. 1-6 Hydroxyalkyl or fluorine. Or, R 12 and R 13 They can combine with the carbon atoms they are attached to form cyclopropyl or cyclobutyl groups. X in formula (I) 1 X 2 and X 3 Each is independently a covalent bond, C 1-6 Alkylene, C 1-6 imide or C 1-6 Alynyl group. The subscript n in formula (I) is an integer from 2 to 10.

[0006] On the other hand, this disclosure provides lipid nanoparticles comprising phospholipids or pharmaceutically acceptable salts thereof, and ionizable lipids or pharmaceutically acceptable salts thereof. The phospholipids comprise one or more unsaturated tails and a head group having a positively charged nitrogen atom. The ionizable lipids have the following formula: (I). R in equation (I) 1 and R 2 Each is independently hydrogen, C 1-4 Alkyl or 2- to 4-membered heteroalkyl, wherein the alkyl and heteroalkyl optionally have one or more substituents, and wherein each of the substituents is independently a hydroxyl, C 1-6 Hydroxyalkyl or fluorine. Or, R 1 and R 2 They combine with the nitrogen atoms to form 3- to 8-membered heterocyclic groups optionally having one or more substituents, wherein each substituent is independently a hydroxyl group, C10-20-30-30-30-4 ... 1-6 Hydroxyalkyl or fluorine. R of formula (I) 3 R 4 and R 5 Each is independently a C that optionally has one or more substituents. 5-20 Alkyl, C 5-20 alkenyl, C 5-20 alkynyl or C 5-12 Cycloalkyl, wherein each of the substituents is independently C10. 1-6 Alkyl or C 2-6Alkenyl. R of formula (I) 6 and R 7 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group. Or, R 6 and R 7 They combine with the carbon atoms to form cyclopropyl or cyclobutyl groups. R in formula (I) 8 and R 9 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group. Or, R 8 and R 9 They combine with the carbon atoms to form cyclopropyl or cyclobutyl groups. R in formula (I) 10 and R 11 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group. Or, R 10 and R 11 They combine with the carbon atoms they are attached to to form cyclopropyl or cyclobutyl groups. 12 and R 13 Each is independently hydrogen or optionally has one or more substituents. 1-6 Alkyl group, wherein each of the substituents is independently a hydroxyl group, C10, or C20. 1-6 Hydroxyalkyl or fluorine. Or, R 12 and R 13 They can combine with the carbon atoms they are attached to form cyclopropyl or cyclobutyl groups. X in formula (I) 1 X 2 and X 3 Each is independently a covalent bond, C 1-6 Alkylene, C 1-6 imide or C 1-6 Alynyl group. The subscript n in formula (I) is an integer from 2 to 10.

[0007] On the other hand, this disclosure provides a composition, such as a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. The pharmaceutical composition also comprises the lipid nanoparticles or compounds disclosed herein.

[0008] On the other hand, this disclosure provides a method for introducing nucleic acids into cells. The method includes contacting cells with lipid nanoparticles disclosed herein, wherein the lipid nanoparticles contain nucleic acids.

[0009] On the other hand, this disclosure provides an in vivo method for delivering nucleic acids to a subject. The method includes administering to a subject lipid nanoparticles disclosed herein, wherein the lipid nanoparticles contain nucleic acids.

[0010] On the other hand, this disclosure provides a method for preventing or treating a disease or condition in a subject. The method includes administering to the subject a measured amount, such as a therapeutically effective amount, of the lipid nanoparticles disclosed herein, the compounds disclosed herein, and / or the pharmaceutical compositions disclosed herein.

[0011] In another embodiment, this disclosure provides a method for editing genomic information in cells. The method includes contacting cells with lipid nanoparticles disclosed herein, wherein the lipid nanoparticles comprise nucleic acids. The nucleic acids and / or proteins encoded by the nucleic acids are components of a gene editing system. Attached Figure Description

[0012] Figure 1 A graph showing tdTomato activity in the livers of mice administered with various lipid nanoparticle formulations according to the provided embodiments, wherein the lipid nanoparticles contain tdTomato mRNA.

[0013] Figure 2 A graph showing the pKa of lipid nanoparticles formulated according to the provided embodiment is displayed. The pKa is measured as the pH at which 50% of the ionizable lipids in the lipid nanoparticles are ionized. Detailed Implementation A. Definition

[0014] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, any methods or materials similar to or equivalent to those described herein may be used in the practice of this disclosure. The following terms are defined for the purposes of this disclosure.

[0015] The abbreviations used herein have their conventional meanings in the chemical and biological fields. The description of the compounds disclosed herein is limited by the principles of chemical bonding known to those skilled in the art. Therefore, when a group can be substituted by one or more substituents, such substituents are chosen to conform to the principles of chemical bonding and to yield compounds that are not inherently unstable and / or that are known to those skilled in the art to be unstable under environmental conditions (e.g., aqueous, neutral, or physiological conditions). When substituents are specified by conventional chemical formulas written from left to right, they also encompass chemically identical substituents obtained by writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.

[0016] Unless otherwise stated, the compounds disclosed herein may also contain atomic isotopes in non-natural proportions at one or more atoms constituting such compounds. For example, the compounds may be labeled with isotopes such as deuterium (…). 2 H), tritium ( 3 H), Iodine-125 ( 125 I), carbon-13 ( 13 C) or carbon-14 ( 14 C). All isotopic variants of the compounds disclosed herein, whether radioactive or not, are included within the scope of this disclosure.

[0017] As used herein, the term "substitution," when applied to chemical substances, refers to the replacement of a hydrogen atom with a non-hydrogen atom or a covalently bonded group of atoms. The atom or group of atoms that replaces the hydrogen atom is called a "substituent."

[0018] As used herein, the term "member" in the context of chemical substances refers to a non-hydrogen atom of a covalently bonded group of atoms (e.g., a compound or its substituents).

[0019] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly indicates otherwise. Thus, for example, references to “a phospholipid” may optionally include combinations of two or more phospholipids, etc.

[0020] As used herein, the terms “about” and “approximately”, when used to modify a quantity specified as a numerical value or range, indicate that the numerical value and reasonable deviations from values ​​known to those skilled in the art (e.g., ±20%, ±10%, or ±5%) are within the intended meaning of the value.

[0021] As used herein, the term “and / or” refers to and includes any and all possible combinations of one or more of the related listed items, as well as combinations that are missing when interpreted in the context of substitution (“or”).

[0022] As used herein, the terms “comprising,” “including,” “having,” “containing,” and variations thereof are inclusive and open-ended, and do not exclude additional, unlisted elements or method steps beyond those expressly enumerated. As used herein, the phrase “consisting of” is closed-ended and does not include any element, step, or ingredient not expressly specified. As used herein, the phrase “substantially constitutes” limits the scope of the described feature to specific materials or steps, and those materials or steps that do not materially affect the essential and novel features of the disclosed feature.

[0023] As used herein, the terms "lipid particle," "lipid nanoparticle," and "LNP" refer to particles containing phospholipids and ionizable lipids. Lipid particles may contain additional lipid components, such as sterols and / or conjugated lipids, and may also contain nucleic acids, wherein the nucleic acids may be encapsulated within the particle. Lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Application Publications Nos. 2004 / 0142025 and 2007 / 0042031 and International Patent Application Publication No. WO 2012 / 000104.

[0024] As used herein, the term "phospholipid" refers to a lipid substance having a hydrophilic "head group" containing phosphoric acid and a hydrophobic portion. The hydrophobic portion may contain one or more hydrophobic groups, most typically two. The hydrophobic groups, also known as hydrophobic "tails," may be derived from fatty acids and linked by alcohol residues (e.g., glycerol). Exemplary structures of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

[0025] As used herein, the term "ionizable lipid" refers to a lipid substance that carries a net positive charge at a selected pH (such as acidic pH or physiological pH). In some cases, ionizable lipids contain ionizable primary, secondary, or tertiary amine head groups (e.g., pH-titrile). In some cases, ionizable lipids promote the encapsulation of negatively charged nucleic acid (e.g., mRNA or siRNA) payloads during particle formation. In some cases, ionizable lipids promote endosome fusion and cytoplasmic release of the payload after cellular uptake of lipid nanoparticles.

[0026] As used herein, the term "conjugated lipid" refers to a lipid conjugated with a polymer, such as a lipid conjugated with a polymer that inhibits lipid particle aggregation. Such lipid conjugations include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugations), polysarcosine-lipid conjugations, polyoxazoline (POZ)-lipid conjugations, PEG-lipid conjugations such as PEG coupled with dialkyloxypropyl, PEG coupled with diacylglycerol, PEG coupled with cholesterol, PEG coupled with phosphatidylethanolamine, PEG conjugated with ceramide (see, for example, U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG may be conjugated directly to lipids or may be linked to lipids via a linker portion. Any linker portion suitable for conjugating a polymer to a lipid may be used, including, for example, linker portions containing non-esters and linker portions containing esters.

[0027] As used herein, the term "salt" refers to an acid or base salt of the compounds disclosed herein. A "pharmaceutically acceptable salt" is a salt that is compatible with other components of a pharmaceutical composition containing the compound and is harmless to its recipient, i.e., the subject. Therefore, it is understood that pharmaceutically acceptable salts do not produce significant adverse toxicological effects on the subject.

[0028] As used herein, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain or branched saturated aliphatic group having the indicated number of carbon atoms. A branched alkyl group may include one or more branches having a geminal, ortho, and / or isolated configuration. For example, an alkyl group may include a geminal methyl group. An alkyl group may contain any number of carbons, such as C10. 1-2 C 1-3 C 1-4 C 1-5 C 1-6 C 1-7 C 1-8 C 1-9 C 1-10 C 2-3 C 2-4 C 2-5 C 2-6 C 3-4 C 3-5 C 3-6 C 4-5 C 4-6 and C 5-6 For example, C 1-6 Alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl. Alkyl groups can also refer to alkyl groups having a maximum of 40 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, and decyl. In some aspects, i.e., as indicated in this disclosure, alkyl groups may be substituted. Unless otherwise stated, a “substituted alkyl group” can be substituted by one or more groups selected from halogens, hydroxyl groups, amino groups, alkylamino groups, amide groups, acyl groups, oxo groups, nitro groups, cyano groups, and alkoxy groups. In some embodiments including one or more halogen substituents, fluorine is preferred.

[0029] As used herein, the term "alkylene" itself, or as part of another substituent, refers to a straight-chain or branched saturated aliphatic group, i.e., a divalent hydrocarbon group, having the indicated number of carbon atoms and connected to at least two other groups. The two parts connected to the alkylene group can be attached to the same or different atoms of the alkylene group. For example, a straight-chain alkylene group can be -(CH2). n- A divalent group, wherein n is 1, 2, 3, 4, 5, or 6. Branched alkylene groups may include one or more branches having a geminal, ortho, and / or isolated configuration. For example, an alkylene group may include a geminal methyl group. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, secondary butylene, pentylene, and hexylene. In some aspects, i.e., as indicated in this disclosure, alkylene groups may be substituted. Unless otherwise stated, a “substituted alkylene group” may be substituted with one or more groups selected from halogens, hydroxyl groups, amino groups, alkylamino groups, amide groups, acyl groups, oxo groups, nitro groups, cyano groups, and alkoxy groups.

[0030] As used herein, the term "alkenyl" itself, or as part of another substituent, refers to an alkyl group having at least one carbon-carbon double bond. Alkenyl groups can contain any number of carbons, such as C2, C3, C4, C5, C6, C7, C8, C9 ... 2-3 C 2-4 C 2-5 C 2-6 C 2-7 C 2-8 C 2-9 C 2-10 C3, C 3-4 C 3-5 C 3-6 C4, C 4-5 C 4-6 C5, C 5-6 And C6. The alkenyl group can have any suitable number of double bonds, including but not limited to 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethene), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl and 1,3,5-hextrienyl. In some aspects, i.e., as indicated in this disclosure, the alkenyl group can be substituted. Unless otherwise stated, a “substituted alkenyl group” can be substituted by one or more groups selected from halogen, hydroxyl, amino, alkylamino, amide, acyl, oxo, nitro, cyano and alkoxy groups. In some embodiments that include one or more halogen substituents, fluorine is preferred.

[0031] As used herein, the term "alkenyl" itself, or as part of another substituent, refers to an alkylene group, i.e., a divalent hydrocarbon group, having at least one carbon-carbon double bond and attached to at least two other groups. The two parts attached to the alkenyl group may be attached to the same or different atoms of the alkenyl group. In some respects, i.e., as indicated in the disclosure, the alkenyl group may be substituted. Unless otherwise stated, a "substituted alkenyl group" may be substituted by one or more groups selected from halogens, hydroxyl groups, amino groups, alkylamino groups, amide groups, acyl groups, oxo groups, nitro groups, cyano groups, and alkoxy groups.

[0032] As used herein, the term "alkynyl" itself, or as part of another substituent, refers to an alkyl group having at least one carbon-carbon triple bond. The alkynyl group can contain any number of carbons, such as C2, C3, C4, C5, C6, C7, C8, C9 ... 2-3 C 2-4 C 2-5 C 2-6 C 2-7 C 2-8 C 2-9 C 2-10 C3, C 3-4 C 3-5 C 3-6 C4, C 4-5 C 4-6 C5, C 5-6 And C6. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butyrynyl, 1-pentynyl, 2-pentynyl, isopentenynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hextriynyl. In some aspects, i.e., as indicated in this disclosure, the alkynyl group may be substituted. Unless otherwise stated, a “substituted alkynyl” group may be substituted with one or more groups selected from halogens, hydroxyl, amino, alkylamino, amide, acyl, oxo, nitro, cyano, and alkoxy groups. In some embodiments including one or more halogen substituents, fluorine is preferred.

[0033] As used herein, the term "ynynyl" itself, or as part of another substituent, refers to an ynynyl group, i.e., a divalent hydrocarbon group, having at least one carbon-carbon triple bond and attached to at least two other groups. The two parts attached to the ynynyl group may be attached to the same or different atoms of the ynynyl group. In some respects, i.e., as indicated in this disclosure, the ynynyl group may be substituted. Unless otherwise stated, a "substituted ynynyl" may be substituted by one or more groups selected from halogens, hydroxyl groups, amino groups, alkylamino groups, amide groups, acyl groups, oxo groups, nitro groups, cyano groups, and alkoxy groups.

[0034] As used herein, the term "alkoxy" refers to a substituted alkyl group as defined above, having an oxygen atom connecting the alkyl group to the junction: alkyl-O-. As for the unsubstituted portion of the alkyl group, the alkoxy group can have any suitable number of carbon atoms, such as C... 1-6 Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.

[0035] As used herein, the term "hydroxyalkyl" or "alkylhydroxyl" refers to an alkyl group as defined above, wherein at least one hydrogen atom is substituted with a hydroxyl group. As for the unsubstituted portion of the alkyl group, the alkylhydroxyl group may have any suitable number of carbon atoms, such as C1. 1-6 Exemplary alkyl hydroxyl groups include, but are not limited to, hydroxymethyl, hydroxyethyl (wherein the hydroxyl group is at position 1 or 2), hydroxypropyl (wherein the hydroxyl group is at position 1, 2 or 3), hydroxybutyl (wherein the hydroxyl group is at position 1, 2, 3 or 4), hydroxypentyl (wherein the hydroxyl group is at position 1, 2, 3, 4 or 5), hydroxyhexyl (wherein the hydroxyl group is at position 1, 2, 3, 4, 5 or 6), 1,2-dihydroxyethyl, etc.

[0036] As used herein, the term "heteroalkyl" itself, or as part of another substituent, refers to an alkyl group having any suitable length and any number (e.g., 1 to 3) of heteroatoms (such as N, O, or S). Heteroatoms may also be oxidized, for example, but not limited to -S(O)- and -S(O)2-. For example, heteroalkyl groups may include ethers, thioethers, and alkylamines. The heteroatom portion of a heteroalkyl group may substitute for the hydrogen atom of the alkyl group to form a hydroxyl, thio, or amino group. Alternatively, the heteroatom portion may be a linking atom or inserted between two carbon atoms. In some aspects, i.e., as indicated in this disclosure, heteroalkyl groups may be substituted. Unless otherwise stated, a "substituted heteroalkyl" group may be substituted by one or more groups selected from halogens, hydroxyl, amino, alkylamino, amide, acyl, oxo, nitro, cyano, and alkoxy groups.

[0037] As used herein, the term "cycloalkyl" itself, or as part of another substituent, refers to a cyclic assembly of saturated or partially unsaturated, monocyclic, fused polycyclic, spirocyclic, or bridged polycyclic rings containing 3 to 12 ring atoms, or the indicated number of atoms. Cycloalkyl groups may contain any number of carbons, such as C1. 3-6 C 4-6 C 5-6 C 3-8 C 4-8 C 5-8 C 6-8 C 3-9 C 3-10 C 3-11 and C 3-12Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, bicyclic [1.1.1]pentane, norbornene, [2.2.2]bicyclic octane, decahydronaphthalene, and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornene. When the cycloalkyl group is a saturated monocyclic C 3-8 When cycloalkyl is used, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. When the cycloalkyl group is a saturated monocyclic C 3-6 When cycloalkyl is used, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In some aspects, i.e., as indicated in this disclosure, the cycloalkyl group may be substituted. Unless otherwise stated, the "substituted cycloalkyl" group may be substituted by one or more groups selected from halogens, hydroxyl, amino, alkylamino, amide, acyl, oxo, nitro, cyano, and alkoxy. In some embodiments including one or more halogen substituents, fluorine is preferred.

[0038] As used herein, the term "heterocyclic group," either on its own or as part of another substituent, refers to a saturated heterocyclic ring system having 3 to 15 membered rings or the number of atoms indicated, a partially unsaturated non-aromatic ring, or a partially unsaturated non-aromatic fused, spirocyclic, or bridged polycyclic system, wherein one or more carbon atoms are each independently substituted by the same or different heteroatoms (such as N, O, or S). Heteroatoms may be oxidized to form moieties, such as, but not limited to, -S(O)- and S(O)2. Heterocyclic groups may include any number of ring atoms, such as C 3-6 C 4-6 C 5-6 C 3-8 C 4-8 C 5-8 C 6-8 C 3-9 C 3-10 C 3-11 C 3-12 Or C 3-15In a heterocyclic group, at least one carbon atom is replaced by a heteroatom. Any suitable number of carbon ring atoms in the heterocyclic group can be replaced by heteroatoms, such as 1, 2, 3 or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4 or 3 to 4. Heterocyclic groups can include, for example, aziridine, aziridine, pyrrolidine, piperidine, aziridine-heptane, aziridine-octane, quinine ring, pyrazolidine, imidazoline, piperazine (1,2-, 1,3- and 1,4-isomers), ethylene oxide, oxacyclobutane, tetrahydrofuran, oxacyclohexane (tetrahydropyran), hexane oxide, cyclothioethane, thiohexane, thiohexane (tetrahydrothiophene), thiohexane (tetrahydrothiaran), oxazolidine, isoxazolidine, thiazoline, isothiazolidine, dioxopentane, dithiohexopentane, morpholine, thiomorpholine, dioxacyclohexane, or dithiohexane. One or more heterocyclic alkyl rings of the heterocyclic group can also be fused with aromatic or non-aromatic rings to form, but not limited to, dihydroindole. Therefore, heterocyclic groups include partially unsaturated ring systems containing one or more double bonds, including fused ring systems with one aromatic ring and one non-aromatic ring, but not fully aromatic ring systems. Examples include dihydroquinoline (e.g., 3,4-dihydroquinoline), dihydroisoquinoline (e.g., 1,2-dihydroisoquinoline), tetrahydroquinoline (e.g., 1,2,3,4-tetrahydroquinoline), tetrahydroisoquinoline, dihydroimidazole, tetrahydroimidazole, etc., isoindoline, isoindoline (e.g., isoindoline-1-one), indigo, dihydrophthalazine, quinolineone, spiro[cyclopropane-1,1'-isoindoline]-3'-one, etc. Heterocyclic groups can have 3-15 members, or 3-12 members, or 3-10 members, or 3-7 members, or 5-6 members. Heterocyclic groups can be linked at any position on the ring. For example, aziridine can be 1- or 2-aziridine, aziridine can be 1- or 2-aziridine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine can be 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3- or 4-pyrazolidine, imidazoline can be 1-, 2-, 3- or 4-imidazoline, piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1- or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 4- or 5-oxazolidine, isoxazolidine can be 2-, 3-, 4- or 5-isooxazolidine, thiazoline can be 2-, 3-, 4- or 5-thiazoline, isothiazolidine can be 2-, 3-, 4- or 5-isothiazoline, and morpholine can be 2-, 3- or 4-morpholine. In some aspects, namely as indicated in this disclosure, the heterocyclic group may be substituted. Unless otherwise stated, the “substituted heterocyclic group” may be substituted by one or more groups selected from halogen, hydroxyl, amino, alkylamino, amide, acyl, oxo, nitro, cyano, and alkoxy groups. In some embodiments including one or more halogen substituents, fluorine is preferred.

[0039] As used herein, the term "hydroxyl group" itself, or as part of another substituent, refers to the -OH moiety.

[0040] As used herein, the terms “halogenated” and “halogen” refer, either on their own or as part of another substituent, to a fluorine, chlorine, bromine, or iodine atom.

[0041] As used herein, the term “oxo” itself, or as part of another substituent, refers to the oxygen atom (i.e., O=) bonded to the double bond of a compound.

[0042] As used herein, the term "nitro" itself, or as part of another substituent, refers to the -NO2 moiety.

[0043] As used herein, the term "cyano" itself, or as part of another substituent, refers to a group consisting of nitrogen bound to a carbon triple bond: -C≡N.

[0044] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing nucleotide analogs or modified backbone residues or bonds that are synthetic, naturally occurring, or non-natural, have binding properties similar to a reference nucleotide, and are metabolized in a similar manner to a reference nucleotide. Non-limiting examples of nucleotide analogs are described, for example, in International Patent Application No. WO 2007 / 024708. Examples of nucleic acids containing such nucleotide analogs, modified backbone residues, or bonds include, but are not limited to, nucleic acids containing phosphate thioesters, aminophosphate esters, methyl phosphonate, chiral methyl phosphonate, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by producing a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine (Batzer et al., (191) Nucleic Acid Res. 19: 5081; Ohtsuka et al., (1985) J. Biol. Chem. 260: 2605; and Rossolini et al., (1994) Mol. Cell. Probes 8:91).

[0045] Non-limiting examples of polynucleotides or nucleic acids include DNA, RNA, coding or non-coding regions of genes or gene segments, intergenic DNA, loci (sites) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), small nucleolar RNA (snoRNA), ribozymes, deoxynucleotides (dNTPs), or dideoxynucleotides (ddNTPs). Polynucleotides may also include complementary DNA (cDNA), which is the DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification. Polynucleotides may also include synthetically produced or amplified DNA molecules, genomic DNA (gDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, or primers. Polynucleotides may include modified nucleotides suitable for reducing RNA immunogenicity, such as methylated nucleotides, nucleotide analogs, and / or nucleosides, as described in International Patent Application No. WO 2007 / 024708. If present, the nucleotide structure may be modified before or after polymer assembly. The nucleotide sequence may be broken down by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeled components. Unless otherwise specified, the provided polynucleotide sequences are listed in a 5' to 3' orientation.

[0046] Nucleic acids or polynucleotides can be double-stranded or triple-stranded nucleic acids, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acids, the nucleic acid strands do not need to be coextensive; for example, double-stranded nucleic acids do not need to be double-stranded along their entire length.

[0047] Nucleic acid modification can include adding chemical groups to individual nucleic acid bases or to nucleic acids as a whole, which introduce additional charges, polarizabilities, hydrogen bonds, electrostatic interactions, and functions. Such modifications include base modifications such as 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, cytosine exocyclic amine modifications, S-bromouracil substitutions, backbone modifications, and anomalous base pairing combinations, such as isobasic cytosine and isoguanidine.

[0048] Nucleic acids can be derived from completely chemically synthesized processes, such as solid-phase mediated chemical synthesis; from biological sources, such as by isolation from any species that produces nucleic acids; or from processes involving manipulation of nucleic acids using molecular biological tools, such as DNA replication, PCR amplification, reverse transcription, in vitro transcription, as described, for example, in International Patent Application Publication No. WO 2007 / 024708; or from combinations of these processes.

[0049] As used herein, in the context of lipid nanoparticles and nucleic acids, the term "completely encapsulated" means that the nucleic acids in the particles do not significantly degrade after exposure to serum or nuclease assays that would significantly degrade free nucleic acids. In a completely encapsulated system, in treatments that typically degrade 100% of free nucleic acids, preferably less than 25% of the particle nucleic acids are degraded, more preferably less than 10%, and most preferably less than 5%. Complete encapsulation also indicates that the particles are serum-stable, meaning that they do not rapidly disintegrate into their constituent parts upon in vivo administration.

[0050] As used herein, in the context of lipid nanoparticles and cells, the term "contact" refers to the direct or indirect delivery of lipid nanoparticles to a cell surface, either in vitro or in vivo, for example, by providing lipid nanoparticles at or near the location of the cells to be contacted. In vitro contact may involve cells, for example, in cell cultures or tissue cultures. Cell cultures or tissue cultures may include suspension cells and / or adherent cells. The cells in contact may be the same cell type or two or more different cell types. For illustration only, different contacting cell types may include, for example, hepatocytes and hepatic stellate cells. In some instances, different cell types are cultured together before and / or during contact with lipid nanoparticles. In other instances, different cell types are cultured separately. In further instances, one of the two or more cell types to be contacted is specifically cultured in the absence of any other cell type among those two or more. In some instances, hepatic stellate cells are cultured alone or in combination with other cell types from the liver, such as hepatocytes, before and / or during contact. In some instances, the tissue culture is a liver tissue culture. In vivo cell contact typically involves administering lipid nanoparticles to a subject, where cells are present within the subject's body. In some instances, administration occurs at a location close to the cells in the subject's body. In other instances, administration occurs at a location distant from the cells, and the lipid nanoparticles migrate to the cellular location, for example, through the particle's targeting capabilities.

[0051] As used herein, the term "subject" refers to a vertebrate, preferably a mammal. Suitable mammalian subjects for the provided compositions include, but are not limited to, mice, rats, apes, humans, farm animals, sporting animals, and pets. In some embodiments, the subject is a human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is over 10 years of age, for example, over 20 years, over 30 years, over 40 years, over 50 years, over 60 years, over 70 years, or over 80 years of age. In some embodiments, the subject is under 80 years of age, for example, under 70 years, under 60 years, under 50 years, under 40 years, under 30 years, under 20 years, or under 10 years of age.

[0052] As used herein, the terms "pharmaceuticalally acceptable excipient" and "pharmaceuticalally acceptable carrier" refer to substances that facilitate the administration and absorption of the active agent by a subject and may be included in the compositions of this disclosure without causing significant adverse toxicological effects on the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, physiological saline solutions, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, etc. Those skilled in the art will recognize that other pharmaceutically acceptable excipients and carriers are also useful in this disclosure.

[0053] As used herein, the term “application” means oral administration, suppository administration, local contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, subcutaneous administration, intrathecal administration, intravenous administration, intracranial administration, subretinal administration, or intravitreal administration, or implantation of a sustained-release device, such as a microosmotic pump, into a subject.

[0054] As used herein, the term “therapeutic effective amount” refers to the amount or dose of a compound, composition, or formulation that produces the therapeutic effect of its application. The exact amount or dose will depend on the purpose of treatment and will be determined by someone skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0055] As used herein, the term "treatment" refers to any process that results in the successful elimination or improvement of any sign of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameters such as relief; alleviation; reduction of symptoms or making the symptoms, injury, pathology, or condition more tolerable for the patient; reduction of the frequency or duration of symptoms or condition; or, in some cases, prevention of the onset of one or more symptoms. Treatment or improvement of symptoms can be based on any objective or subjective parameters, including, for example, the results of a physical examination or laboratory test. B. Lipid nanoparticles

[0056] On the one hand, this disclosure provides various lipid nanoparticles comprising ionizable lipids or pharmaceutically acceptable salts thereof. The ionizable lipids of the lipid nanoparticles are nitrogen-containing silyl ethers and may be any of those further described in detail in Section B.2. Specific selection, composition, and amount (e.g., relative amount) of the ionizable lipids in the lipid nanoparticles provide remarkable improvements to a variety of properties of the lipid nanoparticles, such as properties favorable for nucleic acid delivery. These improved properties include, for example, enhanced targeting, optimized stability, and reduced toxicity and immunostimulation.

[0057] In some embodiments, the provided lipid nanoparticles also comprise phospholipids or pharmaceutically acceptable salts thereof. In some instances, the phospholipids of the lipid nanoparticles include one or more unsaturated tails and a head group having a positively charged nitrogen, and are any of those further described in detail in Section B.1. Specific selection, composition, and amount (e.g., relative amount) of phospholipids in the lipid nanoparticles can further contribute to the advantages of the lipid nanoparticles as delivery carriers.

[0058] In some embodiments, the provided lipid nanoparticles also comprise sterols, such as cholesterol or derivatives thereof. Cholesterol or derivatives thereof may be any of those further described in detail in Section B.3. In some embodiments, the lipid nanoparticles also comprise conjugated lipids, such as lipids conjugated with PEG. Conjugated lipids may be any of those further described in detail in Section B.4. Specific selection, composition, and amount (e.g., relative amount) of cholesterol and / or conjugated lipids in the lipid nanoparticles may further contribute to the advantages of the lipid nanoparticles as a delivery carrier.

[0059] Lipid nanoparticles can be configured to have sizes advantageous for specific applications, such as in vivo or in vivo delivery of nucleic acids to cells or subjects. Provided lipid nanoparticles may have, for example, an average diameter of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. Lipid nanoparticles can be configured to be substantially non-toxic, for example, by selecting their components.

[0060] In some instances, the provided lipid nanoparticles contain ionizable lipids comprising about 30 mol% to about 80 mol% of the total lipids present in the particles (e.g., about 40 mol% to about 70 mol%, about 45 mol% to about 65 mol%, about 45 mol% to about 60 mol%, or about 50 mol% to about 60 mol%) of the total lipids present in the particles; phospholipids comprising about 3 mol% to about 20 mol% of the total lipids present in the particles (e.g., about 5 mol% to about 15 mol% or about 8 mol% to about 12 mol%) of the total lipids present in the particles; cholesterol or its derivatives comprising about 10 mol% to about 60 mol% of the total lipids present in the particles (e.g., about 20 mol% to about 50 mol% or about 30 mol% to about 40 mol%) of the total lipids present in the particles; and conjugated lipids comprising about 0.1 mol% to about 10 mol% of the total lipids present in the particles. (For example, about 0.1 mol% to about 5 mol% or about 0.5 mol% to about 3 mol% of total lipids present in the particles).

[0061] In a specific example, the lipid nanoparticles comprise about 45 mol% to about 60 mol% of the total lipids present in the particles, phospholipids about 8 mol% to about 12 mol% of the total lipids present in the particles, cholesterol or its derivatives about 30 mol% to about 40 mol% of the total lipids present in the particles, and conjugated lipids about 0.5 mol% to about 3 mol% of the total lipids present in the particles.

[0062] In some instances, the lipid nanoparticles comprise about 52 mol% to about 57 mol% of the total lipids present in the particles, about 10 mol% to about 12 mol% of the total lipids present in the particles, about 31 mol% to about 34 mol% of cholesterol or a derivative thereof, and about 1 mol% to about 3 mol% of the total lipids present in the particles, and conjugated lipids.

[0063] In one example, the lipid nanoparticles comprise approximately 54.6 mol% of ionizable lipids, approximately 10.9 mol% of phospholipids, approximately 32.8 mol% of cholesterol or its derivatives, and approximately 1.6 mol% of conjugated lipids, comprising the total lipids present in the particles.

[0064] In one example, the lipid nanoparticles comprise approximately 54.2 mol% of ionizable lipids, approximately 10.8 mol% of phospholipids, approximately 32.5 mol% of cholesterol or its derivatives, and approximately 2.5 mol% of conjugated lipids, comprising the total lipids present in the particles.

[0065] The specific structure of the lipid nanoparticles described herein, particularly the design and combination of their contained phospholipids and ionizable lipids, provides the nanoparticles with pKa properties that are beneficial for, for example, nucleic acid delivery, targeting, stability, degradability, and toxicity. For example, in some embodiments, the lipid nanoparticles have a pKa of about 5.8 to about 6.9, such as about 5.8 to about 6.8, about 5.8 to about 6.7, about 5.8 to about 6.6, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1, about 5.8 to about 6.0, about 5.8 to about 5.9, about 5.9 to about 6.9, about 5.9 to about 6.8, about 5.9 to about 6.7, about 5... 0.9 to about 6.6, about 5.9 to about 6.5, about 5.9 to about 6.4, about 5.9 to about 6.3, about 5.9 to about 6.2, about 5.9 to about 6.1, about 5.9 to about 6.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about 6.0 to about 6.1, about 6.1 to about 6.9, About 6.1 to about 6.8, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.1 to about 6.4, about 6.1 to about 6.3, about 6.1 to about 6.2, about 6.2 to about 6.9, about 6.2 to about 6.8, about 6.2 to about 6.7, about 6.2 to about 6.6, about 6.2 to about 6.5, about 6.2 to about 6.4, about 6.2 to about 6.3, about 6.3 to about 6.9, about 6.3 to about 6.8, about 6.3 to about 6. 7, about 6.3 to about 6.6, about 6.3 to about 6.5, about 6.3 to about 6.4, about 6.4 to about 6.9, about 6.4 to about 6.4 to about 6.8, about 6.4 to about 6.7, about 6.4 to about 6.6, about 6.4 to about 6.5, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.6 to about 6.9, about 6.6 to about 6.8, about 6.6 to about 6.7, about 6.7 to about 6.8, or about 6.8 to about 6.9. In some instances, lipid nanoparticles exhibit favorable targeting, stability, degradability, and toxicity properties, in part because the lipid nanoparticles have a pKa of about 6.2 to about 6.5.

[0066] In a preferred embodiment, the pKa of the lipid nanoparticles described herein is determined using a test measuring ionization at a temperature of about 34°C to about 40°C. In some embodiments, the lipid nanoparticles have a pKa of about 5.8 to about 6.9, as determined using a test performed at a temperature of about 34°C to about 40°C, or within any of the pKa ranges listed above. In a particularly preferred embodiment, the pKa of the lipid nanoparticles described herein is determined using a test measuring ionization at a temperature of about 37°C. In some embodiments, the lipid nanoparticles have a pKa of about 5.8 to about 6.9, as determined using a test performed at a temperature of about 37°C, or within any of the pKa ranges listed above.

[0067] In a preferred embodiment, the pKa of the lipid nanoparticles described herein is determined using an ionization assay performed at more than five different pH levels. In some embodiments, the lipid nanoparticles have a pKa of about 5.8 to about 6.9, or within any of the pKa ranges listed above, as determined by an assay performed at more than five different pH levels. In a particularly preferred embodiment, the pKa of the lipid nanoparticles described herein is determined using an ionization assay performed at more than 15 different pH levels. In some embodiments, the lipid nanoparticles have a pKa of about 5.8 to about 6.9, or within any of the pKa ranges listed above, as determined by an assay performed at more than 15 different pH levels.

[0068] In a preferred embodiment, in-situ fluorescence titration, preferably TNS fluorescence titration, is used to determine the pKa of the provided lipid nanoparticles. This assay typically yields an sigmoid curve showing fluorescence (e.g., TNS fluorescence), where the pKa of the provided lipid nanoparticles is determined on a scale of 0 to 1.0, corresponding to a value of 0.5 normalized fluorescence (e.g., normalized TNS fluorescence). For illustrative, non-limiting embodiments, see [link to illustrative embodiment]. Figure 2 In a preferred embodiment, the pKa of the provided lipid nanoparticles is determined using the test described in Example 7. In some embodiments, the lipid nanoparticles have a pKa of about 5.8 to about 6.9, as determined using the test described in Example 7, or within any of the pKa ranges listed above.

[0069] To determine the pKa of the lipid nanoparticles described herein, the lipid nanoparticles preferably comprise phospholipids or pharmaceutically acceptable salts thereof (preferably comprising about 1 mol% to about 20 mol% of the total lipids present in the lipid nanoparticles), ionizable lipids or pharmaceutically acceptable salts thereof (preferably comprising about 30 mol% to about 70 mol% of the total lipids present in the lipid nanoparticles), cholesterol or derivatives thereof (preferably comprising about 0.1 mol% to about 5 mol% of the total lipids present in the lipid nanoparticles), and conjugated lipids (preferably comprising about 0.1 mol% to about 5 mol% of the total lipids present in the lipid nanoparticles). In a particularly preferred embodiment, to determine the pKa of the lipid nanoparticles described herein, the lipid nanoparticles also comprise nucleic acids.

[0070] The provided lipid nanoparticles have been shown to possess in vivo clearance and degradation properties, which are particularly beneficial when the nanoparticles are used to administer nucleic acids to subjects, for example, to provide treatment to subjects and / or edit the subjects' genetic information. Excessively high clearance and degradation rates may prevent the lipid nanoparticles from protecting cargo, such as nucleic acid cargo, and prevent their successful delivery to the target destination within the subject's body. Alternatively, excessively low clearance and degradation rates may be associated with increased toxicity or adverse off-target effects.

[0071] A useful measure of these beneficial clearance and degradation characteristics includes comparing the amount of ionizable lipids in the liver of a subject at different time points following administration of the lipid nanoparticles. Ionizable lipids are the main component of lipid nanoparticles and are typically used in the highest molar amounts of the various lipids present in the particles. For applications requiring more frequent administration, lipid accumulation in tissues can be exacerbated, leading to potential tolerance challenges. Because the role of lipid nanoparticles in delivering nucleic acid payloads is largely completed within hours after administration, there is no need for the lipid components of the particles to remain in tissues for an extended period. Therefore, ionizable lipids capable of rapid clearance from tissues are preferred. The lipid nanoparticles provided exhibit this preferred clearance property. For example, in some embodiments, after administration of the lipid nanoparticles to a subject, the concentration of ionizable lipids in the liver of the subject is less than about 50% of the concentration of ionizable lipids in the liver 7 days after administration, for example, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some implementations, after the lipid nanoparticles are administered to the subject, the concentration of ionizable lipids in the subject's liver 7 days after administration is less than about 50% of the concentration of ionizable lipids present in the total amount of lipid nanoparticles administered to the subject, for example less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.

[0072] Conversely, for some applications, a clearance rate that is too low for lipid nanoparticles may be detrimental. For example, an excessively rapid clearance rate may indicate that the lipid nanoparticles degrade too quickly to effectively and / or efficiently deliver the payload to the target. Advantageously, in some embodiments, after administration of the provided lipid nanoparticles to a subject, the concentration of ionizable lipids in the subject's liver 7 days after administration is greater than about 5% of the concentration of ionizable lipids in the liver 24 hours after administration, for example, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 40%, or greater than about 45%. In some embodiments, after administration of the lipid nanoparticles to a subject, the concentration of ionizable lipids in the subject's liver 7 days after administration is greater than about 5% of the concentration of ionizable lipids present in the total amount of lipid nanoparticles administered to the subject, for example, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, or greater than about 45%.

[0073] In some embodiments, after the lipid nanoparticles are administered to a subject, the concentration of ionizable lipids in the subject's liver 7 days after administration is about 5% to about 50% of the concentration of ionizable lipids in the liver 24 hours after administration or the concentration of ionizable lipids present in the total amount of lipid nanoparticles administered to the subject, for example, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 50%, about 1 5% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, about 40% to about 45%, or about 45% to about 50%.

[0074] The provided lipid nanoparticles can also exhibit advantageous specificity in delivering, for example, nucleic acids to target sites in the subject when administered to the subject. A useful metric for these advantageous target-specific characteristics includes comparing the activity of a protein in a target organ (e.g., the liver) and comparing the activity of a protein in an organ (e.g., the spleen), for example, when the protein is encoded by the mRNA payload of the lipid nanoparticles. For example, when the lipid nanoparticles are used in a method of delivering nucleic acids that encode a protein that is expected to be active in or secreted by the subject's liver cells, the activity or secretion of that protein in the liver can be significantly higher than in another organ (e.g., the spleen), for example, by about 20 times or more. In some embodiments, after the lipid nanoparticles are administered to a subject, the activity and / or expression of the protein in the subject's liver is at least about 20 times higher than the activity or expression of the protein in the subject's spleen, for example, at least about 30 times, at least about 40 times, at least about 60 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 500 times, at least about 700 times, or at least about 1000 times. 1. Phospholipids

[0075] In some aspects, the provided lipid nanoparticles comprise at least one phospholipid or a pharmaceutically acceptable salt thereof, wherein the phospholipid has one or more unsaturated tails and a head group with a positively charged nitrogen. The phospholipids can be selected or designed to provide lipid nanoparticles with desired particle size, stability, and nucleic acid encapsulation capability. Furthermore, lipid nanoparticles comprising the specific phospholipids described herein exhibit, for example, enhanced targeting, optimized clearance, and reduced toxicity and immunostimulation. In some embodiments, the lipid nanoparticles comprise one phospholipid. In other embodiments, the lipid nanoparticles comprise two or more phospholipids, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more phospholipids.

[0076] In some embodiments, the phospholipids of the provided lipid nanoparticles comprise at least one unsaturated tail. In some instances, the phospholipid comprises two unsaturated tails. In other instances, the phospholipid comprises only one unsaturated tail. In some embodiments, at least one of the one or more unsaturated tails of the phospholipid has only one double bond. In some instances, each tail of the phospholipid has only one double bond. In other instances, only one tail of the phospholipid has only one double bond. In some embodiments, at least one of the one or more unsaturated tails of the phospholipid is a cis-unsaturated tail. In some instances, each tail of the phospholipid is a cis-unsaturated tail. In other instances, only one tail of the phospholipid is a cis-unsaturated tail. In some embodiments, at least one of the one or more unsaturated tails of the phospholipid is C10-20 Alkenyl. In some instances, each tail of the phospholipid is C. 10-20 Alkenyl. In other examples, phospholipids have only one tail that is C. 10-20 Alkenyl group.

[0077] In some implementations, the phospholipids of the provided lipid nanoparticles are phosphatidylcholine.For example, phospholipids can be 1-palmitoyl-2-oleoyl-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-diisooleoyl-sn-glycerol-3-phosphate choline, 1,2-bis[(8Z)octadecenoyl]-sn-glycerol-3-phosphate choline, 1,2-dimyristoleoyl-sn-glycerol-3-phosphate choline, 1,2-ditransmyristoleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-ditranspalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dicarboxyl-sn-glycerol-3-phosphate choline, 1,2-ditransoleo ... 3-Chocolate, 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-Di(eicosenoyl)-sn-glycerol-3-phosphate choline, 1,2-Disorhoyl-sn-glycerol-3-phosphate choline (DEPC), 1,2-Diceryl-sn-glycerol-3-phosphate choline, 1,2-Di(docosahexaenoyl)-sn-glycerol-3-phosphate choline, 1-Pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-docosahexaenoyl Oleyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-myristoyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline, 1-(8Z-octadecenoyl)-2-palmitoyl-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-oleoyl-sn- Glyceryl-3-phosphate choline, 1-O-hexadecyl-2-arachidonico-sn-glyceryl-3-phosphate choline, 1-O-hexadecyl-2-(8Z,11Z,14Z-eicostrienoyl)-sn-glyceryl-3-phosphate choline, 1-O-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicospentanoyl)-sn-glyceryl-3-phosphate choline, 1-O-hexadecyl-2-docosahexaenoyl-sn-glyceryl-3-phosphate choline, 1-O-hexadecyl-2-O-(9Z-octadecenyl)-sn-glyceryl-3-phosphate choline, 1,2-di-O-(9Z-octadecenyl)-sn-glyceryl-3-phosphate choline, or any combination of two or more thereof.In some instances, at least one phospholipid in the lipid nanoparticles is phosphatidylcholine. In some instances, every phospholipid in the lipid nanoparticles is phosphatidylcholine.

[0078] In some embodiments, the phospholipids of the provided lipid nanoparticles are phosphatidylethanolamines. For example, the phospholipids may be 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2- Oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine, 1-O-hexadecyl-2-O-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine, or any combination of two or more thereof. In some examples, at least one phospholipid of the lipid nanoparticle is a phosphatidylethanolamine. In some examples, each phospholipid of the lipid nanoparticle is a phosphatidylethanolamine.

[0079] In some embodiments, the phospholipids of the provided lipid nanoparticles include at least one phosphatidylcholine and at least one phosphatidylethanolamine.

[0080] In addition to the type of phospholipid, the amount of phospholipid contained in the provided lipid nanoparticles also demonstrates an advantageous characteristic for the particles. The phospholipids present in the provided lipid nanoparticles can account for, for example, from about 3 mol% to about 20 mol% of the total lipids present in the particles, for example, from about 5 mol% to about 20 mol%, from about 8 mol% to about 20 mol%, from about 10 mol% to about 20 mol%, from about 3 mol% to about 15 mol%, from about 5 mol% to about 15 mol%, from about 8 mol% to about 15 mol%, from about 10 mol% to about 15 mol%, or from about 8 mol% to about 12 mol%. In some embodiments, the phospholipids present in the lipid nanoparticles account for about 5 mol%, from about 6 mol%, from about 7 mol%, from about 8 mol%, from about 9 mol%, from about 10 mol%, from about 11 mol%, from about 12 mol%, from about 13 mol%, from about 14 mol%, or from about 15 mol% of the total lipids present in the particles. 2. Ionizable lipids

[0081] The provided lipid nanoparticles comprise at least one ionizable lipid or a pharmaceutically acceptable salt thereof, wherein the ionizable lipid is a nitrogen-containing silyl ether. The ionizable lipid can be selected or designed to provide lipid nanoparticles with desired particle size, stability, and nucleic acid encapsulation capability. Furthermore, lipid nanoparticles comprising the specific ionizable lipids described herein exhibit, for example, enhanced targeting, optimized clearance, and reduced toxicity and immunostimulation. In some embodiments, the lipid nanoparticles comprise one ionizable lipid. In other embodiments, the lipid nanoparticles comprise two or more ionizable lipids, for example, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more ionizable lipids.

[0082] The ionizable lipids provided in the lipid nanoparticles typically have the following formula: (I), R in equation (I) 1 and R 2 They can be hydrogen or carbon independently. 1-4 Alkyl or 2- to 4-membered heteroalkyl, wherein the alkyl and heteroalkyl optionally have one or more substituents, and wherein said substituents may each be independently a hydroxyl, C 1-6 Hydroxyalkyl or fluorine. Or, R 1 and R 2 They can combine with the nitrogen to which they are attached to form 3- to 8-membered heterocyclic groups optionally having one or more substituents, wherein each substituent can be an independent hydroxyl group, C... 1-6Hydroxyalkyl or fluorine. R of formula (I) 3 R 4 and R 5 Each can be an C that optionally has one or more substituents, independently. 5-20 Alkyl, C 5-20 alkenyl, C 5-20 alkynyl or C 5-12 Cycloalkyl, wherein the substituents may each be C10 and C20 independently. 1-6 Alkyl or C 2-6 Alkenyl. R of formula (I) 6 and R 7 They can be hydrogen and C independently. 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group. Or, R 6 and R 7 They can combine with the carbon atoms they are attached to form cyclopropyl or cyclobutyl groups. R in formula (I) 8 and R 9 They can be hydrogen and C independently. 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group. Or, R 8 and R 9 They can combine with the carbon atoms they are attached to form cyclopropyl or cyclobutyl groups. R in formula (I) 10 and R 11 They can be hydrogen and C independently. 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne group. Or, R 10 and R 11 They can combine with the carbon atoms they are attached to to form cyclopropyl or cyclobutyl groups. R 12 and R 13 Each C can be hydrogen independently or optionally have one or more substituents. 1-6 Alkyl group, wherein the substituents may each be hydroxyl, C, or C6. 1-6 Hydroxyalkyl or fluorine. Or, R 12 and R 13 They can combine with the carbon atoms they are attached to form cyclopropyl or cyclobutyl groups. X in formula (I) 1 X 2 and X 3 Each can be an independent covalent bond, C 1-6 Alkylene, C 1-6 imide or C 1-6 Alynyl group. The subscript n in formula (I) can be an integer from 2 to 10. In one aspect, this disclosure also provides compounds having the structure of formula (I) or a salt thereof (e.g., a pharmaceutically acceptable salt) that are ionizable lipid compounds.

[0083] In some implementations, R of formula (I) 1 It is hydrogen. In some implementations, R in formula (I) 2 It is hydrogen. In some instances, R 1 and R 2 Each is hydrogen. In some implementations, R 1 It is C 1-4 Alkyl group. In some embodiments, R 2 It is C 1-4 Alkyl group. In some embodiments, R 1 and R 2 One of them is hydrogen, R 1 and R 2 The other one is C. 1-4 Alkyl group. In some instances, R 1 and R 2 Each is C 14 Alkyl group. R 1 and / or R 2 Each of these can be independently, for example, methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, or tert-butyl. In some embodiments, R 1 and R 2 Each is C 1-3 Alkyl group. In some instances, R 1 and R 2 Each can be independently methyl, ethyl, or isopropyl. In some instances, R 1 and R 2 Each is a methyl group.

[0084] In some implementations, R of formula (I) 1 and R 2 They combine with the nitrogen atoms to form 3- to 8-membered heterocyclic groups. In some embodiments, R 1 and R 2 The attached nitrogen atom is the only heteroatom in the 3- to 8-membered heterocyclic group. In some instances, R 1 and R 2 They combine with the nitrogen atoms to form pyrrolidinyl groups. In some instances, R 1 and R 2 They combine with the nitrogen atoms to form nitrogen-containing heterocyclic butyl groups.

[0085] In some implementations, R of formula (I) 3 R 4 and R 5 At least one of them is C 5-20 Alkyl group. In some embodiments, R of formula (I) 3 R 4 and R5 At least two of them are C independently. 5-20 Alkyl group. In some embodiments, R of formula (I) 3 R 4 and R 5 Each is C independently 5-20 Alkyl group. In some embodiments, R of formula (I) 3 R 4 and R 5 At least one of them is C 5-20 Alkenyl. In some embodiments, R of formula (I) 3 R 4 and R 5 At least two of them are C independently. 5-20 Alkenyl. In some embodiments, R of formula (I) 3 R 4 and R 5 Each is C independently 5-20 Alkenyl. In some embodiments, R of formula (I) 3 R 4 and R 5 At least one of them is C 5-20 Alkyne group. In some embodiments, R of formula (I) 3 R 4 and R 5 At least two of them are C independently. 5-20 Alkyne group. In some embodiments, R of formula (I) 3 R 4 and R 5 Each is C independently 5-20 Alkyne group. In some embodiments, R of formula (I) 3 R 4 and R 5 At least one of them is an optional substituted C 5-12 Cycloalkyl. In some embodiments, R of formula (I) 3 R 4 and R 5 At least two of them are independently optional substitutions of C. 5-12 Cycloalkyl. In some embodiments, R of formula (I) 3 R 4 and R 5 Each is an optional substitution of C. 5-12 Cycloalkyl.

[0086] In some implementations, R of formula (I) 3 R 4 and R 5 One or more of them are C 6-12Alkenyl. In some embodiments, R of formula (I) 3 R 4 and R 5 At least one of them is cis-C 6-12 Alkenyl. In some embodiments, R 3 R 4 and R 5 At least two of them are cis C 6-12 Alkenyl. In some embodiments, R 3 R 4 and R 5 Each of them is a cis-C 6-12 Alkenyl. In some embodiments, R of formula (I) 3 R 4 and R 5 At least one of them is a cis-C with only one double bond. 6-12 Alkenyl. In some embodiments, R 3 R 4 and R 5 At least two of them are cis-C with only one double bond. 6-12 Alkenyl. In some embodiments, R 3 R 4 and R 5 Each of them is a cis-C with only one double bond. 6-12 Alkenyl. For R 3 R 4 and / or R 5 Each of these, only one double bond, can be independently located in, for example, cis-C 6-12 The alkenyl group at the 2-, 3-, 4-, or 5-position. In some instances, R 3 R 4 and R 5 At least one of them is cis-non-3-en-1-yl. In some instances, R 3 R 4 and R 5 At least two of them are cis-non-3-en-1-yl. In some instances, R 3 R 4 and R 5 Each of them is cis-non-3-en-1-yl. In some instances, R 3 R 4 and R 5 At least one of them is cis-oct-3-en-1-yl. In some instances, R 3 R 4 and R 5 At least two of them are cis-oct-3-en-1-yl. In some instances, R 3 R4 and R 5 Each of them is cis-oct-3-en-1-yl.

[0087] In some implementations, R of formula (I) 6 R 8 and R 10 At least one of them is hydrogen. In some embodiments, R 6 R 8 and R 10 At least two of them are hydrogen. In some implementations, R 6 R 8 and R 10 Each of them is hydrogen. In some implementations, R 6 R 8 and R 10 At least one of them is C 1-20 Alkyl group. In some embodiments, R 6 R 8 and R 10 At least two of them are C 1-20 Alkyl group. In some embodiments, R 6 R 8 and R 10 Each one in is C 1-20 Alkyl group. In some embodiments, R 6 R 8 and R 10 At least one of them is C 1-4 Alkyl group. In some embodiments, R 6 R 8 and R 10 At least two of them are C 1-4 Alkyl group. In some embodiments, R 6 R 8 and R 10 Each of them is C 1-4 Alkyl group. R 6 R 8 and / or R 10 Each of these can be independently, for example, methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, or tert-butyl. In some embodiments, R 6 R 8 and R 10 Each is independently hydrogen, methyl, ethyl, or butyl. In some embodiments, R 6 R 8 and R 10 At least one of them is C 1-20 Alkenyl. In some embodiments, R 6 R 8 and R10 At least two of them are C 1-20 Alkenyl. In some embodiments, R 6 R 8 and R 10 Each one in is C 1-20 Alkenyl. In some embodiments, R 6 R 8 and R 10 At least one of them is cis-dec-4-en-1-yl. In some embodiments, R 6 R 8 and R 10 At least two of them are cis-dec-4-en-1-yl. In some embodiments, R 6 R 8 and R 10 Each of them is cis-dec-4-en-1-yl.

[0088] In some implementations, R of formula (I) 7 R 9 and R 11 At least one of them is hydrogen. In some embodiments, R 7 R 9 and R 11 At least two of them are hydrogen. In some implementations, R 7 R 9 and R 11 Each of them is hydrogen. In some implementations, R 7 R 9 and R 11 At least one of them is C 1-20 Alkenyl. In some embodiments, R 7 R 9 and R 11 At least two of them are C 1-20 Alkenyl. In some embodiments, R 7 R 9 and R 11 Each one in is C 1-20 Alkenyl. In some embodiments, R 7 R 9 and R 11 At least one of them is C 1-20 Alkyne group. In some embodiments, R 7 R 9 and R 11 At least two of them are C 1-20 Alkyne group. In some embodiments, R 7 R 9 and R 11 Each one in is C1-20 Alkyne group.

[0089] The specific size of the hydrophobic region of the ionizable lipid in the provided lipid nanoparticles has been shown to provide advantageous properties for the particles. In some embodiments, R of formula (I) 3 R 6 and R 7 The total carbon content is between 7 and 12. In some embodiments, R in formula (I) 4 R 8 and R 9 The total carbon content is between 7 and 12. In some embodiments, R in formula (I) 5 R 10 and R 11 The total carbon content is between 7 and 12. In some implementations, R 3 R 6 and R 7 Total carbon in, R 4 R 8 and R 9 The total amount of carbon and R 5 R 10 and R 11 The total number of carbon atoms in each is individually or identically 7 to 12, for example 7 to 11, 7 to 10, 7 to 9, 7 to 8, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 12, 9 to 11, 9 to 10, 10 to 12, 10 to 11, or 11 to 12. In some embodiments, R 3 R 6 and R 7 The total amount of carbon in R 4 R 8 and R 9 The total amount of carbon and R 5 R 10 and R 11 The total number of carbon atoms in each atom is 7, 8, 9, 10, 11, or 12, either independently or in the same way.

[0090] In some implementations, R of formula (I) 12 and R 13 Each is hydrogen. In some implementations, at least one R 12 Or at least one R 13 It is C 1-6 Alkyl group. In some embodiments, R 12 Or R 13 Only one of them is C. 1-6 Alkyl group. R as an alkyl group 12 Or R 13Each of these can be, independently, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, 2-methylbutyl, pent-2-yl, 3-methylbutyl-2-yl, pent-3-yl, neopentyl, tert-pentyl, hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, hex-2-yl, 2,3-dimethylbutyl, 4-methylpent-2-yl, 3-methylpent-2-yl, 2-ethylbutyl, hex-3-yl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, or 2-methylpent-2-yl.

[0091] In some implementations, X of formula (I) 1 X 2 and X 3 At least one of them is a covalent bond. In some implementations, X 1 X 2 and X 3 At least two of them are covalent bonds. In some implementations, X 1 X 2 and X 3 Each of them is a covalent bond. In some implementations, X 1 X 2 and X 3 At least one of them is C 1-6 Alkylene. In some embodiments, X 1 X 2 and X 3 At least two of them are C 1-6 Alkylene. In some embodiments, X 1 X 2 and X 3 Each one in is C 1-6 Alkylene. In one example, X 1 X 2 and X 3 Each of them is a methylene group. In some implementations, X 1 X 2 and X 3 At least one of them is C 1-6 Alkenyl group. In some embodiments, X 1 X 2 and X 3 At least two of them are C 1-6 Alkenyl group. In some embodiments, X of formula (I) 1 X 2 and X 3 Each one in is C 1-6 Alkenyl group. In some embodiments, X 1 X 2 and X 3At least one of them is C 1-6 Ethyne group. In some implementations, X 1 X 2 and X 3 At least two of them are C 1-6 Alynyl group. In some embodiments, X of formula (I) 1 X 2 and X 3 Each one in is C 1-6 Alynyl group.

[0092] The specific length of the hydrophilic head group of the ionizable lipid in the provided lipid nanoparticles is shown to provide advantageous properties for the particles. In some embodiments, the subscript n of formula (I) is 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10. In some instances, n is 2. In some instances, n is 3. In some instances, n is 4. In some instances, n is 5. In some instances, n is 6. In some instances, n is 7. In some instances, n is 8. In some instances, n is 9. In some instances, n is 10. In some instances, n is between 6 and 8.

[0093] In some embodiments, the ionizable lipids of this disclosure comprise a racemic mixture. In other embodiments, the ionizable lipids of this disclosure comprise a mixture of one or more diastereomers. In some embodiments, the ionizable lipids of this disclosure comprise at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% enantiomer excess. In some other embodiments, the ionizable lipids of this disclosure are enriched with one diastereomer, such that the lipid comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomer excess. In some further embodiments, the ionizable lipids of this disclosure are chiral pure (e.g., comprising a single optical isomer). In a further embodiment, the ionizable lipids of this disclosure are enriched with one optical isomer (e.g., an optically active isomer), such that the lipid comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomer excess. This disclosure provides for the synthesis of any ionizable lipid disclosed herein, said ionizable lipid being a racemic mixture or in optically pure form.

[0094] In some embodiments, the ionizable lipids of the provided lipid nanoparticles have the following formula: (Ia), R of equation (Ia) 3 R 4 and R 5 As defined above for formula (I). In one aspect, this disclosure also provides compounds having the structure of formula (Ia) or a salt thereof (e.g., a pharmaceutically acceptable salt) that are ionizable lipid compounds.

[0095] In some embodiments, the ionizable lipids of the provided lipid nanoparticles include one or more of the following: , , , , , , , , , , , , , , , , , , , , , , and . On the one hand, this disclosure provides compounds having any of these structures or salts thereof, such as pharmaceutically acceptable salts.

[0096] In addition to the type of ionizable lipids, the amount of ionizable lipids contained in the provided lipid nanoparticles also demonstrates an advantageous characteristic for the particles. The ionizable lipids present in the provided lipid nanoparticles may account for, for example, about 30 mol% to about 80 mol%, about 40 mol% to about 80 mol%, about 50 mol% to about 80 mol%, about 30 mol% to about 70 mol%, about 40 mol% to about 70 mol%, about 50 mol% to about 70 mol%, about 45 mol% to about 80 mol%, about 45 mol% to about 70 mol%, about 45 mol% to about 65 mol%, about 45 mol% to about 60 mol%, about 45 mol% to about 55 mol%, about 50 mol% to about 65 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 65 mol%. In some embodiments, the ionizable lipids present in the lipid nanoparticles account for about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, or about 65 mol% of the total lipids present in the particles. 3. Cholesterol

[0097] In some embodiments, the provided lipid nanoparticles also comprise cholesterol or one or more derivatives thereof. Cholesterol or cholesterol derivatives can be selected or configured to provide lipid nanoparticles with desired particle size, stability, and nucleic acid encapsulation capability. The lipid nanoparticles may comprise, for example, cholesterol, cholesterol ketone, cholesterol ketone, coprostinol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, cholesterol hydroxyethyl ether, cholesterol hydroxyhexyl ether, cholesterol stearate, cholesterol oleate, 7-β-hydroxycholesterol, 7-α-hydroxycholesterol, 4-β-hydroxycholesterol, cholesterol PEG, β-sitosterol, or any combination thereof. In some instances, the lipid nanoparticles contain cholesterol but substantially no cholesterol derivatives.

[0098] In addition to the type of cholesterol or cholesterol derivatives, the amount contained in the provided lipid nanoparticles also demonstrates an advantageous characteristic for the particles. The cholesterol or cholesterol derivatives present in the provided lipid nanoparticles may account for, for example, about 10 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 60 mol%, about 40 mol% to about 60 mol%, about 20 mol% to about 50 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, about 20 mol% to about 45 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 30 mol% to about 40 mol%. In some instances, cholesterol or cholesterol derivatives present in lipid particles account for approximately 20 mol%, approximately 25 mol%, approximately 30 mol%, approximately 35 mol%, approximately 40 mol%, approximately 45 mol%, or approximately 50 mol% of the total lipids present in the particles. 4. Conjugated lipids

[0099] In some embodiments, the provided lipid nanoparticles further comprise one or more conjugated lipids. The conjugated lipids can be selected or configured to control particle size during particle formation and / or prevent particle aggregation through spatially stable LNPs. The conjugated lipids of the lipid nanoparticles can be located on the particle surface, with the hydrophilic polymer oriented outwards and in contact with the aqueous environment, and the lipid component embedded within the particle to anchor it in place.

[0100] Suitable lipid conjugations for the provided lipid nanoparticles include, but are not limited to, PEG-lipid conjugations, ATTA-lipid conjugations, cationic polymer-lipid conjugations (CPL), polyoxazoline (POZ)-lipids, polysarcosine (pSAR)-lipids, and mixtures thereof. In some embodiments, the lipid nanoparticles comprise a PEG-lipid conjugation or an ATTA-lipid conjugation, optionally together with a CPL.

[0101] In a preferred embodiment, the conjugated lipid is a PEG-lipid. Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyl (PEG-DAA) as described, for example, in International Patent Application Publication No. WO 05 / 026372; PEG coupled to diacylglycerol (PEG-DAG) as described, for example, in U.S. Patent Application Publications Nos. 2003 / 0077829 and 2005 / 0008689; PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE); PEG conjugated to ceramides as described, for example, in U.S. Patent No. 5,885,613; PEG conjugated to cholesterol or its derivatives; mPEG(2000)-N,N-bistetradecylacetamide (ALC-0159); mPEG(2000)-stearate; 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG). This includes any PEG lipids and mixtures thereof described in R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristoyloxypropyl-3-amine (PEG-C-DOMG), International Patent Application Publication No. WO 2022 / 133344. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. The lipid nanoparticles provided may comprise one or more methyl-terminated PEG-lipids, one or more unterminated PEG-lipids, or combinations thereof.

[0102] PEG is a linear, water-soluble polymer of repeating ethylene glycol units. PEG is classified by its molecular weight; for example, PEG2000 has an average molecular weight of about 2,000 Daltons, and PEG 5000 has an average molecular weight of about 5,000 Daltons. Due to its polydispersity, this molecular weight is an average molecular weight. PEG is commercially available from Sigma Chemical Co. and other companies, and includes, for example, the following: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimide succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-tribenzoate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs (e.g., mPEG (20 kDa) amines) such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 are also used to prepare the PEG-lipid conjugates disclosed herein. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. Additionally, monomethoxy polyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0103] The PEG moiety of the PEG-lipid conjugate described herein may contain an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In some instances, the PEG moiety has an average molecular weight of about 750 Daltons to about 5,000 Daltons (e.g., about 1,000 Daltons to about 5,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons to about 3,000 Daltons, about 750 Daltons to about 2,000 Daltons, etc.). In a preferred embodiment, the PEG moiety has an average molecular weight of about 2,000 Daltons or about 750 Daltons.

[0104] In some instances, PEG may optionally be substituted with alkyl, alkoxy, acyl, or aryl groups. PEG may be directly conjugated to lipids or may be linked to lipids via a linker motif. Any linker motif suitable for coupling PEG to lipids may be used, including, for example, non-ester-containing linker motifs and ester-containing linker motifs. In a preferred embodiment, the linker motif is a non-ester-containing linker motif. As used herein, the term "non-ester-containing linker motif" refers to a linker motif that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable non-ester-containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), urethane (NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl ((O)CCH2CH2C(O)-), succinamide (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof (such as linkers containing both urethane and amide linker moieties). In a preferred embodiment, the urethane linker is used to couple PEG to a lipid.

[0105] In other embodiments, the ester-containing linker portion is used to couple PEG to the lipid. Suitable ester-containing linker portions include, for example, carbonates (-OC(O)O-), succinyl groups, phosphate esters (-O-(O)POH-O-), sulfonates, and combinations thereof.

[0106] In addition to the foregoing, those skilled in the art will readily recognize that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, ATTA, cationic polymers, polyoxazoline (POZ), polysarcosine, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, polyethyl ethylene phosphate, and derived celluloses such as hydroxymethyl cellulose or hydroxyethyl cellulose.

[0107] The conjugated lipids of the provided lipid nanoparticles may include one or more of the following: polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, or mixtures thereof. In a preferred embodiment, the nucleic acid-lipid particles comprise PEG-lipid conjugates or ATTA-lipid conjugates. The conjugated lipids may comprise PEG-lipids, including, for example, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dispalmityloxypropyl (C16), PEG-distearateloxypropyl (C18), or mixtures thereof.

[0108] Further PEG-lipid conjugates suitable for the provided lipid nanoparticles include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol ester (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Further PEG-lipid conjugates suitable for this disclosure include, but are not limited to, 1-[8'-(1,2-dimyristoyl-3-propoxy)-formamido-3',6'-dioxaoctyl]carbamoyl-w-methylpoly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0109] In addition to the type of conjugated lipid, the amount of conjugated lipid contained in the provided lipid nanoparticles also shows to provide advantageous characteristics for the particles. The conjugated lipids present in the provided lipid nanoparticles may account for, for example, about 0.1 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 1 mol% to about 10 mol%, about 1.5 mol% to about 10 mol%, about 2 mol% to about 10 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 10 mol%, about 4 mol% to about 10 mol%, about 5 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 0.3 mol% to about 5 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 5 mol%, about 1.5 mol% to about 5 mol%, about 2 mol% to about 5 mol%, about 2.5 mol% to about 5 mol%, about 3 mol% to about 5 mol%, about 0.1 mol% to about 3 mol%, about 0.5 mol% to about 3 mol%, about 1 mol% to about 3 mol%. mol%, about 1.5 mol% to about 3 mol%, about 2 mol% to about 3 mol%, about 2.2 mol% to about 3 mol%, or about 2.5 mol% to about 3 mol%. In some instances, the conjugated lipids present in the lipid nanoparticles account for about 0.1 mol%, about 0.5 mol%, about 1 mol%, about 1.2 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.8 mol%, about 2 mol%, about 2.2 mol%, about 2.5 mol%, or about 3 mol% of the total lipids present in the particles.

[0110] By controlling the composition and concentration of the conjugated lipids in the provided lipid nanoparticles, the rate at which the conjugated lipids exchange out of the lipid nanoparticles can be controlled, thereby controlling the rate of lipid nanoparticle fusion. For example, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as the conjugated lipid, the rate of lipid nanoparticle fusion can be altered, for example, by changing the concentration of the lipid conjugate, by changing the molecular weight of PEG, or by changing the chain length and saturation of the acyl chain on the phosphatidylethanolamine or ceramide. Additionally, other variables, such as pH, temperature, and ionic strength, can be used to alter and / or control the rate of lipid nanoparticle fusion. Other methods for controlling the rate of lipid nanoparticle fusion will become apparent to those skilled in the art upon reading this disclosure. 5. Nucleic acid

[0111] In some embodiments, the provided lipid nanoparticles are bound to nucleic acids. The composition of the lipid nanoparticles can be selected or configured such that the nucleic acids (when present in the lipid nanoparticles) are resistant to degradation by nucleases in aqueous solutions. The nucleic acids can be administered alone in the lipid nanoparticles described herein, or in combination with lipid particles containing peptides, polypeptides, or small molecules such as conventional pharmaceuticals (e.g., co-administration). In some embodiments, at least 50% of the nucleic acid is encapsulated within the lipid nanoparticles; in one embodiment, at least 75% of the nucleic acid is encapsulated within the lipid nanoparticles; in one embodiment, at least 90% of the nucleic acid is encapsulated within the lipid nanoparticles; and in one embodiment, the nucleic acid is completely encapsulated within the lipid nanoparticles.

[0112] In some embodiments, this disclosure provides lipid nanoparticle formulations comprising a plurality of lipid nanoparticles or groups of lipid nanoparticles. In some embodiments, nucleic acids are completely encapsulated within the lipid portion of the lipid nanoparticles, such that approximately 30% to 100%, approximately 40% to 100%, approximately 50% to 100%, approximately 60% to 100%, approximately 70% to 100%, approximately 80% to 100%, approximately 90% to 100%, approximately 30% to 95%, approximately 40% to 95%, approximately 50% to 95%, approximately 60% to 95%, approximately 70% to 95%, approximately 80% to 95%, approximately... The lipid nanoparticles in the plurality of lipid nanoparticles or groups of lipid nanoparticles comprising 85% to 95%, about 90% to 95%, about 30% to 90%, about 40% to 90%, about 50% to 90%, about 60% to 90%, about 70% to 90%, about 80% to 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (or any part or range thereof) have nucleic acids encapsulated therein.

[0113] The nucleic acids present in the lipid nanoparticles described herein may include or be composed of any known form of nucleic acid. The nucleic acids used herein may be single-stranded DNA or RNA (e.g., ssDNA or ssRNA), or double-stranded DNA or RNA (e.g., dsDNA or dsRNA), or DNA-RNA hybrids. Single-stranded nucleic acids include, for example, mRNA, guide RNA (gRNA), antisense oligonucleotides, ribozymes, mature miRNAs, self-amplifying RNA (SAM), and triplet-forming oligonucleotides. Examples of double-stranded DNA include, for example, structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include, for example, siRNA and other RNAi agents, such as aiRNA and precursor miRNA.

[0114] Nucleic acids can have different lengths, typically depending on their specific form. For example, in certain embodiments, the length of mRNA, plasmid, or gene can be from about 1,000 to about 100,000 nucleotides. In certain embodiments, the length of oligonucleotides can range from about 10 to about 100 nucleotides. In various related embodiments, the lengths of single-stranded, double-stranded, and triple-stranded oligonucleotides can all range from about 10 to about 60 nucleotides, about 15 to about 60 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides.

[0115] In some embodiments, the nucleic acids of the provided lipid nanoparticles comprise or consist of modified or substituted polynucleotides or oligonucleotides. In certain cases, modified or substituted polynucleotides and oligonucleotides may be superior to their natural forms due to their properties, such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0116] In some embodiments, the nucleic acid is an RNA molecule containing at least one modified nucleotide. In some preferred embodiments, the RNA molecule contains one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in its double-stranded region. In some cases, the RNA molecule (e.g., siRNA) has a double-stranded region containing about 1% to about 100% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of modified nucleotides in the double-stranded region. In a preferred embodiment, less than about 25% (e.g., less than about 25%, 20%, 15%, 10%, or 5%) or about 1% to about 25% (e.g., about 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, or 10%-20%) of the nucleotides in the double-stranded region contain modified nucleotides.

[0117] In other embodiments, the RNA molecule comprises modified nucleotides, including but not limited to 2'-O-methyl (2'Ome) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxynucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In a preferred embodiment, the RNA comprises 2'Ome nucleotides (e.g., 2'Ome purine and / or pyrimidine nucleotides), such as 2'Ome-guanosine nucleotides, 2'Ome-uridine nucleotides, 2'Ome-adenosine nucleotides, 2'Ome-cytosine nucleotides, and mixtures thereof. In some cases, the RNA does not contain 2'Ome-cytosine nucleotides. In other embodiments, the RNA comprises a hairpin loop structure.

[0118] RNA may contain modified nucleotides in one strand (i.e., sense or antisense) or both strands of the double-stranded region of the RNA molecule. Preferably, uridine and / or guanosine nucleotides are modified at selective sites in the double-stranded region of the RNA double helix. Regarding uridine nucleotide modification, at least one, two, three, four, five, six, or more uridine nucleotides in the sense and / or antisense strands may be modified uridine nucleotides, such as 2'Ome-uridine nucleotides. In some embodiments, each uridine nucleotide in the sense and / or antisense strand is a 2'Ome-uridine nucleotide. Regarding guanosine nucleotide modification, at least one, two, three, four, five, six, or more guanosine nucleotides in the sense and / or antisense strands may be modified guanosine nucleotides, such as 2'Ome-guanosine nucleotides. In some embodiments, each guanosine nucleotide in the sense and / or antisense strand is a 2'Ome-guanosine nucleotide.

[0119] In some embodiments, at least one, two, three, four, five, six, seven, or more 5'-GU-3' motifs in the RNA sequence may be modified, for example, by introducing mismatches to eliminate the 5'-GU-3' motifs and / or by introducing modified nucleotides such as 2'Ome nucleotides. The 5'-GU-3' motifs may be located in the sense strand, antisense strand, or both of the RNA sequence. The 5'-GU-3' motifs may be adjacent to each other, or alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.

[0120] In some embodiments, the nucleic acids of the provided lipid nanoparticles comprise or are composed of oligonucleotides. Oligonucleotides are generally classified as deoxyribonucleotides or ribonucleotides. Deoxyribonucleotides consist of a 5-carbon sugar called deoxyribose, which is covalently linked to a phosphate ester at the 5' and 3' carbons to form an alternating unbranched polymer. Ribonucleotides consist of a similar repeating structure in which the 5-carbon sugar is ribose.

[0121] In certain embodiments, the oligonucleotide (or its chain) specifically hybridizes or complements a target polynucleotide sequence. As used herein, the terms “specifically hybridizable” and “complementary” indicate a sufficient degree of complementarity to result in stable and specific binding between the DNA or RNA target and the oligonucleotide. It should be understood that the oligonucleotide does not need to be 100% complementary to the target nucleic acid sequence to which it is to specifically hybridize. In some embodiments, the oligonucleotide is specifically hybridizable and there is a sufficient degree of complementarity to avoid non-specific binding to non-target sequences under conditions where the desired specific binding is required, i.e., under physiological conditions in vivo assays or therapeutic treatments, or under conditions where it is measured in vitro. Therefore, the oligonucleotide may include one, two, three, or more base substitutions compared to the region of the gene or mRNA sequence to which it is targeted or specifically hybridizes. a) mRNA

[0122] Certain embodiments provide compositions comprising the lipid nanoparticles described herein and methods of using them for expressing one or more mRNA molecules (e.g., mixtures of mRNA molecules) in cells, such as cells within an organism (e.g., a mammal, such as a human). The mRNA molecules encode one or more polypeptides expressed within the cell. Lipid particle formulations comprising the mRNA molecules described herein can be used in a variety of applications, including protein replacement therapy, vaccines, cancer immunotherapy, and gene editing. In a particular embodiment, the lipid nanoparticles comprise mRNA delivered (e.g., specifically delivered) to the liver for expression therein, for example, for editing genomic information within hepatocytes. In some embodiments, the lipid particles described herein are used to treat diseases in which expression of a polypeptide encoded by the mRNA molecule in a diseased organism (e.g., a mammal, such as a human) improves one or more symptoms of the disease. The compositions and methods described herein are particularly useful for treating human diseases caused by the absence or reduction of functional polypeptides in the human body. In other embodiments, the lipid particles described herein are used as vaccines to prevent diseases in which expression of a polypeptide encoded by the mRNA molecule in an organism (e.g., a mammal, such as a human) triggers immunity against the disease. The compositions and methods described herein can be used, for example, to prevent infectious diseases caused by pathogens by expressing antigenic peptides (e.g., mRNA molecules encoding viral proteins such as S (spike) protein, E (enveloping) protein, M (membrane) protein, or N (nucleocapsid) protein, or antigenic fragments thereof) to generate an immune response in an organism (e.g., a mammal, such as a human) by stimulating the adaptive immune system to produce antibodies targeting pathogens such as viruses (e.g., coronaviruses, such as SARS-CoV-2). In other embodiments, the lipid particles described herein are used as vaccines for treating diseases, wherein expression of peptides encoded by mRNA molecules in an organism (e.g., a mammal, such as a human) elicits an immune response against diseased cells. The compositions and methods described herein are particularly suitable for treating cancer by stimulating an adaptive immune response to generate antibodies that target and destroy cancer cells by expressing antigenic peptides (e.g., mRNA molecules encoding tumor-specific antigens, or antigenic fragments thereof).

[0123] In some embodiments, the mRNA molecule is completely encapsulated within lipid particles. In some instances, the provided lipid nanoparticles or populations of provided nanoparticles comprise a mixture of one or more types of mRNA with one or more other types of RNA (e.g., gRNA, siRNA, miRNA, etc.). In some specific instances that can be used for editing genomic information, the lipid nanoparticles or populations of different lipid nanoparticles contain at least one type of mRNA and at least one type of gRNA. In such instances, the mRNA and gRNA may be within the same lipid nanoparticle or within different lipid nanoparticles. Similarly, for formulations containing a mixture of mRNAs, different types of mRNAs present in the mixture (e.g., mRNAs with different sequences) may be co-encapsulated in the same particle, or each type of mRNA present in the mixture may be encapsulated in a separate particle. The mRNA mixture may be formulated in the particles described herein using a mixture of two or more mRNAs (each having a unique sequence) of the same, similar, or different concentrations or molar ratios. In one embodiment, a mixture of mRNAs (corresponding to multiple mRNAs with different sequences) is formulated using the same, similar, or different concentrations or molar ratios of each mRNA species, and different types of mRNAs are co-encapsulated in the same particle. In another embodiment, each type of mRNA present in the mixture is encapsulated in separate particles at the same, similar, or different concentrations or molar ratios of the mRNAs, and the resulting particles (each containing a different mRNA payload) are administered individually (e.g., at different times according to a preventive or therapeutic regimen), or combined and administered together as a single unit dose (e.g., using a pharmaceutically acceptable carrier). In a particular embodiment, the lipid particles are serum stable, resistant to nuclease degradation, and substantially non-toxic to mammals such as humans. (1) Modification of mRNA

[0124] The mRNA molecules present in the provided lipid nanoparticles may contain one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in cells in which it is introduced, compared to the corresponding unmodified mRNA.

[0125] In some embodiments, the modified nucleosides include pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-tauronic acid methyluridine, 1-tauronic acid methyl-2-thio-uridine, 1-tauronic acid methyl-2-thio-uridine, and 1-tauronic acid methyl-2-thio-uridine. 4-thiouridine, 5-methyluridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.

[0126] In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine Isocytidine, 4-thio-1-methyl-1-denitro-pseudo-isocytidine, 1-methyl-l-denitro-pseudo-isocytidine, zabrane, 5-aza-zabrane, 5-methyl-zabrane, 5-aza-2-thio-zabrane, 2-thio-zabrane, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-isocytidine and 4-methoxy-1-methyl-pseudo-isocytidine.

[0127] In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-azaadenine, 7-deadenine-2-aminopurine, 7-deadenine-8-azaa-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N... 6-Isopentenyl adenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyl adenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine.

[0128] In some embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine. The α-thiosubstituted phosphate moiety is provided to confer stability to the RNA polymer via non-natural thiophosphate backbone bonding. Thiophosphate RNA exhibits increased nuclease resistance and thus a longer half-life in the cellular environment. It is anticipated that thiophosphate-linked nucleic acids will also reduce innate immune responses through weaker binding / activation of cellular innate immune molecules.

[0129] In some implementations, for example, where precise protein production timing is required, it is desirable to degrade the modified nucleic acid introduced into the cell within the cell. Therefore, this disclosure provides a modified nucleic acid containing a degradation domain capable of being acted upon in a directed manner within the cell.

[0130] In other embodiments, the modified nucleosides include inosine, 1-methyl-inosine, wyoside, wyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. (2) Optional mRNA components

[0131] In a further embodiment, the mRNA molecule present in the provided lipid nanoparticles may include other optional components. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intronic nucleotide sequences, internal ribosome entry sites (IRES), caps, and poly-A tails. For example, a 5' untranslated region (UTR) and / or a 3' UTR may be included, wherein either or both may independently contain one or more different nucleotide modifications. In such embodiments, nucleotide modifications may also be present in translatable regions. mRNA molecules containing Kozak sequences are also provided. Furthermore, mRNA molecules comprising one or more intronic nucleotide sequences capable of being excised from the mRNA sequence are provided herein. (a) Untranslated Region (UTR)

[0132] The untranslated region (UTR) of a gene is transcribed but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include it; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. Increasing evidence suggests that the UTR plays a regulatory role in the stability and translation of mRNA molecules. Regulatory features of the UTR can be incorporated into the mRNA used in the lipid particles described herein to increase molecular stability. These specific features can also be incorporated to ensure controlled downregulation of the transcript in cases where it is misdirected to unwanted tissue or organ sites. (b) 5' with cap

[0133] The 5' cap structure of mRNA involves nuclear export, thereby increasing mRNA stability, and binds to mRNA cap-binding protein (CBP). This CBP associates with poly(A)-binding protein to form a mature circular mRNA species responsible for mRNA stability and translational capacity within the cell. The cap further aids in the removal of 5' proximal introns during mRNA splicing.

[0134] Endogenous mRNA molecules can be 5' capped, thereby creating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the sense nucleotide transcribed at the 5' end of the mRNA molecule. This 5'-guanosine cap can then be methylated to produce an N7-methyl-guanosine residue. The ribose of the nucleotide transcribed at the 5' end and / or the proximal end of the mRNA can also optionally be 2'-O-methylated. 5'-uncapping via hydrolysis and cleavage of the guanosine cap structure can target the degradation of the mRNA molecule. (c) IRES sequence

[0135] mRNAs containing an internal ribosome entry site (IRES) can also be used in the lipid nanoparticles described herein. The IRES can serve as a single ribosome binding site or as one of multiple ribosome binding sites for the mRNA. mRNAs containing more than one functional ribosome binding site may encode several peptides or polypeptides that are independently translated via ribosomes (“polycistronic mRNA”). When the mRNA has an IRES, a second translatable region is optionally provided further. Examples of IRES sequences include, but are not limited to, those from viruses such as parvoviruses (e.g., FMDV), insect viruses (e.g., CFFV), poliovirus (PV), encephalomyocytovirus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukosis virus (MLV), simian immunodeficiency virus (S1V), and cricket paralysis virus (CrPV). (d) Polymerized A-tail

[0136] During RNA processing, long-chain adenine nucleotides (poly-A tails) can be added to polynucleotide molecules such as mRNA to increase stability. Immediately following transcription, the 3' end of the transcript is cleaved to release the 3' hydroxyl group. Poly-A polymerase then adds the adenine nucleotide chain to the RNA. This process (called polyadenylation) adds poly-A tails of between 100 and 250 residues in length.

[0137] Generally, the polyA-tail is longer than 30 nucleotides. In some embodiments, the polyA-tail is longer than 35 nucleotides (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the polyA-tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the mRNA. In other implementations, the polyA tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total mRNA length. (3) Production of mRNA molecules

[0138] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25: 263-269 (1983); Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition 1989)); the same is true for PCR methods (see, for example, U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., edited, 1990)). Expression libraries are also well known to those skilled in the art. Further foundational articles disclosing the general methods used in this disclosure include Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., edited, 1994). In some embodiments, mRNA molecules are produced by in vitro transcription followed by purification. Standard techniques for purifying mRNA (e.g., mRNA produced through in vivo transcription) are well known in the art.

[0139] In some embodiments, the provided lipid nanoparticles contain mRNA synthesized from a DNA template. Methods for synthesizing mRNA from a DNA template are well known and generally involve the use of an enzyme (e.g., T7 RNA polymerase and / or its derivatives, such as optimized alternative enzymes), nucleotides (optionally including modified nucleotides as described herein), and optional mRNA components (e.g., a 5' cap). In some embodiments, the lipid nanoparticles contain mRNA generated in vitro using a commercially available kit. Commercially available kits suitable for generating mRNA contained in the provided lipid nanoparticles include, for example, the MEGASCRIPT™ T7 Transcription Kit available from ThermoFisher Scientific. Additionally or alternatively, the mRNA used for the provided lipid nanoparticles can be generated using chemical synthesis methods, such as those described by Abe et al., ACS Chem. Biol. 2022, 17, 6, 1308–1314. In a further embodiment, the lipid nanoparticles contain mRNA generated using an mRNA printer, for example, any of the printers reviewed by Sheridan in 2022 (Nature Biotechnol. 2022, 40, 1160–1162).

[0140] In some embodiments, the mRNA produced by any one or more of the methods described herein is purified prior to its use in forming one or more of the provided lipid nanoparticles. mRNA purification techniques suitable for the provided lipid nanoparticles include, but are not limited to, HPLC purification, cellulose purification, tangential flow filtration, bead-based purification (e.g., purification with Dynabeads), silica-based purification, precipitation, and combinations thereof. Non-limiting examples of mRNA purification are described, for example, in International Patent Application Publication No. WO 2018 / 006052 A1. (4) Polypeptides encoded by mRNA

[0141] The mRNA component of the lipid nanoparticles described in this article can be used to express target peptides. Some human diseases are caused by the loss or impairment of functional proteins in cell types where proteins are normally present and active. Functional proteins can be completely or partially lost due to, for example, transcriptional inactivation of the coding gene or due to mutations in the coding gene that render the protein completely or partially nonfunctional. Examples of human diseases caused by complete or partial protein inactivation include: methylmalonic acidemia (caused by a deficiency of methylmalonyl-CoA mutase), glycogen storage disease type 1A (caused by a deficiency of the glucose-6-phosphatase catalytic subunit), glycogen storage disease type 1B (caused by a deficiency of glucose-6-phosphate translocase), Fragile X syndrome (caused by a deficiency of FMR1 protein), urea cycle disorders (caused by a mutation in the ornithine carbamoyltransferase (OTC) gene), Krieger-Najar syndrome type I (caused by a gene mutation leading to a deficiency of bilirubin-UGT), α-1 antitrypsin deficiency (caused by a mutation in the SERPINA1 gene), and thrombotic thrombocytopenic purpura (caused by an integrin-like metalloproteinase 13 containing a type I platelet-binding protein motif). (ADAMTS13) gene mutations, acute intermittent porphyria (caused by mutations in the PBGD gene site), factor IX deficiency hemophilia B (caused by deficiency of factor IX (FIX) protein), X-linked severe combined immunodeficiency disease (X-SCID) (caused by one or more mutations in the gene encoding a common γ chain protein, which is a component of various interleukin receptors and is involved in the development and maturation of B cells and T cells in the immune system), and X-linked adrenoleukodystrophy (X-ALD) (caused by one or more mutations in the gene for a peroxisome membrane transport protein called ABCD1).

[0142] In some embodiments, the mRNA component of the lipid nanoparticles described herein expresses infectious disease antigens, such as viral, bacterial, fungal, protozoan, and / or worm infectious disease antigens. Vaccines comprising lipid particles with antigen-encoding mRNA are particularly useful for the prevention or treatment of infectious diseases. In some embodiments, the infectious disease antigen is a viral infectious disease antigen derived from coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), influenza viruses (e.g., influenza A, influenza B, and influenza C viruses), filoviruses (e.g., Ebola virus, Marburg virus), arenaviruses (e.g., Lassa virus, Junin virus, Machupo virus, Guanareto virus, Sabya virus), Zika virus, rabies virus, rhinovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), hepatitis viruses (e.g., hepatitis C virus), herpes simplex virus, human papillomavirus (HPV), or Epstein-Barr virus. In certain implementations, the infectious disease antigen is a SARS-CoV-2 protein selected from the group consisting of the S (spike) protein, E (enveloped) protein, M (membrane) protein, N (nucleocapsid) protein, and antigenic fragments thereof. In some cases, the development of antigen-specific immunity from mRNA vaccines requires transfection of antigen-presenting cells, such as dendritic cells. Because dendritic cells are densely distributed in skin tissue and skeletal muscle, administration is typically accomplished via intradermal, intramuscular, or subcutaneous injection.

[0143] In some embodiments, the mRNA component of the lipid nanoparticles described herein expresses tumor-associated antigens. In certain cases, following administration of an mRNA cancer vaccine to dendritic cells, cytotoxic T cells can target and destroy tumors. In other embodiments, the mRNA component of the lipid nanoparticles described herein expresses a chimeric antigen receptor (CAR) for CAR T-cell therapy. Typically, a subject's T cells are isolated and transfected in vitro with mRNA encoding a CAR, which is a protein fragment displayed on the surface of T cells and binds to a specific tumor epitope. Upon reintroduction of the modified T cells into the subject, the CAR targets and kills tumor cells.

[0144] In some embodiments, the mRNA component of the lipid particles described herein expresses a gene-editing nuclease. Examples of gene-editing nucleases include CRISPR / Cas nucleases (e.g., Cas9, Cpf1), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases. CRISPR-mediated gene editing requires a Cas nuclease responsible for DNA cleavage and a short guide RNA (gRNA) to direct the Cas nuclease to cleave DNA at a precise location. In some embodiments, the gRNA targets the Cas nuclease to genes in the viral genome. As a non-limiting example, the viral genome is the SARS-CoV-2 genome, with genes selected from the group consisting of orf1ab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, and ORF10. In some cases, the mRNA and gRNA encoding a Cas nuclease such as Cas9 are encapsulated in the same lipid particle. In other cases, the mRNA and gRNA encoding a Cas nuclease are encapsulated in separate lipid particles. b) siRNA

[0145] In some embodiments, the nucleic acids of the provided lipid nanoparticles contain or are composed of siRNA. siRNA is an interfering RNA capable of silencing the expression of a target sequence in vitro and / or in vivo. The length of siRNA can be about 15-60, 15-50, or 15-40 (double-stranded) nucleotides, more typically about 15-30, 15-25, or 19-25 (double-stranded) nucleotides, and preferably about 20-24, 21-22, or 21-23 (double-stranded) nucleotides (e.g., each complementary sequence of the double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides long, preferably about 20-24, 21-22, or 21-23 nucleotides long, and the length of the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs, preferably about 18-22, 19-20, or 19-21 base pairs). A double-stranded siRNA may contain a 3' overhang and a 5' phosphate terminus of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides. Examples of siRNA include, but are not limited to: double-stranded polynucleotide molecules assembled from two separate strands, one of which is a sense strand and the other is a complementary antisense strand; double-stranded polynucleotide molecules assembled from single-stranded molecules, wherein the sense and antisense regions are linked by nucleic acid-based or non-nucleic acid-based linkers; double-stranded polynucleotide molecules having hairpin secondary structures with their own complementary sense and antisense regions; and cyclic single-stranded polynucleotide molecules having two or more loop structures and a stem structure with their own complementary sense and antisense regions, wherein the cyclic polynucleotides can be processed in vivo or in vitro to produce active double-stranded siRNA molecules.

[0146] In some preferred embodiments, the modified siRNA molecule is less immunostimulatory than the corresponding unmodified siRNA sequence. In such embodiments, the modified siRNA molecule with reduced immunostimulatory properties advantageously retains RNAi activity against the target sequence. In another embodiment, the immunostimulatory properties of the modified siRNA molecule and its ability to silence target gene expression can be balanced or optimized by introducing minimal and selective 2'Ome modifications within the siRNA sequence, such as within the double-stranded region of the siRNA duplex. In some instances, the immunostimulatory properties of the modified siRNA are at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than the corresponding unmodified siRNA. Those skilled in the art will readily recognize that the immunostimulatory properties of modified siRNA molecules and their corresponding unmodified siRNA molecules can be determined by measuring INF-α and / or IL-6 levels approximately two to twelve hours after transfection of mammalian responder cells, for example, by systemic administration in mammals or by using an appropriate lipid-based delivery system (such as the LNP delivery system disclosed herein).

[0147] In some embodiments, the modified siRNA molecule has an IC50 value that is less than or equal to ten times that of the corresponding unmodified siRNA. 50 (i.e., the half-maximal inhibitory concentration) (i.e., the modified siRNA has an IC50 value less than or equal to that of the corresponding unmodified siRNA). 50 Ten times the IC 50 In other embodiments, the modified siRNA has an IC50 value that is less than or equal to three times that of the corresponding unmodified siRNA sequence. 50 In other embodiments, the modified siRNA has an IC50 value that is less than or equal to twice that of the corresponding unmodified siRNA. 50 Those skilled in the art will readily recognize that dose-response curves can be generated for modified siRNAs and their corresponding unmodified siRNAs, with IC50 values... 50 The value can be readily determined using methods known to those skilled in the art.

[0148] In another embodiment, the modified siRNA molecule is capable of silencing the expression of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 20%, 80%, 85%, 90%, 95%, or 100% of the target sequence relative to the corresponding unmodified siRNA sequence.

[0149] In some embodiments, the siRNA molecule, for example, does not contain phosphate backbone modifications in the sense and / or antisense strands of the double-stranded region. In other embodiments, the siRNA, for example, contains one, two, three, four, or more phosphate backbone modifications in the sense and / or antisense strands of the double-stranded region. In a preferred embodiment, the siRNA does not contain phosphate backbone modifications. In yet another embodiment, the siRNA, for example, does not contain 2'-deoxynucleotides in the sense and / or antisense strands of the double-stranded region. In yet another embodiment, the siRNA, for example, contains one, two, three, four, or more 2'-deoxynucleotides in the sense and / or antisense strands of the double-stranded region. In a preferred embodiment, the siRNA does not contain 2'-deoxynucleotides.

[0150] In some instances, the nucleotide at the 3' end of the double-stranded region in the sense and / or antisense strand is not a modified nucleotide. In some other instances, the nucleotide near the 3' end of the double-stranded region in the sense and / or antisense strand (e.g., within one, two, three, or four nucleotides at the 3' end) is not a modified nucleotide.

[0151] The siRNA molecules described herein may have one, two, three, four or more nucleotide 3' overhangs on one or both sides of the double-stranded region, or may lack overhangs on one or both sides of the double-stranded region (i.e., have blunt ends). Preferably, the siRNA has two nucleotide 3' overhangs on each side of the double-stranded region. In some instances, the 3' overhang on the antisense strand is complementary to the target sequence, and the 3' overhang on the sense strand is complementary to the complementary strand of the target sequence. Alternatively, the 3' overhang is not complementary to the target sequence or its complementary strand. In some embodiments, the 3' overhangs comprise one, two, three, four or more nucleotides, such as 2'-deoxy (2'H) nucleotides. In some preferred embodiments, the 3' overhangs comprise deoxythymidine (dT) and / or uridine nucleotides. In other embodiments, one or more nucleotides in the 3' overhangs on one or both sides of the double-stranded region comprise modified nucleotides. Non-limiting examples of modified nucleotides are described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxynucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In a preferred embodiment, one, two, three, four, or more nucleotides present at the 3' overhangs on the sense and / or antisense strands of the siRNA comprise 2'Ome nucleotides (e.g., 2'Ome purine and / or pyrimidine nucleotides), such as 2'Ome-guanosine nucleotides, 2'Ome-uridine nucleotides, 2'Ome-adenosine nucleotides, 2'Ome-cytosine nucleotides, and mixtures thereof.

[0152] The siRNA may contain at least one or a mixture of unmodified and / or modified siRNA sequences (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) that silence the expression of a target gene. The mixture of siRNAs may contain sequences targeting the same region or domain (e.g., a “hotspot”) and / or different regions or domains targeting one or more target genes. In some instances, the mixture contains one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNA sequences that silence the expression of a target gene. In some other instances, the mixture contains one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence the expression of a target gene.

[0153] In some embodiments, the antisense strand of the siRNA molecule comprises or is composed of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule comprises or is composed of a sequence that is 100% complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule comprises or is composed of a sequence that specifically hybridizes to the target sequence or a portion thereof.

[0154] In other embodiments, the sense strand of the siRNA molecule comprises or is composed of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to or constitutes a portion thereof of the target sequence. In yet another embodiment, the sense strand of the siRNA molecule comprises or is composed of a sequence that is 100% identical to or constitutes a portion thereof of the target sequence. (1) Selection of siRNA sequence

[0155] Suitable siRNA sequences can be identified using any method known in the art. Typically, the methods described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20: 6877-6888 (2001) are combined with reasonable design rules enumerated in Reynolds et al., Nature Biotech., 22(3): 326-330 (2004). Generally, the 3' dinucleotide sequence of the AUG start codon from the transcript of the target gene is scanned (e.g., AA, NA, CC, GG, or UU, where N = C, G, or U) (see, for example, Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotide immediately adjacent to the dinucleotide sequence at the 3' is identified as a possible siRNA sequence (i.e., the target sequence or sense sequence). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately adjacent to the 3' end of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides immediately adjacent to the AA or NA dinucleotide are identified as potential siRNA sequences. siRNA sequences are typically spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of the siRNA sequence, potential siRNA sequences can be analyzed to identify sites, for example, in the target cell or organism that do not contain regions homologous to other coding sequences. For example, a suitable siRNA sequence with approximately 21 base pairs will typically not have more than 16-17 adjacent base pairs homologous to the coding sequence in the target cell or organism. If the siRNA sequence will be expressed from the RNA Pol III promoter, siRNA sequences lacking more than 4 adjacent A's or T's are selected.

[0156] Once a potential siRNA sequence has been identified, a complementary sequence (i.e., an antisense sequence) can be designed. Various standards known in the art can also be used to analyze potential siRNA sequences. For example, to enhance silencing efficiency, sequences with one or more of the following characteristics can be identified for analysis using a well-designed algorithm: (1) a G / C content of about 25% to about 60%; (2) at least three A / U pairs at positions 15-19 of the sense strand; (3) no internal repeats; (4) an A pair at position 19 of the sense strand; (5) an A pair at position 3 of the sense strand; (6) a U pair at position 10 of the sense strand; (7) no G / C pairs at position 19 of the sense strand; and (8) no G pairs at position 13 of the sense strand. Those skilled in the art will understand that sequences having one or more of the above characteristics can be selected for further analysis and testing as potential siRNA sequences.

[0157] Additionally, possible siRNA sequences having one or more of the following criteria may often be omitted as siRNA: (1) extended sequences containing rows of four or more identical bases; (2) homopolymers containing G (i.e., sequences to reduce possible nonspecific effects due to the structural features of these polymers); (3) sequences containing tribasic motifs (e.g., GGG, CCC, AAA, or TTT); (4) extended sequences containing rows of seven or more G / Cs; and (5) sequences containing direct repeats of four or more bases within candidates leading to internal foldback structures. However, those skilled in the art will understand that sequences having one or more of the above characteristics may still be selected for further analysis and testing as possible siRNA sequences.

[0158] In some implementations, possible siRNA sequences can be further analyzed based on the siRNA duplex asymmetry described, for example, in Khvorova et al., Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003). In other implementations, possible siRNA sequences can be further analyzed based on the secondary structures at the target site, as described, for example, in Luo et al., Biophys.Res.Commun., 318:303-310 (2004). For example, the Mfold algorithm (available at www.bioinfo.rpi.edu / applications / mfold / rna / forml.cgi) can be used to model the secondary structures at the target site to select siRNA sequences that favor entry at the target site, where there are fewer base-pairing and stem-loop secondary structures.

[0159] Once a potential siRNA sequence has been identified, the presence of any immunostimulatory properties of the sequence can be analyzed, for example, using in vitro cytokine assays or in vivo animal models. Motifs in the sense and / or antisense strands of the siRNA sequence, such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.), can also provide an indication of whether the sequence is likely to be immunostimulatory. Once an siRNA molecule is found to be immunostimulatory, it can then be modified as described herein to reduce its immunostimulatory properties. As a non-limiting example, the siRNA sequence can be exposed to mammalian responding cells under conditions that induce a detectable immune response in the cells to determine whether the siRNA is immunostimulatory or non-immunostimulatory. The mammalian responding cells can be derived from nascent mammals (i.e., mammals that have not previously been exposed to the gene product of the siRNA sequence). The mammalian responding cells can be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. Detectable immune responses may include the production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or combinations thereof. The immunostimulatory properties of siRNA molecules identified as immunostimulatory can then be reduced by replacing at least one nucleotide on the sense and / or antisense strands with a modified nucleotide. For example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-stranded region of the siRNA duplex can be replaced with a modified nucleotide such as a 2'Ome nucleotide. The modified siRNA can then be exposed to mammalian responding cells as described above to confirm a reduction or elimination of its immunostimulatory properties.

[0160] Suitable in vitro assays for detecting immune responses include, but are not limited to, the biclonal antibody sandwich immunoassay technique of David et al. (US Patent No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the “Western blotting” method of Gordon et al. (US Patent No. 4,452,901); immunoprecipitation with labeled ligands (Brown et al., J. Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assay (ELISA) as described, for example, by Raines et al., J. Biol. Chem., 257:5154-5160 (1982); and immunocytochemical techniques, including the use of fluorescent dyes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980)); and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassays described above, many other immunoassays are available, including those described in U.S. Patent Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074 and 4,098,876. The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0161] Non-limiting examples of in vivo models for detecting immune responses include in vivo mouse cytokine induction assays as described, for example, Judge et al., Mal. Ther., 13: 494-505 (2006). In some embodiments, the assays can be performed as follows: (1) siRNA can be administered by standard intravenous injection in the lateral tail vein; (2) blood can be collected by cardiac puncture and processed into plasma for cytokine analysis approximately 6 hours after administration; and (3) cytokines can be quantified using a sandwich ELISA kit according to the manufacturer's instructions (e.g., mouse and human IFN-α (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF-α (eBioscience; San Diego, Calif); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, Calif)).

[0162] Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from a variety of sources and can be produced using methods known in the art (see, for example, Kohler et al., Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). The production of monoclonal antibodies has been previously described and can be achieved by any means known in the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, the monoclonal antibody is labeled (e.g., using any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) to facilitate detection. (2) Generation of siRNA sequences

[0163] siRNA can be provided in several forms, including, for example, as one or more isolated small interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. siRNA can be chemically synthesized. Oligonucleotides containing siRNA molecules can be synthesized using a variety of techniques known in the art, such as any of the techniques described in: Usman et al., J. Am. Chem. Soc., 109: 7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res., 23: 2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59 (1997).

[0164] RNA populations can be used to provide long precursor RNAs, or long precursor RNAs that are substantially or completely identical to the selected target sequence can be used to manufacture siRNAs. RNA can be isolated from cells or tissues, synthesized, and / or cloned according to methods well known to those skilled in the art. RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, subtracted, selected, etc.) or can represent a single target sequence. RNA can be naturally occurring (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerases and PCR products or cloned cDNA), or chemically synthesized.

[0165] To form long dsRNA, for the synthetic RNA, in vitro transcription and complement hybridization are also performed to form dsRNA. If a naturally occurring RNA group is used, complement is also provided, for example, by transcription of cDNA corresponding to the RNA group or by using an RNA polymerase (e.g., to form dsRNA for digestion by E. coli RNase III or cleavage enzyme). The precursor RNA is then hybridized to form double-stranded RNA for digestion. The dsRNA can be administered directly to the subject or can be digested in vitro prior to administration.

[0166] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, creating and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25: 263-269 (1983); Sambrook et al., ibid.; Ausubel et al., ibid.), as are PCR methods (see, U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., ed., 1990)). Expression libraries are also well known to those skilled in the art. Further foundational articles disclosing the general methods used in the provided embodiments include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition, 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., ed., 1994). The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0167] Preferably, siRNA is chemically synthesized. siRNA can also be produced by cleaving longer dsRNAs (e.g., dsRNAs longer than about 25 nucleotides) using E. coli RNase III or cleavage enzyme. These enzymes process dsRNA into biologically active siRNA (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99: 9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99: 14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31: 981-987 (2003); Knight et al., Science, 293: 2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides long and about 100, 200, 300, 400, or 500 nucleotides long. The dsRNA can be up to 1000, 1500, 2000, 5000 nucleotides long or longer. The dsRNA can encode an entire gene transcript or a portion of a gene transcript. In some instances, the plasmid can encode siRNA (e.g., a sequence transcribed into a double strand that folds automatically into a hairpin loop).

[0168] siRNA molecules can also be synthesized via tandem synthesis, in which both strands are synthesized as single, continuous oligonucleotide fragments or strands separated by a cleavable linker, which is then cleaved to provide separate fragments or strands for hybridization to form a siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem synthesis of siRNA is readily adaptable to porous / multiplate synthesis platforms as well as large-scale synthesis platforms using batch reactors, synthesis towers, etc. Alternatively, siRNA molecules can be assembled from two dissimilar oligonucleotides, one containing the sense strand of the siRNA and the other containing the antisense strand. For example, each strand can be synthesized separately and linked together by hybridization or ligation after synthesis and / or deprotection. In some other instances, siRNA molecules can be synthesized as single, continuous oligonucleotide fragments, in which self-complementary sense and antisense regions hybridize to form a siRNA duplex with a hairpin secondary structure. (3) Modification of siRNA sequence

[0169] In some aspects, the siRNA molecule comprises a double-stranded structure with two strands and at least one modified nucleotide in the double-stranded region, wherein each strand is about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than the corresponding unmodified siRNA sequence, but still retains the ability to silence the expression of the target sequence. In a preferred embodiment, the degree of chemical modification introduced into the siRNA molecule strikes a balance between reducing or eliminating the immunostimulatory properties of the siRNA and preserving RNAi activity. As a non-limiting example, the siRNA molecule targeting the gene of interest may be minimally modified (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5% modification) on selective uridine and / or guanosine nucleotides within the siRNA double-stranded structure to eliminate the immune response generated by the siRNA while retaining its ability to silence the expression of the target gene.

[0170] Examples of modified nucleotides include, but are not limited to, ribonucleotides having a 2'-O-methyl (2'Ome), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group. Modified nucleotides with a Northern conformation, such as those described, for example, in Saenger, Principles of Nucleic Acid Structure, Springer-Verlag (eds.) (1984), are also suitable for use in siRNA molecules. Such modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides (e.g., 2'-O nucleotides, 4'-C-methylene-(D-furanose) nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thioethyl nucleotides, 2'-deoxy-2'-fluoro(2'F) nucleotides, 2'-deoxy-2'-chloro(2'Cl) nucleotides, and 2'-azidonucleotides. In some instances, the siRNA molecules described herein include one or more G-clamp nucleotides. G-clamp nucleotides refer to modified cytosine analogs in which the modification confers hydrogen bonding capability on both the Watson-Crick and Hoogsteen faces of the complementary guanine nucleotide within the duplex (see, for example, Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotides with nucleotide base analogues such as C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamide, and nitropyrrole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, for example, Loakes, Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated into siRNA molecules.

[0171] In some implementations, the siRNA molecule may also contain one or more chemical modifications, such as a terminal cap portion, phosphate backbone modifications, etc. Examples of terminal cap portions include, but are not limited to: reverse deoxygenated debase residues, glycerol modifications, 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-dehydrated hexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threopentose threonose nucleotides, acyclic 3',4'-closed nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-reverse nucleotide moiety, 3'-3'-reverse debasement moiety, 3'-2'-reverse nucleotide moiety, 3'-2'-reverse debasement moiety, 5'-5'-reverse nucleotide moiety, etc. 5'-5'-reverse debasing moiety, 3'-5'-reverse deoxy-basing moiety, 5'-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-aminophosphate, 5'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-thiophosphate, 5'-thiophosphate, dithiophosphate, and bridging or unbridging methyl phosphonate or 5'-mercapto group (see, for example, U.S. Patent No. 5,998,203; Beaucage et al., Tetrahedron 49:1925 (1993)). Non-limiting examples of phosphate backbone modifications (i.e., those that produce modified internucleotide bonds) include thiophosphates, dithiophosphates, methyl phosphonates, triphosphates, morpholino compounds, amidates, carbamates, carboxymethyl compounds, acetamides, polyamides, sulfonates, sulfonamides, aminosulfonates, formacetals, thiomethyl acetals, and alkylsilyl substitutions (see, for example, Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). Such chemical modifications can occur at the sense strand, antisense strand, or the 5' and / or 3' ends of both strands of the siRNA.

[0172] In some embodiments, the sense and / or antisense strands of the siRNA molecule may further include a 3'-terminal overhang having about one to about four (e.g., one, two, three, or four) 2'-deoxyribonucleotides and / or any combination of modified and unmodified nucleotides. Further examples of the types of modified nucleotides and chemical modifications that may be introduced into the siRNA molecule are described, for example, in UK Patent No. GB ​​2,397,818 B and US Patent Application Publications Nos. 2004 / 0192626, 2005 / 0282188, and 2007 / 0135372.

[0173] The siRNA molecules described herein may optionally contain one or more nonnucleotides in one or both strands of the siRNA. As used herein, the term "nonnucleotide" refers to any group or compound that may be incorporated into the nucleic acid chain in place of one or more nucleotide units, including sugar and / or phosphate ester substitutions, and allows the remaining bases to exhibit their activity. Such groups or compounds are debased because they do not contain well-known nucleotide bases such as adenosine, guanine, cytosine, uracil, or thymine and are therefore lacking a base at the 1' position.

[0174] In other embodiments, chemical modification of the siRNA includes attaching a conjugate to the siRNA molecule. The conjugate may be attached to the 5'-end and / or 3'-end of the sense and / or antisense strand of the siRNA via covalent attachment, such as a biodegradable linker. The conjugate may also be attached to the siRNA, for example, via a carbamate group or other linking group (see, for example, U.S. Patent Application Publications 2005 / 0074771, 2005 / 0043219, and 20050158727). In some instances, the conjugate is a molecule that facilitates the delivery of the siRNA into the cell. Examples of conjugate molecules suitable for attachment to siRNA include, but are not limited to, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and their derivatives), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands of cellular receptors capable of mediating cellular uptake, and combinations thereof (see, for example, U.S. Patent Application Publications 2003 / 0130186, 2004 / 0110296 and 2004 / 0249178; U.S. Patent No. 6,753,423). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, small groove binders, cleavage agents, and crosslinking agent conjugates described in U.S. Patent Application Publication Nos. 2005 / 0119470 and 2005 / 0107325. Other examples include 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cation-modified pyrimidines, cationic peptides, guanidino groups, amidine groups, and cationic amino acid conjugates described in U.S. Patent Application Publication No. 2005 / 0153337. Other examples include hydrophobic groups, membrane-active compounds, cell-penetrating compounds, cell-targeting signaling compounds, interaction modifiers, and steric stabilizer conjugates described in U.S. Patent Application Publication No. 2004 / 0167090. Further examples include conjugates described in U.S. Patent Application Publication No. 2005 / 0239739. The type of conjugate used and the extent to which it is conjugated to the siRNA molecule can be evaluated based on the improved pharmacokinetic characteristics, bioavailability, and / or stability of the siRNA while preserving RNAi activity. Therefore, those skilled in the art can use any of the various well-known in vitro cell cultures or in vivo animal models to screen siRNA molecules with various conjugates attached thereto in order to identify siRNA molecules with improved properties and full RNAi activity. c)aiRNA

[0175] In some embodiments, the provided lipid nanoparticles comprise an asymmetric interfering RNA (aiRNA) molecule that silences the expression of a target gene. In some instances, the aiRNA molecule comprises a double-stranded (double-stranded) region of about 10 to about 25 (base-paired) nucleotides in length, wherein the aiRNA molecule includes an antisense strand containing 5' and 3' overhangs, and wherein the aiRNA molecule is capable of silencing the expression of the target gene.

[0176] In some instances, the aiRNA molecule comprises a double-stranded (double-stranded) region of about 12-20, 12-19, 12-18, 13-17, or 14-17 (base-paired) nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 (base-paired) nucleotides in length. In some other instances, the 5' and 3' overhangs on the antisense strand contain sequences complementary to the target RNA sequence and may optionally further contain non-target sequences. In some embodiments, the 5' and 3' overhangs on the antisense strand each contain 1, 2, 3, 4, 5, 6, 7, or more nucleotides, or consist of 1, 2, 3, 4, 5, 6, 7, or more nucleotides.

[0177] In other embodiments, the aiRNA molecule comprises a modified nucleotide selected from the group consisting of: 2'oMe nucleotide, 2'F nucleotide, 2'-deoxynucleotide, 2'-OMOE nucleotide, LNA nucleotide, and mixtures thereof. In a preferred embodiment, the aiRNA molecule comprises a 2'oMe nucleotide. As a non-limiting example, the 2'Ome nucleotide may be selected from the group consisting of: 2'oMe-guanosine nucleotide, 2'oMe-uridine nucleotide, and mixtures thereof.

[0178] Like siRNA, asymmetric interfering RNA (aiRNA) recruits the RNA-induced silencing complex (RISC) and causes effective silencing of various genes in mammalian cells by mediating sequence-specific cleavage of the target sequence between nucleotides 10 and 11 at the 5' end of the antisense strand (Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, aiRNA molecules comprise a short RNA duplex with sense and antisense strands, wherein the duplex contains overhangs at the 3' and 5' ends of the antisense strand. aiRNA is generally asymmetric because the sense strand is shorter at both ends compared to the complementary antisense strand. In some embodiments, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, the methods described above for selecting siRNA sequences can be used to select and generate aiRNA sequences.

[0179] In another embodiment, aiRNA duplexes of various lengths (e.g., about 10–25, 12–20, 12–19, 12–18, 13–17, or 14–17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) with overhangs at the 3' and 5' ends of the antisense strand can be designed to target mRNA. In some instances, the sense strand of the aiRNA molecule is about 10–25, 12–20, 12–19, 12–18, 13–17, or 14–17 nucleotides long, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In some other instances, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides long, more typically about 15-30, 15-25, or 19-25 nucleotides long, and preferably about 20-24, 21-22, or 21-23 nucleotides long.

[0180] In some embodiments, the 5' antisense overhang contains one, two, three, four, or more non-targeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In other embodiments, the 3' antisense overhang contains one, two, three, four, or more non-targeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In some aspects, the aiRNA molecule described herein may, for example, include one or more modified nucleotides in the double-stranded (double-stranded) region and / or in the antisense overhang. As a non-limiting example, the aiRNA sequence may contain one or more of the modified nucleotides described for the siRNA sequence. In a preferred embodiment, the aiRNA molecule contains a 2'oMe nucleotide, such as 2'oMe-guanosine nucleotide, 2'oMe-uridine nucleotide, or a mixture thereof.

[0181] In some embodiments, the aiRNA molecule may contain an antisense strand corresponding to the antisense strand of an siRNA molecule, such as one of the siRNA molecules described herein. In other embodiments, the aiRNA molecule may be used to silence the expression of any of the target genes described above in the context of siRNA molecules, such as genes associated with viral infection and survival, genes associated with metabolic diseases and conditions, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immunomodulatory genes (such as genes associated with inflammatory and autoimmune responses), ligand receptor genes, and genes associated with neurodegenerative diseases. As a non-limiting example, antisense oligonucleotides may be used to silence the expression of SARS-CoV-2 genes encoding S (spike) protein, E (enveloping) protein, M (membrane) protein, or N (nucleocapsid) protein. d) miRNA

[0182] In some embodiments, the provided lipid nanoparticles comprise microRNA (miRNA) molecules that silence the expression of a target gene. In some instances, the miRNA molecule comprises a length of about 15 to about 60 nucleotides, wherein the miRNA molecule is capable of silencing the expression of the target gene. In some instances, the miRNA molecule comprises a length of about 15-50, 15-40, or 15-30 nucleotides, more typically about 15-25 or 19-25 nucleotides, and preferably about 20-24, 21-22, or 21-23 nucleotides. In a preferred embodiment, the miRNA molecule is a mature miRNA molecule that targets a target RNA sequence.

[0183] In some embodiments, the miRNA molecule comprises a modified nucleotide selected from the group consisting of: 2'oMe nucleotide, 2'F nucleotide, 2'-deoxynucleotide, 2'-OMOE nucleotide, LNA nucleotide, and mixtures thereof. In a preferred embodiment, the miRNA molecule comprises a 2'oMe nucleotide. As a non-limiting example, the 2'Ome nucleotide may be selected from the group consisting of: 2'oMe-guanosine nucleotide, 2'oMe-uridine nucleotide, and mixtures thereof.

[0184] Typically, microRNAs (miRNAs) are single-stranded RNA molecules of approximately 21-23 nucleotides in length that regulate gene expression. miRNAs are encoded by genes that transcribe their DNA, but they are not translated into proteins (non-coding RNAs); instead, each initial transcript (initial miRNA) is processed into a short stem-loop structure called a precursor miRNA and eventually becomes a functional mature miRNA. Mature miRNA molecules are partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression. The identification of miRNA molecules has been described, for example, in Lagos-Quintana et al., Science, 294:853-858; Lau et al., Science, 294:858-862; and Lee et al., Science, 294:862-864.

[0185] The genes encoding miRNAs are much longer than the processed mature miRNA molecules. First, the miRNA is transcribed into an initial transcript or a naïve miRNA with a cap and a poly-A tail, and then processed in the nucleus into a short stem-loop structure of approximately 70 nucleotides called a precursor miRNA. This processing occurs in animals via a protein complex called a microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA-binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)). These precursor miRNAs are then processed into mature miRNAs in the cytoplasm through interaction with the endonuclease cleatase, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001)). The sense or antisense strand of DNA can be used as a template for miRNA production.

[0186] When cleavage enzymes cleave the precursor miRNA stem-loop, two complementary short RNA molecules are formed, but only one is incorporated into the RISC complex. This strand, called the guide strand, is selected based on the stability of its 5' end by the catalytically active RNase Argonaute protein within the RISC complex (Preall et al., Curr. Biol., 16:530-535 (2006)). The remaining strand, called the anti-guide or transit strand, is degraded into the RISC complex substrate (Gregory et al., Cell, 123:631-640 (2005)). After incorporation into the active RISC complex, the miRNA base-pairs with its complementary mRNA molecule and induces the degradation of the target mRNA and / or translational silencing.

[0187] Mammalian miRNA molecules are typically complementary to sites in the 3' UTR of target mRNA sequences. In some instances, miRNA annealing with target mRNA inhibits protein translation by blocking the protein translation mechanism. In other instances, miRNA annealing with target mRNA facilitates the cleavage and degradation of target mRNA through a mechanism similar to RNA interference (RNAi). miRNAs can also target methylation at genomic sites corresponding to the targeted mRNA. Generally, the complement-related functions of miRNAs are collectively referred to as miRNPs.

[0188] In some embodiments, the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length. In some embodiments, the miRNA molecule comprises one or more modified nucleotides. As a non-limiting example, the miRNA sequence may comprise one or more modified nucleotides described for the siRNA sequence. In a preferred embodiment, the miRNA molecule comprises a 2'oMe nucleotide, such as 2'oMe-guanosine nucleotide, 2'oMe-uridine nucleotide, or a mixture thereof.

[0189] In some implementations, miRNA molecules can be used to silence the expression of any of the target genes described above in the context of siRNA molecules, such as genes associated with viral infection and survival, genes associated with metabolic diseases and symptoms, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immunomodulatory genes (such as genes associated with inflammatory and autoimmune responses), ligand receptor genes, and genes associated with neurodegenerative diseases. As a non-limiting example, miRNAs can be used to silence the expression of SARS-CoV-2 genes encoding the S (spike) protein, E (enveloping) protein, M (membrane) protein, or N (nucleocapsid) protein.

[0190] In other embodiments, the lipid particles described herein are used to administer one or more agents that block the activity of miRNAs targeting the target mRNA. Examples of blocking agents include, but are not limited to, steric silencing oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blocking agents may bind directly to the miRNA or to a miRNA-binding site on the target mRNA. e) Self-amplifying RNA

[0191] In some embodiments, the nucleic acids of the provided lipid nanoparticles comprise or consist of one or more self-amplifying RNA molecules. Self-amplifying RNA (sa-RNA) may also be referred to as self-replicating RNA, replicative RNA, replicon, or RepRNA. RepRNA (called self-amplifying mRNA), when derived from a positive-sense virus, is produced from a viral genome lacking at least one structural gene; it can be translated and replicated (hence “self-amplifying”) without producing infectious progeny viruses. In some embodiments, RepRNA technology can be used to insert gene cassettes encoding desired target antigens. For example, an alphavirus genome is divided into two open reading frames (ORFs): the first ORF encodes a protein of an RNA-dependent RNA polymerase (replicaase), and the second ORF encodes a structural protein. In a sa-RNA vaccine construct, the ORF encoding the viral structural protein can be replaced with any chosen antigen, while the viral replicaase remains an integral part of the vaccine and drives intracellular amplification of the RNA after immunization. f) Antisense oligonucleotides

[0192] In some embodiments, the nucleic acids of the provided lipid nanoparticles comprise or are composed of antisense oligonucleotides relating to or containing a target gene or target sequence. As used herein, the term "antisense oligonucleotide" or "antisense" includes an oligonucleotide complementary to a targeted polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA complementary to a selected sequence. Antisense RNA oligonucleotides prevent the translation of the complementary RNA strand by binding to RNA. Antisense DNA oligonucleotides can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In some embodiments, the antisense oligonucleotide comprises about 10 to about 60 nucleotides, or about 15 to about 30 nucleotides. The term also covers antisense oligonucleotides that may not be precisely complementary to the desired target gene. Therefore, the lipid nanoparticles described herein can be used in instances where antisense is used to discover non-target-specific activity, or in specific uses where an antisense sequence containing one or more mismatches with the target sequence is most preferred. Antisense oligonucleotides may include natural nucleotides as well as non-natural or modified nucleotides (e.g., modified nucleobases, modified nucleoside bonds, and / or modified sugars, as described herein).

[0193] Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and therefore can be used to specifically inhibit protein synthesis by targeting genes. The efficacy of antisense oligonucleotides in inhibiting protein synthesis is well established. For example, antisense oligonucleotides involving their corresponding mRNA sequences inhibit the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptors (see, U.S. Patents 5,739,119 and 5,759,829). Furthermore, examples of antisense inhibition have been demonstrated for nucleoprotein cyclins, multidrug resistance genes (MDR1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor, and human EGF (see Jaskulski et al., Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun., 1:225-32 (1989); Penis et al., Brain Res., 15; 57:310-20 (1998); and U.S. Patent Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288). In addition, antisense constructs have been described for inhibition and can be used to treat various abnormal cell proliferations, such as cancer (see U.S. Patent Nos. 5,747,470; 5,591,317; and 5,783,683). The publicly available content of these references is incorporated into this paper in its entirety by reference for all purposes.

[0194] Methods for generating antisense oligonucleotides are known in the art and readily adaptable to generate antisense oligonucleotides targeting any polynucleotide sequence. The selection of a target-sequence-specific antisense oligonucleotide sequence is based on the analysis of the selected target sequence and the determination of its secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in the host cell. Highly preferred target regions for mRNA include those regions located at or near the AUG translation start codon and those sequences substantially complementary to the 5' region of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using OLIGO primer analysis software version 4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0195] In some embodiments, the antisense oligonucleotide component of the lipid particles described herein can be used to inhibit the expression or replication of target genes. Target genes described in the context of siRNA molecules above include, but are not limited to: genes associated with viral infection and survival; genes associated with metabolic diseases and conditions (e.g., liver diseases and conditions); genes associated with tumorigenesis and cell transformation (e.g., cancer); angiogenesis genes; immunomodulatory genes such as those associated with inflammatory and autoimmune responses; ligand receptor genes; and genes associated with neurodegenerative diseases. As a non-limiting example, the antisense oligonucleotide can hybridize with SARS-CoV-2 genes (e.g., orf1ab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, or ORF10) and inhibit gene expression or replication. g) Ribozyme

[0196] In some embodiments, the nucleic acids of the provided lipid nanoparticles contain or are composed of ribozymes. Ribozymes are RNA-protein complexes with specific catalytic domains possessing endonuclease activity (see Kim et al., Proc. Natl. Acad. Sci. USA., 84:8788-92 (1987); and Forster et al., Cell, 49:211-20 (1987)). For example, large numbers of ribozymes accelerate highly specific phosphate transesterification reactions, often cleaving only one of several phosphate esters in oligonucleotide substrates (see Cech et al., Cell, 27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990); Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity is attributed to the need for the substrate to bind to the internal guide sequence (“IGS”) of the ribozyme via specific base pairing interactions prior to the chemical reaction.

[0197] At least six basic types of naturally occurring enzymatic RNA molecules are currently known. Each can catalyze the hydrolysis of the phosphodiester bond in trans RNA under physiological conditions (and thus cleave other RNA molecules). Generally, enzymatic nucleic acids function by first binding to a target RNA. This binding occurs through the target-binding portion of the enzymatic nucleic acid, which is kept in close proximity to the enzymatic portion of the molecule that cleaves the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds to the target RNA through complementary base pairing, and once bound to the appropriate site, it enzymatically cleaves the target RNA. Strategically cleaving the target RNA disrupts its ability to guide the synthesis of the encoded protein. After the enzymatic nucleic acid has bound to and cleaved its RNA target, it is released from that RNA to search for another target, and can repeatedly bind to and cleave new targets.

[0198] Enzymatic nucleic acid molecules can be formed in motifs such as hammerhead, hairpin, hepatitis delta virus, group I introns, or RNase pRNA (associated with the RNA guide sequence) or Neurospora VSRNA. Specific examples of hammerhead motifs are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are described, for example, in EP 0360257; Hampel et al., Biochemistry, 28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990); and U.S. Patent No. 5,631,359. Examples of hepatitis delta virus motifs are described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). Examples of RNase P motifs are described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of Neurospora VS RNase motifs are described, for example, in Savville et al., Cell, 61:685-96 (1990); Savville et al., Proc. Natl. Acad. Sci. USA, 88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Examples of group I introns are described, for example, in U.S. Patent No. 4,987,071. An important characteristic of enzymatic nucleic acid molecules is that they have specific substrate-binding sites complementary to one or more target gene DNA or RNA regions, and that they have nucleotide sequences within or around the substrate-binding sites that exert RNA cleavage activity on the molecule. Therefore, ribozyme constructs need not be limited to the specific motifs mentioned herein.

[0199] Methods for generating ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed and synthesized as described, for example, in PCT publications WO 93 / 23569 and WO 94 / 02595, for in vitro and / or in vivo testing as described therein.

[0200] Ribozyme activity can be optimized by altering the length of the ribozyme-binding arm or by chemically synthesizing ribozymes modified to prevent their degradation by serum ribonucleases (see, for example, PCT Publications WO 92 / 07065, WO 93 / 15187, WO 91 / 03162 and WO94 / 13688; EP 92110298.4; and U.S. Patent No. 5,334,711, which describes various chemical modifications that can be made to the sugar moiety of enzymatic RNA molecules), by enhancing their efficacy in cells, and by removing stem II bases to shorten RNA synthesis time and reduce chemical requirements. h) Immunostimulatory oligonucleotides

[0201] In some embodiments, the nucleic acids of the provided lipid nanoparticles include or consist of immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) capable of inducing an immune response when administered to a subject, who may be a mammal such as a human. ISSs include, for example, certain palindromic sequences that result in hairpin secondary structures (see, Yamamoto et al., J. Immunol., 148:4072-6 (1992)) or CpG motifs, as well as other known ISS features (such as multiG domains; see PCT Publication No. WO96 / 11266).

[0202] Immunostimulatory nucleic acids are considered non-sequence-specific when they do not require specific binding to target sequences or reduction of target sequence expression to elicit an immune response. Therefore, some immunostimulatory nucleic acids may contain sequences corresponding to regions of naturally occurring genes or mRNAs, but they can still be considered non-sequence-specific immunostimulatory nucleic acids.

[0203] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide having a methylated cytosine. In one embodiment, the nucleic acid comprises a single CpG dinucleotide wherein the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides wherein at least one cytosine in the CpG dinucleotide is methylated. In another embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In yet another embodiment, the nucleic acid comprises multiple CpG dinucleotides wherein at least one CpG dinucleotide comprises a methylated cytosine. In some embodiments, the oligonucleotide used in the compositions and methods described herein has a phosphodiester (“PO”) backbone or a phosphate thioester (“PS”) backbone, and / or has at least one methylated cytosine residue in the CpG motif. i)shRNA

[0204] In some embodiments, the nucleic acids of the provided lipid nanoparticles include or consist of shRNA. shRNA molecules contain short RNA sequences forming tight hairpin bends, which can be used to silence gene expression via RNA interference. The shRNA molecules disclosed herein can be chemically synthesized or transcribed from a transcription cassette in a DNA plasmid. The shRNA hairpin structure is cleaved into siRNA by cellular mechanisms and then binds to the RNA-induced silencing complex (RISC).

[0205] The length of the shRNA molecules disclosed herein is typically about 15-60, 15-50, or 15-40 (double-stranded) nucleotides, more typically about 15-30, 15-25, or 19-25 (double-stranded) nucleotides, and preferably about 20-24, 21-22, or 21-23 (double-stranded) nucleotides (e.g., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides, preferably about 20-24, 21-22, or 21-23 nucleotides, and the length of the double-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs, preferably about 18-22, 19-20, or 19-21 base pairs). The shRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and / or a 5'-phosphate terminus on the sense strand. In some embodiments, the shRNA comprises a sense strand and / or an antisense strand sequence of about 15 to about 60 nucleotides (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30 or 15-25 nucleotides), preferably about 19 to about 40 nucleotides (e.g., about 19-40, 19-35, 19-30 or 19-25 nucleotides), and more preferably about 19 to about 23 nucleotides (e.g., 19, 20, 21, 22 or 23 nucleotides).

[0206] Non-limiting examples of shRNA include double-stranded polynucleotide molecules assembled from single-stranded molecules, wherein the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and double-stranded polynucleotide molecules having a hairpin secondary structure having self-complementary sense and antisense regions. In a preferred embodiment, the sense and antisense strands of the shRNA are linked by a loop structure comprising about 1 to about 25 nucleotides, about 2 to about 20 nucleotides, about 4 to about 15 nucleotides, about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

[0207] Suitable shRNA sequences can be identified, synthesized, and modified using any means known in the art for designing, synthesizing, and modifying siRNA sequences. Further embodiments relating to the shRNA molecules of this disclosure, as well as methods for designing and synthesizing such shRNA molecules, are described in U.S. Patent Application Publication No. 2011 / 0071208, the disclosure of which is incorporated herein by reference in its entirety for all purposes. j)asRNA

[0208] In some embodiments, the nucleic acids of the provided lipid nanoparticles include or consist of asRNA. asRNA molecules, also known in the art as antisense transcripts, are naturally occurring or synthetically produced single-stranded RNA molecules that are complementary to, hybridize with, and thereby block the translation of mRNA into protein. Antisense transcripts are classified into short (less than 200 nucleotides) and long (more than 200 nucleotides) non-coding RNAs (ncRNAs).

[0209] The asRNA molecule may include a sequence complementary to a genomic sequence 100, 80, 60, 40, 20, or 10 kb upstream of the transcription start site of the target gene and downstream of the transcription termination site. In some embodiments, the asRNA includes a sequence complementary to a genomic sequence 1 kb upstream of the transcription start site of the target gene and 1 kb downstream of the transcription termination site of the target gene. In other embodiments, the asRNA includes a sequence complementary to a genomic sequence 500, 250, or 100 nucleotides upstream of the transcription start site of the target gene and 500, 250, or 100 nucleotides downstream of the transcription termination site of the target gene.

[0210] asRNA molecules may include sequences complementary to genomic sequences containing the coding region of a target gene. asRNA may also include sequences complementary to genomic sequences aligned with the promoter region of the target gene on the template strand. The gene may have multiple promoter regions. In this case, the asRNA may be aligned with one, two, or more promoter regions. Online databases of annotated gene sites can be used to identify gene promoter regions.

[0211] The alignment region between the asRNA molecule and the promoter region of the target gene can be as short as a single nucleotide, or at least 15 or at least 20 nucleotides, or at least 25 nucleotides, or at least 30, 35, 40, 45 or 50 nucleotides, or at least 55, 60, 65, 70 or 75 nucleotides, or at least 100 nucleotides.

[0212] The primary natural function of asRNA molecules involves regulating gene expression, and synthetic versions have been widely used as research tools for gene knockout and therapeutic applications. asRNA molecules and their functions have been described in this field (see, for example, Weiss et al., (1999) Cell. Mol. Life Sci. 55:334-358; Wahlstedt (2013) Nat. Rev. Drug Disc. 12:433-446; Pelechano and Steinmetz (2013) Nat. Rev. Genet. 14:880-893). k)DsiRNA

[0213] In some embodiments, the nucleic acids of the provided lipid nanoparticles comprise or consist of DsiRNA. The DsiRNA molecules are processed in vivo by a cleavage enzyme to produce active siRNA, which is then incorporated into the RISC complex to interfere with the target gene via RNA. In some embodiments, the DsiRNA is of sufficient length to be processed by the cleavage enzyme to produce siRNA. According to these embodiments, the DsiRNA comprises (i) a first oligonucleotide sequence (also called a sense strand) of about 25 to about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferably about 25 to about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotide sequence (also called an antisense strand) which is annealed to the first sequence under biological conditions (e.g., conditions found in the cytoplasm of cells). The second oligonucleotide sequence can be about 25 to about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides), and preferably about 25 to about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides). Additionally, one of the DsiRNA sequences, particularly the region of the antisense strand, has a sequence of at least about 19 nucleotides in length, for example, about 19 to about 60 nucleotides (e.g., about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25 nucleotides), preferably about 19 to about 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides), which is sufficiently complementary to the nucleotide sequence of the RNA produced by the target gene to trigger an RNAi response.

[0214] In some embodiments, the DsiRNA is long enough to be processed by a chytokinase to produce siRNA and has at least one of the following properties: (i) the DsiRNA is asymmetric, for example, having a 3'-protrusion on the antisense strand; and / or (ii) the DsiRNA has a modified 3'-terminus on the sense strand to guide the direction of chytokinase binding and the processing of the DsiRNA into active siRNA. According to the latter embodiment, the sense strand contains about 22 to about 28 nucleotides, and the antisense strand contains about 24 to about 30 nucleotides.

[0215] In some embodiments, the DsiRNA has a 3'-end overhang on the antisense strand. In some embodiments, the sense strand is modified with a suitable modifier located at the 3'-end of the sense strand for chytosuccinate binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, acyclic nucleotides, etc., and sterically hindered molecules such as fluorescent molecules. When nucleotide modifiers are used, they replace the ribonucleotides in the DsiRNA such that the length of the DsiRNA remains unchanged. In some embodiments, the DsiRNA has a 3'-end overhang on the antisense strand and the sense strand is modified for chytosuccinate processing. In some embodiments, the 5'-end of the sense strand has a phosphate ester. In some embodiments, the 5'-end of the antisense strand has a phosphate ester. In some embodiments, the antisense strand or the sense strand, or both, has one or more nucleotides modified with 2'-O-methyl (2'OMe). In some embodiments, the antisense strand contains a 2'OMe-modified nucleotide. In some embodiments, the antisense strand contains a 3' overhang comprising a 2'OMe-modified nucleotide. The antisense strand may also contain additional 2'OMe-modified nucleotides. Both the sense and antisense strands are annealed under biological conditions (such as those found in the cytoplasm of cells). Additionally, one of the sequences of the DsiRNA, particularly the region of the antisense strand, has a sequence of at least about 19 nucleotides in length, wherein these nucleotides are located in a 21-nucleotide region adjacent to the 3'-terminus of the antisense strand and are fully complementary to the nucleotide sequence of the RNA produced by the target gene. Furthermore, according to these embodiments, the DsiRNA may also have one or more of the following additional properties: (a) the antisense strand has a right shift relative to the typical 21-mer (i.e., the antisense strand contains nucleotides on the right side of the molecule compared to the typical 21-mer); (b) the strands may not be perfectly complementary (i.e., the strands may contain simple mismatches); and (c) the 5' end of the sense strand may contain base modifications, such as locked nucleic acids.

[0216] In some embodiments, the sense strand comprises about 25 to about 28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the two nucleotides at the 3' end of the sense strand are deoxyribonucleotides. The 5' end of the sense strand contains a phosphate ester. The antisense strand comprises about 26 to about 30 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides) and contains a 3' overhang of 1-4 nucleotides. The nucleotide containing the 3' overhang is modified with a 2'OMe-modified ribonucleotide. The antisense strand contains alternating 2'OMe-modified nucleotides, which begin in the first monomer of the antisense strand adjacent to the 3' overhang and extend 15-19 nucleotides from the first monomer adjacent to the 3' overhang. For example, for a 27-nucleotide antisense strand, and with the first base at the 5'-terminus of the antisense strand calculated as position number 1, the 2'OMe modification would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, and 27.

[0217] In some embodiments, the DsiRNA is long enough to be processed by chytozase to produce siRNA and has at least one of the following properties: (i) the DsiRNA is asymmetric, for example, having a 3'-protrusion on the sense strand; and (ii) the DsiRNA has a modified 3'-terminus on the antisense strand to guide the direction of chytozase binding and the processing of the DsiRNA into active siRNA. According to these embodiments, the sense strand contains about 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) and the antisense strand contains about 22 to about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the DsiRNA has a protrusion at the 3'-terminus of the sense strand. In some embodiments, the antisense strand is modified with a suitable modifier located at the 3'-terminus of the antisense strand for chytozase binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides and acyclic nucleotides, as well as sterically hindered molecules such as fluorescent molecules. When nucleotide modifiers are used, they replace the ribonucleotides in the DsiRNA, so that the length of the DsiRNA remains unchanged. In some embodiments, the DsiRNA has a 3'-end overhang in the sense strand and the antisense strand is modified for cleavage enzyme processing. In some embodiments, the antisense strand has a 5'-phosphate ester. The sense strand and antisense strand are annealed under biological conditions (such as those found in the cytoplasm of cells). Additionally, one of the sequences of the DsiRNA, particularly the region of the antisense strand, has a sequence of at least 19 nucleotides in length, wherein these nucleotides are adjacent to the 3'-end of the antisense strand and are sufficiently complementary to the nucleotide sequence of the RNA produced by the target gene. In addition, according to this implementation, DsiRNA may also have one or more of the following additional properties: (a) the antisense strand has a left shift relative to the typical 21-mer (i.e., the antisense strand contains nucleotides on the left side of the molecule compared to the typical 21-mer); and (b) the strand may not be perfectly complementary, i.e. the strand may contain simple mismatch pairings.

[0218] In a preferred embodiment, the DsiRNA has an asymmetric structure, with a sense strand of 25 base pairs and an antisense strand of 27 base pairs with a 3' overhang of 2 bases. In some cases, this asymmetric DsiRNA also contains two deoxyribonucleotides at the 3' end of the sense strand, replacing two ribonucleotides. In some other cases, this asymmetric DsiRNA also contains 2'OMe modifications at positions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand (where the first base at the 5' end of the antisense strand is position 1). In some further cases, this asymmetric DsiRNA also contains a 3' overhang on the antisense strand containing 1, 2, 3, or 4 2'OMe nucleotides (e.g., 3' overhangs of 2'OMe nucleotides at positions 26 and 27 of the antisense strand).

[0219] In another embodiment, dsiRNA can be designed by first selecting an antisense siRNA sequence of at least 19 nucleotides in length. In some cases, the antisense siRNA is modified to include about 5 to about 11 ribonucleotides at the 5' end to provide a length of about 24 to about 30 nucleotides. When the antisense strand has a length of 21 nucleotides, 3 to 9, preferably 4 to 7, or more preferably 6 nucleotides can be added to the 5' end. Although the added ribonucleotides may be complementary to the target gene sequence, complete complementarity between the target sequence and the antisense siRNA is not required. That is, the resulting antisense siRNA is sufficiently complementary to the target sequence. A sense strand of about 22 to about 28 nucleotides is then generated. The sense strand is substantially complementary to the antisense strand to anneal with the antisense strand under biological conditions. In one embodiment, the sense strand is synthesized to include a modified 3' end to guide cleatosylase processing of the antisense strand. In another embodiment, the antisense strand of the dsRNA has a 3' overhang. In a further embodiment, the sense strand is synthesized to include a modified 3'-terminus for cleavage and processing, and the antisense strand of the dsRNA has a 3'-protrusion.

[0220] In related embodiments, the antisense siRNA may be modified to include about 1 to about 9 ribonucleotides at its 5' end, providing a length of about 22 to about 28 nucleotides. When the antisense strand has a length of 21 nucleotides, 1 to 7, preferably 2 to 5, or more preferably 4 ribonucleotides may be added to the 3' end. The added ribonucleotides may have any sequence. Although the added ribonucleotides may be complementary to the target gene sequence, complete complementarity between the target sequence and the antisense siRNA is not required. That is, the resulting antisense siRNA is sufficiently complementary to the target sequence. A sense strand having about 24 to about 30 nucleotides is then generated. The sense strand is substantially complementary to the antisense strand to anneal with the antisense strand under biological conditions. In one embodiment, the antisense strand is synthesized to include a modified 3' end to guide cleavage enzyme processing. In another embodiment, the sense strand of the dsRNA has a 3' overhang. In a further embodiment, the antisense strand is synthesized to include a modified 3'-terminus for clematase binding and processing, and the sense strand of the dsRNA has a 3'-protruding end.

[0221] Further embodiments of the DsiRNA molecule relating to this disclosure, as well as methods for designing and synthesizing such DsiRNA molecule, are described in U.S. Patent Application Publications 2005 / 0244858, 2005 / 0277610, 2007 / 0265220, and 2011 / 0071208, the disclosures of which are incorporated herein by reference in their entirety for all purposes. l)piRNA

[0222] In some embodiments, the nucleic acid of the provided lipid nanoparticles comprises or is composed of piRNA. The piRNA molecule, which binds to a protein-bound piRNA molecule, can be about 10 to 50 nucleotides long, about 25 to 39 nucleotides long, or about 26 to 31 nucleotides long. See, for example, U.S. Patent Application Publication No. 2009 / 0062228.

[0223] piRNA molecules represent the largest class of small non-coding RNA molecules. piRNAs can be substantially complementary to target genes and can selectively form RNA-protein complexes through interactions with the Piwi or Aubergine subclasses of Argonaute proteins. These piRNA complexes are involved in epigenetic and posttranscriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly those involved in spermatogenesis. They differ from microRNAs (miRNAs) in size (typically 24–31 nt instead of 21–24 nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, particularly transposon silencing. Most piRNAs are antisense to transposon sequences, indicating that transposons are targets of piRNAs. In mammals, the activity of piRNAs in transposon silencing appears to be most important during embryonic development; in *C. elegans* and humans, piRNAs are essential for spermatogenesis. piRNAs play a role in RNA silencing by forming RNA-induced silencing complexes (RISCs). m) Downregulates target genes of nucleic acids

[0224] In some embodiments, the nucleic acid components (e.g., siRNA) of the lipid nanoparticles described herein can be used to downregulate or silence the translation (i.e., expression) of target genes. Target genes include, but are not limited to: genes associated with viral infection and survival; genes associated with metabolic diseases and conditions (e.g., liver diseases and conditions); genes associated with tumorigenesis and cell transformation (e.g., cancer); angiogenesis genes; immunomodulatory genes such as those associated with inflammatory and autoimmune responses; ligand receptor genes; and genes associated with neurodegenerative diseases. In some embodiments, the target gene is expressed in hepatocytes.

[0225] Genes associated with viral infection and survival include those expressed by the virus to bind to, enter, and replicate within cells. Of particular interest are viral sequences associated with chronic viral diseases.Viral sequences of particular interest include those of the following viruses: filamentous viruses, such as Ebola and Marburg (see, for example, Geisbert et al., J. Infect.Dis., 193: 1650-1657 (2006)); arenaviruses such as Lassa, Junin, Machupo, Guanareto, and Sabya (Buchmeier et al., Arenaviridae: the viruses and their replication, In: FIELDS VIROLOGY, Knipe et al. (eds.), 4th edition, Lippincott-Raven, Philadelphia, (2001)); and influenza viruses such as influenza A, influenza B, and influenza C (see, for example, Steinhauer et al., Annu Rev Genet., 36:305-332 (2002); and Neumann et al., J Gen Virol., 83:2635-2662). (2002)); Hepatitis viruses (see, for example, Hamasaki et al., FEBSLett., 543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Wilson et al., Proc.Natl.Acad.Sci.USA, 100:2783 (2003); Kapadia et al., Proc.Natl.Acad.Sci.USA, 100:2014 (2003); and FIELDSVIROLOGY, Knipe et al. (eds.), 4th edition, Lippincott-Raven, Philadelphia (2001)); Human immunodeficiency virus (HIV) (Banerjea et al., Mal.Ther., 8:62 (2003); Song et al., J. Viral., 77:7174 (2003); Stephenson, JAMA, 289:1494 (2003); Qin et al., Proc. Natl. Acad. Sci. USA, 100: 183 (2003)); herpesvirus (Jia et al., J. Virol., 77:3301 (2003)); and human papillomavirus (Hall et al., J. Virol., 77:6066 (2003); Jiang et al., Oncogene, 21:6041 (2002)).

[0226] Exemplary silencing filamentous virus nucleic acid sequences include, but are not limited to, nucleic acid sequences encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein (GP), VP24). Complete genome sequences for Ebola virus are listed in, for example, GenBank accessions NC-002549; AY769362; NC-006432; NC-004161; AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001; and AF086833. Ebola virus VP24 sequences are listed in, for example, GenBank accessions U77385 and AY058897. Ebola virus Lpol sequences are listed in, for example, GenBank accession X67110. The Ebola virus VP40 sequence is listed, for example, in Genbank accession number AY058896. The Ebola virus NP sequence is listed, for example, in Genbank accession number AY058895. The Ebola virus GP sequence is listed, for example, in Genbank accession number AY058898; Sanchez et al., VirusRes., 29:215-240 (1993); Will et al., J. Viral., 67:1203-1210 (1993); Volchkov et al., FEBS Lett., 305:181-184 (1992); and U.S. Patent No. 6,713,069. Additional Ebola virus sequences are listed, for example, in Genbank accession numbers LI 1365 and X61274. The complete genome sequence for Marburg virus is listed in, for example, GenBank accession numbers NC-001608; AY430365; AY430366 and AY358025. Marburg virus GP sequences are listed in, for example, GenBank accession numbers AF005734; AF005733 and AF005732. Marburg virus VP35 sequences are listed in, for example, GenBank accession numbers AF005731 and AF005730. Additional Marburg virus sequences are listed in, for example, GenBank accession numbers X64406; Z29337; AF005735 and Z12132. Non-limiting examples of siRNA molecules targeting Ebola virus and Marburg virus nucleic acid sequences include those described in U.S. Patent Application Publication No. 2007 / 0135370, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0227] Exemplary influenza virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding nucleoproteins (NPs), matrix proteins (M1 and M2), non-structural proteins (NS1 and NS2), RNA polymerases (PA, PBI, PBI2), neuraminidase (NA), and hemagglutinin (HA). Influenza A virus NP sequences are available in, for example, Genbank accession numbers NC-004522; AY818138; AB166863; AB188817; AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493; AY651494; AY651495; and AY651496. AY651497; AY651498; AY651499; AY651500; AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507; AY651509; AY651528; AY770996; AY790308; and AY818140 are listed. Influenza A virus PA sequences are listed, for example, in Genbank accession number AY818132; A The following are listed: Y790280; AY646171; AY818132; AY818133; AY646179; AY818134; AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611; AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615; AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995; and AY724786. Non-limiting examples of siRNA molecules targeting influenza virus nucleic acid sequences include those described in U.S. Patent Application Publication No. 2007 / 0218122, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0228] Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P) and nucleic acid sequences encoding structural proteins (e.g., core proteins including Cp proteins and Cp-related proteins; capsid proteins and envelope proteins including S proteins, M proteins, and / or L proteins, or fragments thereof) (see, for example, FIELDS VIROLOGY, ibid.). Exemplary hepatitis C virus (HCV) nucleic acid sequences that can be silenced include, but are not limited to, 5'-untranslated region (5'-UTR), 3'-untranslated region (3'-UTR), multiprotein translation start codon region, internal ribosome entry site (IRES) sequence, and / or nucleic acid sequences encoding the following proteins: core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protease / helicase, NS4A protein, NS4B protein, NS5A protein, and / or NS5B RNA-dependent RNA polymerase. HCV genome sequences are listed in, for example, Genbank accession numbers NC-004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC-009823 (HCV genotype 2), NC-009824 (HCV genotype 3), NC-009825 (HCV genotype 4), NC-009826 (HCV genotype 5), and NC-009827 (HCV genotype 6). The nucleic acid sequences of hepatitis A virus are listed, for example, in GenBank accession number NC-001489; the nucleic acid sequences of hepatitis B virus are listed, for example, in GenBank accession number NC-003977; the nucleic acid sequences of hepatitis D virus are listed, for example, in GenBank accession number NC-001653; the nucleic acid sequences of hepatitis E virus are listed, for example, in GenBank accession number NC-001434; and the nucleic acid sequences of hepatitis G virus are listed, for example, in GenBank accession number NC-001710. Silencing the sequences encoding genes associated with viral infection and survival can be conveniently combined with the administration of conventional agents used to treat viral symptoms. Non-limiting examples of siRNA molecules targeting hepatitis virus nucleic acid sequences include those described in U.S. Patent Application Publications Nos. 2006 / 0281175, 2005 / 0058982, and 2007 / 0149470; U.S. Patent No. 7,348,314; and U.S. Provisional Application No. 61 / 162,127, filed March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0229] Genes associated with metabolic diseases and conditions (e.g., conditions in which the liver is a target, and liver diseases and conditions) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors such as LXRa and LXR~ (Genbank accession number NM-007121), farnesoid X receptor (FXR) (Genbank accession number NM-005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), apolipoprotein B (APOB) (Genbank accession number 20 NM-000384), apolipoprotein CIII (APOC3) (Genbank accession numbers NM-000040 and NG-008949 REGION: 5001.8164) and apolipoprotein E (APOE) (Genbank accession numbers NM-000041 and NG-007084). REGION: 5001.8612); and genes expressed in diabetes (e.g., glucose-6-phosphatase) (see, for example, Forman et al., Cell, 81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995); Zavacki et al., Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272: 12778-12785 (1997); Willy et al., Genes Dev., 9: 1033-1045 (1995); Lehmann et al., J. Biol. Chem., 272:3137-3140). (1997); Janowski et al., Nature, 383:728-731 (1996); and Peet et al., Cell, 93:693-704 (1998). Those skilled in the art will appreciate that genes associated with metabolic diseases and conditions (e.g., diseases and conditions in which the liver is a target, and liver diseases and conditions) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. Silencing sequences encoding genes associated with metabolic diseases and conditions can be conveniently combined with the administration of conventional agents used to treat diseases or conditions. Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in U.S. Patent Application Publication No. 2006 / 0134189, the disclosure of which is incorporated herein by reference in its entirety for all purposes.Non-limiting examples of siRNA molecules targeting the ApoC3 gene include those described in U.S. Provisional Application No. 61 / 147,235, filed January 26, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0230] Examples of gene sequences associated with tumorigenesis and cellular transformation (e.g., cancer or other tumor formation) include mitotic kinases such as Eg5 (KSP, KIF11; Genbank accession number NM-004523); serine / threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank accession number NM-005030; Barr et al., Nat. Rev. Mal. Cell. Biol., 5:429-440 (2004)); tyrosine kinases such as WEE1 (Genbank accession numbers NM-003390 and NM-001143976); apoptosis inhibitors such as XIAP (Genbank accession number NM-001167); COP9 signaling subunits such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM-006837); CSN6, CSN7 A, CSN7B, and CSN8; ubiquitin ligases such as COPl (RFWD2; Genbank accession numbers NM-022457 and NM-001001740); and histone deacetylases such as HDAC1, HDAC2 (Genbank accession number NM-001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. Non-limiting examples of siRNA molecules targeting the Eg5 and XIAP genes include those described in U.S. Patent Application Serial No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Application Publications 2005 / 0107316 and 2007 / 0265438; and U.S. Patent Application Serial No. 12 / 343,342, filed December 23, 2008, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Non-limiting examples of siRNA molecules targeting the CSN5 gene include those described in U.S. Provisional Application No. 61 / 045,251, filed April 15, 2008, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0231] Other examples of gene sequences associated with tumorigenesis or cell transformation include translocation sequences such as MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-fLil, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al., Cancer Res. 63:1515 (2003)), and cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev., 16:2923 (2002)), β-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), telomerase gene (Kosciolek et al., Mal Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM-005228, NM-201282, NM-201283 and NM-201284; see also Nagy et al. Exp. Cell Res., 285:39-49 (2003), ErbB2 / HER-2 (Genbank accession numbers NM-004448 and NM-001005862), ErbB3 (Genbank accessions NM-001982 and NM-001005915) and ErbB4 (Genbank accessions NM-005235 and NM-001042599); as well as mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, Mal. Interventions, 2:158 (2002)). Non-limiting examples of siRNA molecules targeting the EGFR gene include those described in U.S. Patent Application Serial No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0232] Silencing sequences encoding DNA repair enzymes can be combined with the administration of chemotherapy agents (Collis et al., Cancer Res., 63: 1550 (2003)). Genes encoding proteins associated with tumor migration can also target sequences of interest, such as integrins, selection proteins, and metalloproteinases. The above examples are not exclusive. Those skilled in the art will understand that any whole or part of a gene sequence that is favorable to or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0233] Angiogenesis genes can promote the formation of new blood vessels. Of particular interest are vascular endothelial growth factor (VEGF) (Reich et al., Mal. Vis., 9:210 (2003)) or VEGFR. siRNA sequences targeting VEGFR are listed, for example, in GB 2396864; U.S. Patent Application Publication No. 2004 / 0142895; and CA 2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0234] Anti-angiogenic genes can inhibit the formation of new blood vessels. These genes are particularly useful for treating cancers in which angiogenesis plays a role in the pathological development of the disease. Examples of anti-angiogenic genes include, but are not limited to, endostatin (see, for example, U.S. Patent No. 6,174,861), angiostatin (see, for example, U.S. Patent No. 5,639,725), and VEGFR2 (see, for example, Decaussin et al., J. Pathol., 188: 369-377 (1999)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0235] Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory genes include, but are not limited to, cytokines such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas genes and Fas ligand genes are also target sequences for immunomodulation (Song et al., Nat. Med., 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic cells and lymphoid cells are also included in this disclosure, such as Tee family kinases like Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).

[0236] Cellular receptor ligands include those capable of binding to cell surface receptors (e.g., insulin receptor, EPO receptor, G protein-coupled receptor, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to regulate (e.g., inhibit, activate, etc.) physiological pathways involved by the receptors (e.g., glucose level regulation, blood cell development, mitosis, etc.). Examples of cellular receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc. Templates for amplification of trinucleotide repeat sequences (e.g., CAG repeat sequences) can be used to silence pathogenic sequences in neurodegenerative diseases caused by amplification of trinucleotide repeat sequences, such as spinal-medullobulal muscular atrophy and Huntington's disease (Caplen et al., Hum. Mal. Genet., 11:175 (2002)).

[0237] Other target genes whose expression can be downregulated or silenced by nucleic acids (e.g., siRNA) include, but are not limited to, actin, α2, smooth muscle, Aorta (ACTA2), alcohol dehydrogenase 1A (ADRIA), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), Afamin (AFM), angiotensinogen (AGT), serine-pyruvate aminotransferase (AGXT), α-2-HS-glycoprotein (AHSG), aldehyde-ketone reductase family I member C4 (AKR1C4), serum albumin (ALB), α-1-microglobulin / dikunin precursor (AMBP), angiopoietin-associated protein 3 (ANGPTL3), serum amyloid P fraction (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C3 (APOC3), apolipoprotein C-IV (APOC4), apolipoprotein F (APOF), β-2-glycoprotein 1 (APOH), and aquaporin 9. (AQP9), bile acid-CoA: amino acid N-acyltransferase (BAAT), C4b-binding protein β chain (C4BPB), putative uncharacterized protein encoded by LINC01554 (C5orf27), complement factor 3 (C3), complement factor 5 (C5), complement component C6 (C6), complement component C8 α chain (CSA), complement component C8 β chain (C8B), complement component C8 γ chain (C8G), complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), CXC motif chemokine 2 (CXCL2), Cytochrome P450 1A2 (CYP1A2), Cytochrome P450 2A6 (CYP2A6), Cytochrome P450 2C8 (CYP2C8), Cytochrome P450 2C9 (CYP2C9), Cytochrome P450 family 2 subfamily D member 6 (CYP2D6), Cytochrome P450 2E1 (CYP2E1), Phloroquinone ω-hydroxylase CYP4F2 (CYP4F2), 7-α-hydroxycholest-4-en-3-one 12-α-hydroxylase (CYP8B1), Dipeptidyl peptidase 4 (DPP4), Coagulation factor 12 (F12), Coagulation factor II (thrombin) (F2), Coagulation factor IX(F9), fibrinogen α chain (FGA), fibrinogen β chain (FGB), fibrinogen γ chain (FGG), fibrinogen-like 1 (FGL1), flavin-containing monooxygenase 3 (FMO3), flavin-containing monooxygenase 5 (FMO5), group-specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronic acid-binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxy acid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-associated protein; haptoglobin (HPR), hemoglobin-binding protein (HPX), histidine-rich glycoprotein (HRG), hydroxysteroid (11-5β) dehydrogenase 1 (HSD11Bl), hydroxysteroid (17-β) dehydrogenase 13 (HSD17Bl3), interstitial α-trypsin inhibitor heavy chain H1 (ITIH1), interstitial α-trypsin inhibitor heavy chain H2 (ITIH2), interstitial α-trypsin inhibitor heavy chain H3 (ITIH3), interstitial α-trypsin inhibitor heavy chain H4 (ITIH4), prokalopeptide-releasing enzyme (KLKB1), lactate dehydrogenase A (LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte-derived chemokine 2 (LECT2), lipoprotein(a) (LPA), mannan-binding lectin tadase 2 (MASP2), S-adenosylmethionine synthase isoform 1 (MAT1A), NADPH oxidase 4 (NOX4), poly[ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC), retinol dehydrogenase 16 (RDH16), serum amyloid A4, constitutive (SAA4), serine dehydratase (SDS), Serpin family A member 1 (SERPINA1), Serpin A11 (SERPINA11), kallikrein-statin (SERPINA4), corticosteroid-binding globulin (SERPINA6), antithrombin-III (SERPINC1), heparin cofactor 2 (SERPIND1), Serpin family H member 1 (SERPINH1), solute transporter family 5 member 2 (SLC5A2), sodium / bile acid cotransporter (SLC10A1), solute transporter family 13 member 5 (SLC13A5), solute transporter family 22 member 1 (SLC22A1), solute transporter family 25 member 47 (SLC25A47), solute transporter family 2, glucose transporter member 2 (SLC2A2), sodium-coupled neutral amino acid transporter 4(SLC38A4), solute transporter family 1B1 (SLCO1B1), sphingomyelin phosphodiesterase 1 (SMPD1), bile salt sulfonyltransferase (SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO2), UDP glucuronyltransferase 2 family, peptide B10 (UGT2B10), UDP glucuronyltransferase 2 family, peptide B15 (UGT2B15), UDP glucuronyltransferase 2 family, peptide B4 (UGT2B4), and vilinkin (VTN).

[0238] In addition to their use in silencing the expression of any of the genes described above for therapeutic purposes, certain nucleic acids (e.g., siRNAs) described herein also have applications in research and development, as well as diagnostic, preventative, prognostic, clinical, and other healthcare applications. As a non-limiting example, certain nucleic acids (e.g., siRNAs) can be used in target validation studies to test whether a gene of interest might be a potential therapeutic target. Certain nucleic acids (e.g., siRNAs) can also be used in target identification studies aimed at discovering genes that may be potential therapeutic targets. C. Pharmaceutical compositions and formulations

[0239] On the other hand, this disclosure provides a pharmaceutical composition comprising one or more lipid nanoparticles as described in Section B herein, or one or more ionizable lipids as described in Section B.2 herein. In some embodiments, the pharmaceutical composition further comprises a therapeutically effective amount of a pharmaceutically acceptable excipient. Additionally or alternatively, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0240] Pharmaceutically acceptable carriers (e.g., physiological saline or phosphate-buffered saline) can be selected based on the route of administration and standard pharmaceutical practice. Physiologically buffered saline (e.g., 135–150 mM NaCl) is typically used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and glycoproteins such as albumin, lipoproteins, and globulins, which enhance stability. Other suitable carriers are described, for example, in Remington: The Science and Practice of Pharmacy, 23rd Edition (2020). Pharmaceutically acceptable carriers are typically added after particle formation. Therefore, after particle formation, the particles can be diluted with a pharmaceutically acceptable carrier such as physiologically buffered saline.

[0241] Examples of suitable excipients for use with the provided pharmaceutical compositions include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, and polyacrylic acid, such as Carbopol, for example Carbopol 941, Carbopol 980, Carbopol 981, etc.

[0242] The provided pharmaceutical composition may additionally include lubricants such as talc, magnesium stearate, and mineral oil; humectants; emulsifiers; suspending agents; preservatives such as methylparaben, ethylparaben, and propylparaben (i.e., para-hydroxybenzoates); pH adjusters such as inorganic and organic acids and bases; sweeteners; colorants; and flavoring agents. The composition may also include biodegradable polymer beads, dextran, and cyclodextrins, inclusion complexes.

[0243] Pharmaceutical compositions that are liquid compositions, whether they are solutions, suspensions, or other similar forms, may also contain one or more of the following substances: sterile diluents, such as water for injection, saline solutions (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixing oils, such as synthetic mono- or diglycerides of glycerol, polyethylene glycol, glycerol, cyclodextrin, propylene glycol, or other solvents that can be used as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as amino acids, acetates, citrates, or phosphates; detergents, such as nonionic surfactants, polyols; and agents for adjusting tension, such as sodium chloride or glucose. Liquid compositions may include aqueous or oily suspensions or emulsions containing sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, glucose, or sterile aqueous solutions and similar drug carriers. Saline solutions are also commonly used for injection. Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives and vegetable oils can also be used. For example, by using coatings, such as lecithin, to maintain the desired particle size in the case of dispersions, and by using surfactants, appropriate flowability can be maintained.

[0244] The provided pharmaceutical composition for administration is preferably sterile. Antimicrobial activity can be achieved through various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The composition for administration can be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials.

[0245] For oral administration, the provided pharmaceutical composition may be in the form of tablets, lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays, powders, and sustained-release formulations. Suitable excipients for oral administration include pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0246] For topical application, the provided pharmaceutical composition may be in the form of emulsion, lotion, gel, cream, jelly, solution, suspension, ointment, and transdermal patch. For inhalation delivery, the composition may be delivered in dry powder form or in liquid form via a nebulizer. For parenteral administration, the composition may be in the form of sterile injectable solution and sterile packaged powder. Preferably, the injectable solution is formulated at a pH of about 4.5 to about 7.5.

[0247] The concentration of lipid nanoparticles in the provided pharmaceutical formulation can vary widely, for example from less than about 0.05% by weight, typically or at least about 2% by weight to about 5% by weight, up to about 10% by weight to about 90% by weight, and can be selected primarily by fluid volume, viscosity, etc., depending on the specific route of administration chosen. For example, the concentration can be increased to reduce the fluid load associated with treatment. This is particularly desirable in patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, lipid nanoparticles, which consist mainly of irritating lipids, can be diluted to a low concentration to reduce inflammation at the application site.

[0248] The provided pharmaceutical compositions can be sterilized using conventional, well-known sterilization techniques. Aqueous solutions can be packaged for use or filtered and lyophilized under aseptic conditions; the lyophilized formulation is mixed with the sterile aqueous solution prior to administration. When near-physiological conditions are required, the compositions may contain pharmaceutically acceptable excipients, such as pH adjusters and buffers, tonic modifiers, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Additionally, lipid nanoparticle suspensions may include lipid protectants to protect lipids from free radical and lipid peroxidation damage during storage. Lipophilic free radical quenchers such as α-tocopherol and water-soluble iron-specific chelating agents such as ferrichlor are suitable. D. Reagent kit

[0249] On the other hand, this disclosure provides kits to facilitate and / or standardize the use of the materials described herein (i.e., the lipid nanoparticles described in Section B, the ionizable lipids described in Section B.2, and / or the pharmaceutical compositions described in Section C), and to promote the methods described herein (i.e., the methods described in Section E). Materials and reagents for implementing these different methods may be provided in kit form to facilitate the implementation of these methods.

[0250] The provided kits may contain chemical reagents and other ingredients. Furthermore, kits containing the provided lipid nanoparticles, ionizable lipids, and / or pharmaceutical compositions may include, but are not limited to, instructions for kit users. For ease of storage and safe transport, the kits may also be packaged, for example, as ampoules or other vials in a box with a lid.

[0251] The provided kit may contain one or more containers, each partitioned to contain various components of the lipid nanoparticles (e.g., nucleic acids and individual lipid components of the nanoparticles). In some embodiments, the kit may also contain an endosome membrane destabilizer (e.g., calcium ions). The kit typically contains the lipid nanoparticle composition of this disclosure, preferably in dehydrated form, along with instructions for its rehydration and application. E. Method

[0252] In other respects, this disclosure provides several methods for manufacturing and / or using the lipid nanoparticles described in Section B, the ionizable lipids described in Section B.2, and / or the pharmaceutical compositions described in Section C. The methods disclosed herein benefit from the improved properties of the materials provided, such as properties favorable for nucleic acid delivery. These improved properties include, for example, enhanced targeting, optimized stability, and reduced toxicity and immunostimulation. 1. Introducing nucleic acids into cells

[0253] Some methods disclosed herein can be used to introduce nucleic acids into cells. These methods generally involve contacting cells with one or more lipid nanoparticles as described in Section B. The nucleic acid introduced into cells using lipid nanoparticles can be, for example, any nucleic acid described in Section B.5. Once formed, the lipid nanoparticles described herein are particularly suitable for introducing nucleic acids such as mRNA and / or siRNA into cells. The method is carried out in vitro or in vivo by first forming particles and then contacting the particles with cells for a period of time sufficient to deliver the nucleic acid into the cells.

[0254] The lipid nanoparticles described herein can be absorbed into virtually any cell type that mixes with or comes into contact with them. Once absorbed, the particles can be endocytosed by a portion of the cell, exchange lipids with the cell membrane, or fuse with the cell. The transfer or incorporation of the nucleic acid portion of the particle can occur through any of these pathways. Specifically, when fusion occurs, the particle membrane is incorporated into the cell membrane and the contents of the particle combine with intracellular fluid.

[0255] For in vitro applications, nucleic acids (e.g., mRNA and / or siRNA) can be delivered to any cultured and growing cells, whether plant or animal-derived, vertebrate or invertebrate, and of any tissue or type. In some embodiments, the cells are animal cells, such as mammalian cells, like human cells.

[0256] When performed in vitro, the contact between cells and lipid nanoparticles typically occurs in a biocompatible medium. The concentration of the particles can vary widely depending on the specific application, but is typically from about 1 μmol to about 10 mmol. Cell treatment with lipid particles is usually performed at physiological temperatures (about 37°C) for about 1 hour to about 48 hours. In some instances, the contact between cells and lipid nanoparticles lasts from about 2 to about 4 hours.

[0257] In some embodiments, a suspension of lipid nanoparticles is added to coated cells that are 60-80% confluent, at a cell density of approximately 102. 3 To about 10 5 cells / ml, for example, about 2 × 10⁻⁶ cells / ml 4 Cells / ml. The concentration of the suspension added to the cells can be from about 0.01 to 0.2 μg / ml, for example, about 0.1 μg / ml.

[0258] Endosomal release parameter (ERP) assays can be used to optimize the delivery efficiency of lipid particles. ERP assays are described in U.S. Patent Application Publication No. 20030077829. More specifically, the purpose of an ERP assay is to differentiate the roles of various cationic lipids and cooperating lipid components based on their relative effects on endosome membrane binding / absorption or fusion / destabilization. This assay allows for the quantitative determination of how each component of a lipid particle affects delivery efficiency, thereby optimizing the lipid particle. Typically, ERP assays measure the expression of reporter proteins (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.), and in some instances, lipid particle formulations optimized for expression plasmids are also suitable for encapsulating other types of nucleic acids such as mRNA. In some instances, ERP assays can be used to measure the downregulation of transcription or translation of target sequences in the presence or absence of interfering RNA (e.g., siRNA). In other instances, ERP assays can be used to measure the expression of target proteins in the presence or absence of mRNA. By comparing the ERP of each of various lipid particles, optimized systems, such as lipid particles with maximum uptake in cells, can be readily identified.

[0259] The compositions and methods described herein are used for in vivo and in vitro treatment of various cell types. Suitable cells include, for example, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, immune cells, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, dormant lymphocytes, terminally differentiated cells, slow-circulating or non-circulating primary cells, parenchymal cells, lymphoid cells, epithelial cells, osteoblasts, etc. In some embodiments, lipid particles containing nucleic acids (e.g., mRNA) are delivered to immune cells, such as antigen-presenting cells (e.g., dendritic cells, macrophages, B cells) and T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells). In some implementations, lipid particles containing nucleic acids (e.g., siRNA) are delivered to cancer cells, such as lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, stomach cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, kidney cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteosarcoma tumor cells, and leukemia cells.

[0260] In vivo delivery of liposome particles, such as LNPs encapsulating one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA), is suitable for targeting cells of any cell type. The methods and compositions can be used in cells of a variety of vertebrates, including mammals such as dogs, cats, horses, cattle, sheep, goats, rodents (e.g., mice, rats, and guinea pigs), rabbits, pigs, and primates (e.g., monkeys, chimpanzees, and humans).

[0261] Depending on the extent that cell culture may be required, it is well known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Edition, Wiley-Liss, New York (1994); Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross Inc. (1977), and the references cited therein provide general guidance on cell culture. Cultured cell systems are often presented as cell monolayers, but cell suspensions are also used. 2. Delivery of nucleic acid to the subject

[0262] Some methods provided in this disclosure can be used to deliver nucleic acids to a subject. These methods generally involve administering to a subject one or more lipid nanoparticles or one or more pharmaceutical compositions, wherein the lipid nanoparticles include those described in Section B, and the pharmaceutical compositions include those described in Section C. The nucleic acid delivered to the subject can be any nucleic acid, such as that described in Section B.5. In some embodiments, the lipid nanoparticles described herein are administered to the subject via systemic delivery, such as via a body system like circulation to distal target cells. In some embodiments, this disclosure provides fully encapsulated lipid particles that protect the nucleic acid from degradation by nucleases in serum, are non-immunogenic, small in size, and suitable for repeated administration.

[0263] In some embodiments, the ionizable lipids and lipid nanoparticles provided in this disclosure are particularly useful for preferentially delivering nucleic acids (e.g., RNA) to the liver of a subject. Specific configurations of the ionizable lipids and lipid nanoparticles described in Section B advantageously result in improved liver targeting of the material relative to other organs of the subject to which the material is administered. For example, in some embodiments, the provided ionizable lipids and lipid nanoparticles can be used to deliver nucleic acids encoding proteins to the liver of a subject such that the activity or secretion of the protein in the liver is greater than about 20-fold, greater than about 30-fold, greater than about 40-fold, greater than about 60-fold, greater than about 100-fold, greater than about 200-fold, greater than about 300-fold, greater than about 500-fold, greater than about 700-fold, or greater than about 1000-fold.

[0264] For in vivo administration, it can be administered in any manner known in the art, such as by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. Administration can be performed via a single dose or in multiple doses. The pharmaceutical composition can be administered parenterally, i.e., intra-articular, intravenous, intraperitoneal, subcutaneous, or intramuscularly. In some embodiments, the pharmaceutical composition is administered via bolus intravenous or intraperitoneal administration (see, for example, U.S. Patent No. 5,286,634). Intracellular nucleic acid delivery is also discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993). Other methods of administering lipid-based therapeutic agents are described, for example, in U.S. Patent Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. Lipid nanoparticles can be administered by direct injection at the disease site or by injection at a site remote from the disease site (see, for example, Culver, HUMAN GENE THERAPY, MaryAnn Liebert Inc., Publishers, New York. pp. 70-71 (1994)).

[0265] The compositions described herein, alone or in combination with other suitable components, can be formulated into aerosol formulations for administration via inhalation (e.g., intranasal or intratracheal) (i.e., they can be “nebulized”) (see, Brigham et al., Am. J. Sci., 298:278 (1989)). Aerosol formulations can be contained in pressurizable propellants such as dichlorodifluoromethane, propane, nitrogen, etc.

[0266] In some embodiments, the pharmaceutical composition can be delivered via nasal spray, inhalation, and / or other aerosol delivery media. Methods for directly delivering nucleic acid compositions to the lungs via nasal aerosol sprays have been described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticle resins and lysophosphatidylglycerol compounds (U.S. Patent 5,725,871) is well known in the pharmaceutical field. Likewise, transmucosal drug delivery in the form of a polytetrafluoroethylene carrier matrix is ​​described in U.S. Patent No. 5,780,045.

[0267] Preparations suitable for parenteral administration, such as those administered via intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In specific embodiments, the composition is administered intravenously (e.g., by intravenous infusion), intramuscularly, intrapulmonaryly, orally, topically, intranasally, intracerebrally, intraperitoneally, intravesically, or intrathecally.

[0268] Typically, when administered intravenously, liposome formulations are formulated with a suitable drug carrier. Many pharmaceutically acceptable carriers can be used in the compositions and methods described herein. Suitable formulations are found, for example, in Adejare, A. (Ed.). (2020). Remington: The Science and Practice of Pharmacy (23rd edition), Elsevier. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, etc., and may include glycoproteins such as albumin, lipoprotein, globulin, etc., to enhance stability. Physiologically buffered saline (135 mM–150 mM NaCl) is typically used as a pharmaceutically acceptable carrier, but other suitable carriers will also be acceptable. These compositions can be sterilized using conventional liposome sterilization techniques such as filtration. When near-physiological conditions are required, the composition may contain pharmaceutically acceptable excipients such as pH adjusters and buffers, tonicants, humectants, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc. These compositions can be sterilized using the techniques mentioned above, or alternatively, they can be produced under aseptic conditions. The resulting aqueous solution can be packaged for use or filtered and lyophilized under aseptic conditions. The lyophilized formulation is combined with the sterile aqueous solution prior to administration.

[0269] In some applications, the lipid particles described herein can be delivered to subjects via oral administration. The particles can be combined with excipients and used in the form of ingestible tablets, lozenges, troches, capsules, pills, sugar tablets, elixirs, mouthwashes, suspensions, oral sprays, syrups, crystals, etc. (see, for example, U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451). These oral dosage forms may also contain: binders, gelatin; excipients, lubricants, and / or flavorings. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the materials described above. Various other materials may be present as a coating or otherwise modify the physical form of the dosage unit. Of course, any materials used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts used.

[0270] Typically, these oral formulations may contain at least about 0.1% or more lipid particles, although the percentage of particles can vary and is generally between about 1% or about 2% and about 60% or about 70% or more of the total weight or volume of the formulation. Naturally, the amount of particles in each therapeutically useful composition can be prepared in a manner suitable for the dosage obtained at any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will take into account factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore may require a variety of dosages and treatment regimens.

[0271] Formulations suitable for oral administration may consist of: (a) liquid solutions, such as an effective amount of lipid particles containing nucleic acids (e.g., mRNA) suspended in a diluent such as water, saline, or PEG 400; (b) capsules, small capsules, or tablets, each containing a predetermined amount of lipid particles containing nucleic acids (e.g., mRNA) in liquid, solid, granular, or gelatinous form; (c) suspensions in suitable liquids; and (d) suitable emulsions. Tablet forms may contain one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silica, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, humectants, preservatives, flavorings, dyes, disintegrants, and pharmaceutically compatible carriers. Sugar tablets may contain lipid particles (containing nucleic acids, such as mRNA) in a flavoring agent (e.g., sucrose) or soft tablets containing lipid particles in an inert matrix (such as gelatin and glycerol or sucrose and gum arabic emulsions, gels, etc.), which, in addition to lipid particles, also contain a carrier known in the art.

[0272] In another instance of its use, lipid particles can be incorporated into a wide range of topical dosage forms. For example, suspensions containing lipid particles can be formulated and applied as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, etc.

[0273] When preparing pharmaceutical formulations of the lipid particles described herein, it is preferable to use a large quantity of purified particles to reduce or eliminate empty particles or particles with nucleic acids bound to the outer surface.

[0274] The methods described herein can be practiced in a variety of hosts. Preferred hosts include mammals such as primates (e.g., humans, chimpanzees, and other non-human primates), dogs, cats, horses, cattle, sheep, goats, rodents (e.g., rats and mice), rabbits, and pigs.

[0275] The amount of granules administered will depend on the ratio of nucleic acid (e.g., mRNA) to lipids, the specific nucleic acid used, the disease or condition being treated, the subject's age, weight, and condition, and the clinician's judgment, but is generally from about 0.01 mg / kg body weight to about 50 mg / kg body weight, from about 0.1 mg / kg body weight to about 5 mg / kg body weight, or about 10 mg / kg body weight per administration (e.g., injection). 8 -10 10 Each particle. 3. To treat diseases or symptoms

[0276] Some methods provided in this disclosure can be used to prevent or treat a disease or condition in a subject. These methods generally involve administering to a subject one or more lipid nanoparticles, one or more ionizable lipids, or one or more pharmaceutical compositions, wherein the lipid nanoparticles include those described in Section B, the ionizable lipids include those described in Section B.2, and the pharmaceutical compositions include those described in Section C. In some embodiments, treatment of a subject's disease includes reducing or eliminating one or more signs or symptoms of the disease.

[0277] In some embodiments, the methods described herein are used for the prevention or treatment of liver diseases or conditions. Due to the enhanced liver targeting of the ionizable lipids and lipid nanoparticles provided, for example, as described in Section E.2, these materials are particularly useful for the prevention or treatment of liver diseases, and / or for the prevention or treatment of diseases that provide desired therapeutic effects by modulating the expression and / or activity of one or more proteins in the liver. Non-limiting examples of liver diseases or conditions suitable for treatment with the provided methods include fatty liver diseases (e.g., alcoholic fatty liver disease (AFLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH)), cirrhosis, liver fibrosis, diseases or conditions characterized by elevated liver enzymes (e.g., alanine aminotransferase (ALT) or aspartate aminotransferase (AST)), simple steatosis, steatohepatitis, solid liver diseases, viral hepatitis, hepatocellular carcinoma, and any complications of these conditions (including, but not limited to, cardiac or metabolic diseases associated with NASH or NAFLD, portal hypertension or thrombosis, esophageal or gastric varices or bleeding from these varices, and other complications associated with liver diseases).

[0278] In certain implementations, the methods described herein are used to prevent or treat cancer. Cancer generally includes various malignant tumors characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new body sites. Non-limiting examples of different types of cancer that can be treated using the provided methods include ovarian cancer, breast cancer, lung cancer, bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, colorectal cancer, colon cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, rectal cancer, appendix cancer, small bowel cancer, stomach cancer, kidney cancer (i.e., renal cell carcinoma), central nervous system cancer, skin cancer, oral squamous cell carcinoma, choriocarcinoma, head and neck cancer, bone cancer, osteosarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, leukemia (e.g., acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or hairy cell leukemia), lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, B-cell lymphoma, or Burkitt lymphoma), and multiple myeloma. In some implementations, the cancer is an αvβ6 integrin-mediated disease or disorder. In some implementations, the cancer is lung cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, cutaneous squamous cell carcinoma, gastric cancer, or endometrial cancer. In some cases, the subject has a primary lesion (e.g., a primary tumor). In some cases, the subject has metastases (e.g., metastatic forms of any cancer type described herein). In some cases, the subject has both a primary lesion and metastases. In some implementations, the subject has pancreatic cancer, such as locally advanced or metastatic pancreatic cancer, locally advanced, unresectable or metastatic pancreatic cancer, or pancreatic ductal adenocarcinoma (PDAC).

[0279] In another implementation, the provided method can be used to treat infectious or contagious diseases caused by, for example, viruses, bacteria, fungi, parasites, or any other infectious agents. Non-limiting examples of contagious diseases suitable for treatment using the provided method include acquired immunodeficiency syndrome (AIDS / HIV) or HIV-related conditions, Albers syndrome, anthrax, bovine spongiform encephalopathy (mad cow disease), chickenpox, cholera, conjunctivitis, Creutzfeldt-Jakob disease (CJD), dengue fever, Ebola, elephantiasis, encephalitis, fatal familial insomnia, fifth disease, Gerstmann-Straussler-Scharinkel syndrome, Hantavirus, Helicobacter pylori, hepatitis (hepatitis A, hepatitis B, hepatitis C), herpes, influenza (e.g., avian influenza A), kuru, leprosy, Lyme disease. Diseases, malaria, hemorrhagic fevers (e.g., Rift Valley fever, Crimean-Congo hemorrhagic fever, Lassa fever, Marburg virus disease, and Ebola hemorrhagic fever), measles, meningitis (viral, bacterial), mononucleosis, nosocomial infections, otitis media, pelvic inflammatory disease (PID), plague, pneumonia, poliomyelitis, prions, rabies, rheumatic fever, erysipelas, Ross River virus infection, rubella, salmonellosis, septic arthritis, sexually transmitted diseases (STDs), herpes zoster, smallpox, streptococcal pharyngitis, tetanus, toxic shock syndrome, toxoplasmosis, trachoma, tuberculosis, tularemia, typhoid fever, grain fever, pertussis, and yellow fever.

[0280] In some embodiments, the provided method further includes obtaining a test sample from a subject. The test sample may include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof. In some embodiments, the provided method further includes determining the level of one or more biomarkers in the obtained test sample. As a non-limiting example, determining the presence or level of a biomarker can be used to determine a response to treatment or to select a suitable composition for the prevention or treatment of a disease.

[0281] In instances where the provided lipid nanoparticles or pharmaceutical compositions comprise nucleic acids encoding a target protein or a fragment thereof, the measured biomarker may be an indicator of the expression level of the protein or fragment in the target cell or target organism (e.g., the cell or organism to which the lipid nanoparticles or pharmaceutical composition are administered). For example, the biomarker may be the protein or fragment itself, such that the measured level of the protein or fragment indicates its expression level. When the target protein is an antigen, the measured biomarker may include or be composed of one or more components of the immune system of the target cell or target organism. For example, the biomarker may include one or more T cells, such as CD8. + T cells or cells composed of them. In these ways, determining the levels of biomarkers in test samples can provide information related to the efficiency and / or effectiveness of administering lipid nanoparticles to prevent or treat diseases or conditions.

[0282] In some embodiments, the provided method further includes comparing the measured levels of one or more biomarkers in the obtained test sample with the levels of the same one or more biomarkers in a reference sample. For example, a reference sample may be obtained from the subject prior to obtaining the test sample, such as before administering a therapeutically effective amount of the provided material to the subject. In this way, the reference sample can provide information about baseline levels of the biomarker in the pre-treatment sample, and the test sample can provide information about post-treatment biomarker levels.

[0283] Alternatively, reference samples can be obtained from, for example, different subjects, including those who have not received treatment according to the provided method. In this way, reference samples can provide information about baseline levels of untreated biomarkers, and test samples can provide information about levels of treated biomarkers. Reference samples can also be obtained from, for example, a group of subjects, including those who have not received treatment according to the provided method. In this way, reference samples can provide population-average information about baseline levels of untreated biomarkers, and test samples can provide information about levels of treated biomarkers.

[0284] Reference samples can also be obtained from individuals or groups of individuals treated according to the provided methods, and can be used as, for example, positive control samples. In some embodiments, reference samples are obtained from normal tissue. In some embodiments, reference samples are obtained from abnormal tissue.

[0285] Depending on the biomarker, an increase or decrease relative to a normal control or reference sample can indicate the presence of a disease or a response to treatment. In some embodiments, an increase in the biomarker level in the test sample is determined when the biomarker level is at least 1.1 times higher than the control, for example, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times higher, and thus the presence of a disease (e.g., an infectious disease or cancer), an increased risk of disease, or a response to treatment. In other embodiments, a reduction in the biomarker level in the test sample is determined when the biomarker level is at least 1.1 times lower than the control, for example, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times lower, and thus the presence of disease, an increased risk of disease, or a response to treatment is determined.

[0286] Biomarker levels can be detected using any method known in the art, including the use of antibodies specific to the biomarker. Exemplary methods include, but are not limited to, polymerase chain reaction (PCR), Western blotting, dot blotting, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, such as those using Luminex or fluorescent microbeads. In some cases, nucleic acid sequencing is used.

[0287] In some implementations, in immunoassays or PCR reactions (e.g., quantitative PCR), the presence of a decrease or increase in the level of one or more biomarkers is indicated by a detectable signal (e.g., blot, fluorescence, chemiluminescence, color, or radioactivity). This detectable signal can be compared with a signal or threshold from a reference sample.

[0288] In some implementations, the results of biomarker level determination are recorded in a tangible medium. For example, the results of diagnostic assays, such as the observation of the presence, reduction, or increase of one or more biomarkers, the presence of an increased risk or disease (e.g., an infectious disease or cancer), or a diagnosis of whether a subject has responded to treatment, may be recorded on, for example, paper or electronic media, such as audio tapes, computer disks, CD-ROMs, or flash drives.

[0289] In some implementations, the provided method also includes the step of providing the subject with diagnostic and / or treatment results. 4. Editing genome information

[0290] Some methods disclosed herein can be used to edit genetic information in cells, such as the genome. These methods typically involve contacting cells with one or more lipid nanoparticles, said lipid nanoparticles including those described in Section B, and said lipid nanoparticles containing one or more nucleic acids of the type described in Section B.5. In particular, nucleic acids or proteins encoded by nucleic acids can be components of a gene editing system.

[0291] Gene editing systems having one or more components can be any known in the art, said components being nucleic acids delivered by provided lipid nanoparticles, or proteins encoded by such nucleic acids. Exemplary gene editing systems include systems using sequence-specific nucleases, such as zinc finger nucleases, engineered or natural meganucleases, TALE-endonucleases, Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), and Natronobacterium gregoryi Argonaute (NgAgo)), or RNA-guided endonucleases (e.g., clustered regularly scattered short palindromic repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / CasX systems, CRISPR / CasY systems, or CRISPR / Cascade systems). In some instances, gene editing systems use single-stranded oligonucleotides to introduce precise base pair modifications into the genome.

[0292] In some embodiments, the provided gene editing method includes lipid nanoparticles containing nucleic acid-coding site-specific nucleases, any associated proteins, template sequences, and / or desired modification sequences for gene modification. In some embodiments, the method includes contacting cells with an expression cassette containing additional nucleic acids associated with additional components of the gene editing system.

[0293] In some embodiments, the gene editing system provided by the method is a system for site-directed integration. Several site-directed integration methods are known in the art involving different sequence-specific nucleases (or protein complexes or guide RNAs or both) that cleave genomic DNA to create double-strand breaks (DSBs) or gaps at desired genomic sites or loci. As is known in the art, in the process of repairing DSBs or gaps introduced by nucleases, donor template DNA, transgenes, or expression cassettes can be integrated into the genome at the site of the DSB or gap. In some embodiments, breaks or gaps in the target DNA sequence are repaired by natural processes of homologous recombination (HR) or non-homologous end joining (NHEJ). In some embodiments, sequence modifications occur at or near the cleavage or gap site, which may include deletions or insertions resulting in multinucleotide modifications, or integration of exogenous nucleic acids via homologous recombination or NHEJ. The presence of homologous arms in the DNA to be integrated can facilitate the insertion of the insert sequence and targeting of the plant genome during the repair process via homologous recombination (homologically directed recombination (HDR)), although the insertion event may occur via non-homologous end joining (NHEJ).

[0294] In some embodiments, site-specific genome-modifying enzymes modify the genome by inducing single-strand breaks. In some embodiments, site-specific genome-modifying enzymes modify the genome by inducing double-strand breaks. In some embodiments, recombination is promoted by providing a single-strand break inducer. In some embodiments, recombination is promoted by providing a double-strand break inducer. In some embodiments, recombination is promoted by providing a strand separation inducing agent. In one aspect, the site-specific genome-modifying enzyme is selected from endonucleases, recombinases, transposases, deaminases, helicases, or any combination thereof. In some embodiments, recombination occurs between chromosome B. In some embodiments, recombination occurs between chromosome B and chromosome A. In some embodiments, the site-specific genome-modifying enzyme includes cytidine deaminase. In some embodiments, the site-specific genome-modifying enzyme includes adenine deaminase. In some embodiments, the site-specific genome-modifying enzyme is a sequence-specific nuclease. a) RNA-guided nucleases

[0295] In some embodiments, the provided methods for editing genomic information in cells include contacting cells with lipid nanoparticles having nucleic acids associated with a gene editing system using RNA-guided nucleases. In some embodiments, the gene editing system uses at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten RNA-guided nucleases. In some embodiments, the site-specific gene editing system is a CRISPR / Cas system. CRISPR systems are based on RNA-guided engineered nucleases that use complementary base pairing to recognize DNA sequences at target sites. CRISPR (clustered, regularly spaced short palindromic repeats) / Cas (CRISPR-associated) systems are an alternative to synthesizing proteins whose DNA-binding domains enable them to modify genomic DNA at specific sequences (e.g., ZFNs and TALENs). In some instances, the site-specific gene editing system described herein may contain any RNA-guided Cas nuclease (non-restricted examples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas7, Cas8, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (also known as Csn1 and Csx12), Cas10, Cas10d, CasF, CasG, CasH, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (CsC5), Cas13, Cas13a). (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, C2c4, C2c8, C2c9, Csy1, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx11, Csx16, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cul966, Mad7, GSU0054, their homologues, or modified forms thereof; and optionally, the guide RNA (gRNA) necessary to target the corresponding nuclease.In some implementations, the CRISPR / Cas9 system, CRISPR / Cas12a system, CRISPR / Cas12e system, or CRISPR / Cas12d system can be used to generate modifications to nucleic acids. For example, the use of lipid nanoparticles to deliver Cas9 and guide RNA is described in International Patent Application Publication WO 2016 / 197133.

[0296] The CRISPR / Cas system is part of the adaptive immune system of bacteria and archaea, protecting them from nucleic acids such as viruses by cutting foreign DNA in a sequence-dependent manner. Immunity is achieved by integrating a short fragment of invading DNA (called a spacer sequence) between two adjacent repetitive sequences proximal to the CRISPR locus. The CRISPR array, including the spacer sequence, is transcribed upon subsequent encounter with invading DNA and processed into a small interfering CRISPR RNA (crRNA) approximately 40 nt in length. This crRNA binds to a trans-activating CRISPR RNA (tracrRNA), activating and directing the Cas9 nuclease. This cleaves a homologous double-stranded DNA sequence in the invading DNA, known as the protospacer sequence. A prerequisite for cleavage is the presence of a conserved protospacer sequence neighboring motif (PAM) downstream of the target DNA, typically with the sequence 5'-NGG-3', but less commonly NAG. Specificity is provided by a so-called "seed sequence" of approximately 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cas12a functions similarly to Cas9, but Cas12a does not require tracrRNA.

[0297] In some embodiments, the site-specific gene editing system includes a dCas9-Fok1 fusion protein. In other embodiments, the site-specific gene editing system includes a dCas9-recombinase fusion protein. As used herein, "dCas9" refers to a Cas9 endonuclease protein having one or more amino acid mutations that result in the Cas9 protein lacking endonuclease activity but retaining RNA-guided site-specific DNA binding. As used herein, "dCas9-recombinase fusion protein" is a dCas9 protein fused to dCas9 in a manner that allows the recombinase to catalyze DNA. In some embodiments, the site-specific gene editing system includes a dCas9-cytosine deaminase fusion protein. In another embodiment, the site-specific gene editing system includes a dCas9-adenine deaminase fusion protein. In some embodiments, the site-specific gene editing system includes a dCas12a-cytosine deaminase fusion protein. In other embodiments, the site-specific gene editing system includes a dCas12a-adenine deaminase fusion protein.

[0298] In some embodiments, the provided method can be used to introduce one or more gRNA molecules described herein into cells by contacting cells with the lipid nanoparticles described herein. The provided lipid nanoparticles can be used, for example, for therapeutic delivery of gRNAs that silence the expression of one or more target genes. In some embodiments, a mixture of gRNAs targeting different regions (e.g., overlapping and / or non-overlapping sequences) of a target gene or transcript is formulated into identical or different nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a human) requiring such treatment. In some embodiments, the mammal has been diagnosed with a specific disease or condition. In certain circumstances, a therapeutically effective amount of the nucleic acid-lipid particles can be administered to the mammal.

[0299] In some embodiments, gRNA can be produced by enzymatic or partial / full organic synthesis, and the modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. In some cases, gRNA is chemically prepared. Methods for synthesizing nucleic acid molecules are known in the art, such as the chemical synthesis methods described in Verma and Eckstein (1998) or described herein.

[0300] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, creating and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., ibid.; Ausubel et al., ibid.), and PCR methods are similarly well known in the art (see, U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., edited, 1990)). Expression libraries are also well known to those skilled in the art. Further foundational articles disclosing the general methods used in the provided embodiments include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition, 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., edited, 1994). The publicly available content of these references is incorporated into this paper in its entirety by reference for all purposes.

[0301] Preferably, the gRNA is chemically synthesized. Oligonucleotides comprising the gRNA molecules of this disclosure can be synthesized using any of the various techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of the oligonucleotides utilizes common nucleic acid protecting groups and coupling groups, such as dimethoxytriphenylmethyl at the 5'-terminus and phosphoramidite at the 3'-terminus. As a non-limiting example, small-scale synthesis can be performed on an Applied Biosystems synthesizer using a 0.2 pmol scale scheme. Alternatively, synthesis at a scale of 0.2 pmol can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA). However, synthesis at larger or smaller scales is also within the scope of this disclosure. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art. b) Recombinase

[0302] In some embodiments, the provided methods for editing genomic information in cells include contacting cells with lipid nanoparticles having nucleic acids associated with a gene editing system that uses a recombinase. Non-limiting examples of recombinases include tyrosine recombinases attached to the DNA recognition motifs provided herein, which are Cre recombinases, Gin recombinases, Flp recombinases, or Tnp1 recombinases. In some embodiments, the Cre or Gin recombinase is bound to a zinc finger DNA-binding domain. The Flp-FRT site-directed recombination system is derived from a 2 μ plasmid of *Saccharomyces cerevisiae*. In this system, the Flp recombinase (a flipper enzyme) recombines sequences between flipper enzyme recognition target (FRT) sites. Each FRT site contains 34 nucleotides. Flp binds to an “arm” of the FRT site (one arm is reversed) and cleaves the FRT site at either end of the inserted nucleic acid sequence. After cleavage, Flp recombines the nucleic acid sequence between the two FRT sites. Cre-lox is a site-directed recombination system derived from bacteriophage P1, similar to the Flp-FRT recombination system. Cre-lox can be used to reverse, delete, or translocate nucleic acid sequences. In this system, the Cre recombinase recombines a pair of lox nucleic acid sequences. The lox site contains 34 nucleotides, with the first 13 nucleotides and the last 13 nucleotides (arms) being palindromic. During recombination, the Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves them at the lox sites. The cleaved nucleic acids are spliced ​​together (reciprocal translocation), and recombination is complete. In other embodiments, the lox sites provided herein are loxP, lox 2272, loxN, lox 511, lox5171, lox71, lox66, M2, M3, M7, or M11 sites.

[0303] In some embodiments, the serine recombinase attached to the DNA recognition motif is a PhiC31 integrase, an R4 integrase, or a TP-901 integrase. In other embodiments, the DNA transposase attached to the DNA-binding domain is a TALE-piggyBac or a TALE-Mutator. c) Zinc finger nucleases

[0304] In some embodiments, methods provided for editing genomic information in cells include contacting cells with lipid nanoparticles containing nucleic acids associated with a gene-editing system using zinc finger nucleases (ZFNs). ZFNs are synthetic proteins composed of engineered zinc finger DNA-binding domains fused to the cleavage domains of a Fold restriction nuclease. ZFNs can be engineered to cleave virtually any long double-stranded DNA for modifying the zinc finger DNA-binding domains. ZFNs form dimers from monomers consisting of non-specific DNA-cleaving domains of a Fold nuclease fused to an array of zinc fingers engineered to bind target DNA sequences. The DNA-binding domains of ZFNs typically consist of an array of 3-4 zinc fingers. The amino acids at positions -1, +2, +3, and +6 relative to the initiation of the zinc finger helix contribute to site-specific binding to the target DNA and can be altered and customized to fit a specific target sequence. Other amino acids form a universal backbone, resulting in ZFNs with different sequence specificities. The rules for selecting ZFN target sequences are known in the art. Fold nuclease domains require dimerization to cleave DNA, thus requiring two ZFNs with C-terminal regions to bind to the opposing DNA strands (5-7 nt apart) at the cleavage site. If the two ZF binding sites are palindromic, the ZFN monomer can cleave the target site. As used herein, the term ZFN is broad and includes monomeric ZFNs that can cleave double-stranded DNA without the assistance of another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs designed to work together to cleave DNA at the same site. The use of lipid nanoparticles to deliver ZFN-associated nucleic acids is described, for example, in Conway et al., Mol Ther. 2019 Apr 10; 27(4):866-877.

[0305] Unrestricted by any scientific theory, the DNA-binding specificity of zinc finger domains can, in principle, be reengineered using a variety of methods, theoretically allowing for the construction of custom ZFNs to target virtually any gene sequence. Publicly available methods for engineering zinc finger domains include context-associated assembly (CoDA), oligopool engineering (OPEN), and modular assembly. d) Meganuclease

[0306] In some implementations, methods for editing genomic information in cells include contacting cells with lipid nanoparticles containing nucleic acids associated with a gene-editing system that uses megnucleases. Megnucleases, typically identified in microorganisms, are unique enzymes with highly active and long recognition sequences (>14 nt) that lead to site-specific digestion of target DNA. Engineered forms of naturally occurring megnucleases often have extended DNA recognition sequences (e.g., 14 to 40 nt). The engineering of megnucleases can be more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of megnucleases are intertwined within a single domain. Specialized mutagenesis and high-throughput screening methods have been used to generate novel megnuclease variants that recognize unique sequences and possess improved nuclease activity. e) Transcription activator-like effector nucleases

[0307] In some embodiments, the provided methods for editing genomic information in cells include contacting cells with lipid nanoparticles having nucleic acids associated with a gene editing system that uses transcription activator-like effector nucleases (TALENs). TALENs are artificial restriction enzymes produced by fusing a transcription activator-like effector (TALE) DNA-binding domain with a nuclease domain. In one aspect, the nuclease is selected from the group consisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. As used herein, the term TALEN is broad and includes monomeric TALENs that can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site. Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any DNA sequence, such as target sequences in nucleic acids encoding AUX / IAA proteins. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus *Xanthomonas*. During infection, pathogen X secretes TALE into host plant cells. TALE moves to the nucleus, where it recognizes and binds to specific DNA sequences in the promoter regions of specific genes within the host genome. TALE has a central DNA-binding domain comprising 13–28 repeating monomers, each monomer containing 33–34 amino acids. Except for the highly variable amino acid residues at positions 12 and 13, the amino acids in each monomer are highly conserved. These two variable amino acids are called repeating variable diresidues (RVDs). The amino acids of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine for NI, NG, HD, and NN, respectively. Adjusting RVDs allows for the recognition of consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allows for the engineering of specific DNA-binding domains by selecting combinations of repeating fragments containing appropriate RVDs. f) Other gene editing systems

[0308] In some implementations, methods for editing genomic information in cells include contacting cells with lipid nanoparticles containing nucleic acids associated with a gene editing system that uses base editing. Base editing is a genome editing method that produces precise point mutations directly in genomic DNA or cellular RNA without directly generating DSBs, without requiring a DNA donor template, and without relying on cellular HDR. The application of lipid nanoparticles in mediating base editing is described, for example, in Rothgangl et al., Nat Biotechnol. 2021 Aug; 39(8):949-957.

[0309] In some implementations, methods provided for editing genomic information in cells include contacting cells with lipid nanoparticles containing nucleic acids associated with a gene editing system that uses prime editing. Prime editing is a “search and replace: genome editing technique” that introduces all base-pair transitions, as well as small insertions and deletions, without requiring double-strand breaks (DSBs) or a donor DNA template. It involves reverse transcription to introduce corrections to the DNA genome. The application of lipid nanoparticles in mediating prime editing is described, for example, in Herrera-Barrera et al., AAPS J. 2023 Jun 28;25(4):65.

[0310] In some implementations, methods for editing genomic information in cells include contacting cells with lipid nanoparticles containing nucleic acids associated with a gene-editing system that uses programmable genome integration (PGI) and / or programmable addition of site-specific targeting elements (PASTE). These techniques utilize programmable genome integration of large, diverse DNA cargoes without exposing DNA double-strand breaks for DNA repair.

[0311] In some implementations, methods for editing genomic information in cells include contacting cells with lipid nanoparticles containing nucleic acids associated with a gene editing system, which uses transgene addition via cDNA synthesis insertion. 5. Preparation of lipid nanoparticles

[0312] Some methods provided in this disclosure can be used to prepare one or more lipid nanoparticles, including those described in Section B. Lipid nanoparticles can be prepared by, for example, sequential mixing methods and / or direct dilution methods. In some embodiments, the methods include selecting, designing, and / or preparing one or more components of the lipid nanoparticles being prepared. For example, the methods may include selecting, designing, and / or preparing phospholipids, ionizable lipids, cholesterol or derivatives thereof, conjugated lipids, and / or nucleic acids.

[0313] In some embodiments, the lipid nanoparticles described herein are produced by a continuous mixing method, for example, a process comprising the following steps: providing an aqueous solution containing a nucleic acid (e.g., any nucleic acid described in Section B.5) in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution is mixed with the aqueous solution, thereby substantially instantaneously producing lipid particles encapsulating the nucleic acid. This process and apparatus for carrying out the process are described in U.S. Patent Application Publication No. 2004 / 0142025.

[0314] In some instances, the organic lipid solution is an alcohol solution comprising an alcoholic organic solvent and lipids that will form lipid nanoparticles. These lipids may include one or more conjugated lipids, one or more ionizable lipids or pharmaceutically acceptable salts thereof, one or more sterols or derivatives thereof, and one or more phospholipids or pharmaceutically acceptable salts thereof. For example, conjugated lipids may be any of those described in Section B.4, ionizable lipids may be any of those described in Section B.2, sterols may be cholesterol or any derivatives thereof described in Section B.3, and phospholipids may be any of those described in Section B.1. In some embodiments, the alcoholic organic solvent of the alcohol solution comprises or is composed of ethanol. In some instances, the alcohol solution is a product of a combination of an alcoholic organic solvent and a phospholipid solution, wherein the phospholipid solution comprises a non-alcoholic organic solvent and one or more phospholipids or pharmaceutically acceptable salts thereof. In some embodiments, the non-alcoholic organic solvent comprises or is composed of tetrahydrofuran.

[0315] The continuous introduction of lipids and buffer solutions into a mixing environment, such as in a mixing chamber, causes the lipid solution to be continuously diluted with the buffer solution, thereby generating lipid particles essentially instantaneously upon mixing. As used herein, the phrase “continuous dilution of the lipid solution with a buffer solution” (and variations) generally refers to a sufficiently rapid dilution of the lipid solution during hydration with forces sufficient to achieve vesicle generation. In the presence of a buffer solution (i.e., an aqueous solution), the organic lipid solution undergoes continuous, stepwise dilution to generate lipid particles by mixing an aqueous solution containing nucleic acids with an organic lipid solution.

[0316] Lipid nanoparticles formed using a continuous mixing method typically have sizes of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. Therefore, the formed particles do not aggregate and the size is optionally set to achieve a uniform particle size.

[0317] In some embodiments, the lipid nanoparticles described herein are produced by a direct dilution method, which includes forming a lipid particle solution and immediately and directly introducing the lipid particle solution into a collection container containing a controlled amount of dilution buffer. In some embodiments, the collection container includes one or more elements configured to agitate the contents of the collection container to facilitate dilution. In one embodiment, the amount of dilution buffer present in the collection container is substantially equal to the volume of the lipid particle solution introduced therein. As a non-limiting example, introducing a solution of lipid particles in 45% ethanol into a collection container containing an equal volume of dilution buffer will advantageously produce smaller particles.

[0318] In some embodiments, the lipid nanoparticles described herein are produced via a direct dilution method, wherein a third reservoir containing a dilution buffer is fluidly connected to a second mixing zone. In this embodiment, the lipid particle solution formed in the first mixing zone is immediately and directly mixed with the dilution buffer in the second mixing zone. In some embodiments, the second mixing zone includes a T-connector arranged such that the lipid particle solution and the dilution buffer flow meet at opposite 180° flows; however, connectors providing a smaller angle, such as about 27° to about 180°, can be used. A pump mechanism delivers a controlled flow rate of buffer to the second mixing zone. In one embodiment, the flow rate of the dilution buffer supplied to the second mixing zone is controlled to be substantially equal to the flow rate at which the lipid particle solution is introduced from the first mixing zone. This embodiment advantageously allows for greater control over the flow rate of the dilution buffer mixed with the lipid particle solution in the second mixing zone, and therefore also control over the concentration of the lipid particle solution in the buffer throughout the second mixing process. This control over the flow rate of the dilution buffer advantageously allows for the formation of small particle sizes at reduced concentrations. U.S. Patent No. 9,005,654 describes examples of these processes and apparatus for implementing these direct dilution methods.

[0319] Lipid particles formed using direct dilution methods typically have sizes of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 nm to about 80 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0320] If desired, the size of the lipid particles described herein can be adjusted using any method available to those skilled in the art. Size can be set to achieve a desired size range and a relatively narrow particle size distribution. Various techniques are available for adjusting particles to the desired size, such as those described in Lam et al. 2023, Adv. Mat., 35:26, 2211420. One method for setting the size of particles for liposomes and equally applicable to the particles of this invention is described in U.S. Patent No. 4,737,323. Ultrasonication of the particle suspension by bath or probe ultrasound produces particles with a gradually decreasing size to less than about 50 nm. Homogenization is another method that relies on shear energy to break larger particles into smaller ones. In a typical homogenization process, particles flow through a standard emulsion homogenizer until a selected particle size, typically between about 60 nm and about 80 nm, is observed. In both methods, the particle size distribution can be monitored by conventional laser beam particle size identification or QELS.

[0321] Extruding particles through a microporous polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle size to a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired particle size distribution is achieved. Gradual size reduction can be achieved by extruding particles through a membrane with progressively smaller pores.

[0322] In some instances, the lipid particle solution formed using a direct dilution method is optionally concentrated. Concentration can be performed, for example, using ultrafiltration (e.g., tangential flow dialysis). Alternatively or additionally, concentration can be performed using centrifugation. The concentration of lipid particles can be increased, for example, by about 1-100 times, 3-70 times, about 5-50 times, or about 10-20 times. Alternatively or additionally, some or all of the organic solvent, such as alcoholic organic solvents, can be removed from the solution formed using the direct dilution method. Solvent removal can include, for example, exchanging the solvent with an aqueous solution (e.g., an aqueous buffer). In some embodiments, this buffer exchange is performed using dialysis. Optional solvent removal can be performed before or after optional concentration of the lipid particles. Alternatively or additionally, the solution formed using the direct dilution method can be filtered, wherein optional filtration can be performed before or after optional concentration of the lipid particles or optional removal of the organic solvent. Examples of these processes and apparatuses for carrying out these processes are described in U.S. Patent No. 9,005,654.

[0323] In some embodiments, the ratio (mass / mass ratio) of nucleic acid to lipids in the formed lipid particles is in the range of about 0.01 to about 0.2, about 0.02 to about 0.1, about 0.03 to about 0.1, or about 0.01 to about 0.08. The ratio of starting materials may also be within this range. In other embodiments, lipid particles are prepared using about 400 µg nucleic acid / 10 mg total lipids, or a nucleic acid to lipid mass ratio of about 0.01 to about 0.08, for example about 0.04, which corresponds to 1.25 mg total lipids / 50 µg nucleic acid. In some embodiments, the particles have a nucleic acid:lipid mass ratio of about 0.08.

[0324] In other embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio) in the formed lipid particles is about 1 (1:1) to about 100 (100:1), about 5 (5:1) to about 100 (100:1), about 1 (1:1) to about 50 (50:1), about 2 (2:1) to about 50 (50:1), about 3 (3:1) to about 50 (50:1), about 4 (4:1) to about 50 (50:1), about 5 (5:1) to about 50 (50:1), about 1 (1:1) to about 25 (25:1), about 2 (2:1) to about 25 (25:1), about 3 (3:1) to about 25 (25:1), about 4 (4:1) to about 25 (25:1), about 5 (5:1) to about 25 (25:1), about 5 (5:1) to about 20 (20:1), about 5 (5:1) to about 15 (15:1), about 5 (5:1) to about 10 (10:1), about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9 (9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1) or 15 (15:1). The ratio of starting materials can also be within this range. F. Exemplary Implementation

[0325] The following implementation schemes are envisioned. All combinations of features and implementation schemes are envisioned.

[0326] Implementation Scheme 1: A lipid nanoparticle comprising: a phospholipid or a pharmaceutically acceptable salt thereof, said phospholipid comprising one or more unsaturated tails and a head group having a positively charged nitrogen; and an ionizable lipid or a pharmaceutically acceptable salt thereof, said ionizable lipid having the following formula: (I), Where R 1 and R 2 Each is independently hydrogen, C 1-4Alkyl or 2- to 4-membered heteroalkyl groups, or combined with the nitrogen to which they are attached to form 3- to 8-membered heterocyclic groups, wherein the alkyl, heteroalkyl, and heterocyclic groups optionally have one or more substituents, and wherein each of the substituents is independently a hydroxyl group, C10 group, C2 ... 1-6 Hydroxyalkyl or fluorine; R 3 R 4 and R 5 Each is independently a C that optionally has one or more substituents. 5-20 Alkyl, C 5-20 alkenyl, C 5-20 alkynyl or C 5-12 Cycloalkyl, wherein each of the substituents is independently C10. 1-6 Alkyl or C 2-6 alkenyl; R 6 and R 7 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne groups, or groups that combine with the carbon atoms they are attached to to form cyclopropyl or cyclobutyl groups; R 8 and R 9 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne groups, or groups that combine with the carbon atoms they are attached to to form cyclopropyl or cyclobutyl groups; R 10 and R 11 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 Alkyne groups, or groups that combine with the carbon atoms they are attached to to form cyclopropyl or cyclobutyl groups; R 12 and R 13 They combine with the carbon atoms to form cyclopropyl or cyclobutyl groups, or each is independently hydrogen or optionally has one or more substituents. 1-6 Alkyl group, wherein each of the substituents is independently a hydroxyl group, C10, C2 ... 1-6 Hydroxyalkyl or fluorine; X 1 X 2 and X 3 Each is independently a covalent bond, C 1-6 Alkylene, C 1-6 imide or C 1-6 The alkynyl group; and n is an integer from 2 to 10.

[0327] Implementation Scheme 2: According to the implementation scheme of Implementation Scheme 1, each of the one or more unsaturated tails of the phospholipid has only one double bond.

[0328] Implementation Scheme 3: According to the implementation scheme of Implementation Scheme 1 or 2, each of the one or more unsaturated tails of the phospholipid is a cis-unsaturated tail.

[0329] Implementation Scheme 4: According to any one of Implementation Schemes 1-3, each of the one or more unsaturated tails of the phospholipid is C 10-20 Alkenyl group.

[0330] Implementation Scheme 5: According to any one of Implementation Schemes 1-4, wherein the phospholipid comprises phosphatidylcholine.

[0331] Implementation Scheme 6: According to the implementation scheme of Implementation Scheme 5, wherein the phospholipids include 1-palmitoyl-2-oleoyl-glycerol-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-diisooleoyl-sn-glycerol-3-phosphate choline, 1,2-bis[(8Z)octadecenoyl]-sn-glycerol-3-phosphate choline, 1,2-dimyristoleoyl-sn-glycerol-3-phosphate choline, 1,2-ditransmyristoleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-ditranspalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dicarboxyl-sn- Glyceryl-3-phosphate choline, 1,2-ditransoleoyl-sn-glyceryl-3-phosphate choline, 1,2-dilinoleoyl-sn-glyceryl-3-phosphate choline (DLPC), 1,2-dilinolenoyl-sn-glyceryl-3-phosphate choline, 1,2-bis(eicosenoyl)-sn-glyceryl-3-phosphate choline, 1,2-disorcinyl-sn-glyceryl-3-phosphate choline (DEPC), 1,2-diceryl-sn-glyceryl-3-phosphate choline, 1,2-bis(docohexanoyl)-sn-glyceryl-3-phosphate choline, 1-pentadecanoyl-2-oleoyl-sn-glyceryl-3-phosphate choline, 1-palmitoyl-2-linoleoyl-sn-glyceryl-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphate choline, Arachidonico-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-arachidonico-sn-glycerol-3-phosphate choline, 1-stearoyl-2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-myristoyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-stearoyl-sn-glycerol-3-phosphate choline, 1-(8Z-octadecenoyl) -2-palmitoyl-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-arachidonicyl-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-(8Z,11Z,14Z-eicoserienoyl)-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosalienoyl)-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1-O-hexadecyl-2-O-(9Z-octadecenyl)-sn-glycerol-3-phosphate choline, 1,2-Di-O-(9Z-octadecenyl)-sn-glycerol-3-phosphate choline, or combinations thereof.

[0332] Implementation Scheme 7: According to any one of Implementation Schemes 1-6, wherein the phospholipid comprises phosphatidylethanolamine.

[0333] Implementation Scheme 8: According to the implementation scheme of Implementation Scheme 7, wherein the phospholipids include 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-ditransoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di(docosahexaenoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1-pentadecanoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine, and 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine. Ethanolamine phosphate, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-arachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine, 1-O-hexadecyl-2-O-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine, or combinations thereof.

[0334] Implementation Plan 9: Based on any one of Implementation Plans 1-8, where R... 1 and R 2 Each is independently either hydrogen or C. 1-4 Alkyl groups, or those bonded to nitrogen atoms, to form 3- to 8-membered heterocyclic groups.

[0335] Implementation Plan 10: Based on the implementation plan of Implementation Plan 9, where R 1 and R 2 Each is C independently 1-3 alkyl.

[0336] Implementation Scheme 11: Based on the implementation scheme of Implementation Scheme 10, where R 1 and R 2 Each can be methyl, ethyl, or isopropyl.

[0337] Implementation Scheme 12: According to the implementation scheme of Implementation Scheme 11, where R1 and R 2 Each is a methyl group.

[0338] Implementation Scheme 13: Based on the implementation scheme of Implementation Scheme 9, where R 1 and R 2 They combine with the nitrogen atoms they are attached to to form 3- to 8-membered saturated heterocyclic groups, in which nitrogen is the only heteroatom.

[0339] Implementation Scheme 14: An implementation scheme according to any one of Implementation Scheme 13, wherein R 1 and R 2 They combine with the nitrogen atoms to form pyrrolidinyl or nitrogen-containing heterocyclic butyl groups.

[0340] Implementation Scheme 15: An implementation scheme based on any one of Implementation Schemes 1-14, wherein R 3 R 4 and R 5 Each is an independent cis-C with only one double bond. 5-20 Alkenyl group.

[0341] Implementation Scheme 16: According to the implementation scheme of Implementation Scheme 15, where R 3 R 4 and R 5 Each is an independent cis-C with only one double bond. 6-12 Alkenyl group.

[0342] Implementation Scheme 17: According to the implementation scheme of Implementation Scheme 15 or 16, wherein the double bond is at the 2, 3, 4 or 5 position of the alkenyl group.

[0343] Implementation Scheme 18: Based on the implementation scheme of Implementation Scheme 17, where R 3 R 4 and R 5 They are both cis-non-3-en-1-yl or cis-oct-3-en-1-yl.

[0344] Implementation Scheme 19: An implementation scheme based on any one of Implementation Schemes 1-18, wherein R 6 R 8 and R 10 Each is independently either hydrogen or C. 1-20 alkyl.

[0345] Implementation Scheme 20: Based on the implementation scheme of Implementation Scheme 19, where R 6 R 8 and R 10 Each of them is hydrogen, methyl, ethyl, or butyl.

[0346] Implementation Scheme 21: According to any one of Implementation Schemes 1-18, where R 6R 8 and R 10 Each is C independently 1-20 Alkenyl group.

[0347] Implementation Scheme 22: According to the implementation scheme of Implementation Scheme 21, where R 6 R 8 and R 10 Each is cis-dec-4-en-1-yl.

[0348] Implementation Scheme 23: According to any one of Implementation Schemes 1-22, where R 7 R 9 and R 11 They are both hydrogen.

[0349] Implementation Scheme 24: An implementation scheme based on any one of Implementation Schemes 1-23, wherein R 3 R 6 and R 7 The total amount of carbon in it; R 4 R 8 and R 9 The total amount of carbon in it; and R 5 R 10 and R 11 The total carbon content in each of them is independently between 7 and 12.

[0350] Implementation Scheme 25: An implementation scheme based on any...

Claims

1. A compound having the following formula or a pharmaceutically acceptable salt thereof, (I), in: R 1 and R 2 Each is independently hydrogen or C 1-4 Alkyl groups, or those bonded to nitrogen atoms to form 3- to 8-membered heterocyclic groups; R 3 R 4 and R 5 Each is independently a cis-C 5-20 alkenyl or C 5-20 Each alkynyl group has only one unsaturated bond; and n is an integer from 4 to 8.

2. The lipid nanoparticles according to claim 1, wherein R 3 R 4 and R 5 Each is an independent cis-C with only one double bond. 5-20 Alkenyl group.

3. The compound according to claim 1 or 2, wherein R 1 and R 2 Each is C independently 1-3 alkyl.

4. The compound according to claim 3, wherein R 1 and R 2 Each can be methyl, ethyl, or isopropyl.

5. The compound according to claim 4, wherein R 1 and R 2 Each is a methyl group.

6. The compound according to claim 1 or 2, wherein R 1 and R 2 They combine with the nitrogen atoms they are attached to to form 3- to 8-membered saturated heterocyclic groups, in which nitrogen is the only heteroatom.

7. The compound according to claim 6, wherein R 1 and R 2 They combine with the nitrogen atoms to which they are attached to form pyrrolidinyl or nitrogen-containing heterocyclic butyl groups.

8. The compound according to any one of claims 1-7, wherein R 3 R 4 and R 5 Each is an independent cis-C with only one double bond. 6-12 Alkenyl group.

9. The compound according to claim 8, wherein the double bond is at the 2, 3, 4 or 5 position of the alkenyl group.

10. The compound according to claim 9, wherein R 3 R 4 and R 5 They are both cis-non-3-en-1-yl or cis-oct-3-en-1-yl.

11. The compound according to any one of claims 1-10, wherein n is 5, 6, 7 or 8.

12. The compound according to claim 11, wherein n is 6 or 8.

13. The compound according to claim 12, wherein n is 6.

14. The compound according to claim 1, wherein the compound is: , , , , , , , , , , , , , , , , , , , , , , or .

15. A lipid nanoparticle comprising the compound according to any one of claims 1-14.

16. The lipid nanoparticles of claim 15, wherein the lipid nanoparticles have a pKa of 5.8 to 6.

9.

17. A lipid nanoparticle comprising an ionizable lipid or a pharmaceutically acceptable salt thereof, said ionizable lipid having the following formula: (I)， The lipid nanoparticles described herein have a pKa of 5.8 to 6.9, and wherein: R 1 and R 2 Each is independently hydrogen, C 1-4 Alkyl or 2- to 4-membered heteroalkyl groups, or combined with the nitrogen to which they are attached to form 3- to 8-membered heterocyclic groups, wherein the alkyl, heteroalkyl, and heterocyclic groups optionally have one or more substituents, and wherein each of the substituents is independently a hydroxyl group, C10 group, C2 ... 1-6 Hydroxyalkyl or fluorine; R 3 R 4 and R 5 Each is independently a C that optionally has one or more substituents. 5-20 Alkyl, C 5-20 alkenyl, C 5-20 alkynyl or C 5-12 Cycloalkyl, wherein each of the substituents is independently C10. 1-6 Alkyl or C 2-6 alkenyl; R 6 and R 7 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 The alkynyl group, or the alkynyl group, can combine with the carbon atoms to which they are attached to to form a cyclopropyl or cyclobutyl group, provided that R... 6 and R 7 No more than one of them is hydrogen; R 8 and R 9 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 The alkynyl group, or the alkynyl group, can combine with the carbon atoms to which they are attached to to form a cyclopropyl or cyclobutyl group, provided that R... 8 and R 9 No more than one of them is hydrogen; R 10 and R 11 Each is independently hydrogen, C 1-20 Alkyl, C 1-20 alkenyl or C 1-20 The alkynyl group, or the alkynyl group, can combine with the carbon atoms to which they are attached to to form a cyclopropyl or cyclobutyl group, provided that R... 10 and R 11 No more than one of them is hydrogen; R 12 and R 13 They combine with the carbon atoms to form cyclopropyl or cyclobutyl groups, or each is independently hydrogen or optionally has one or more substituents. 1-6 Alkyl group, wherein each of the substituents is independently a hydroxyl group, C10, C2 ... 1-6 Hydroxyalkyl or fluorine; X 1 X 2 and X 3 Each is independently a covalent bond, C 1-6 Alkylene, C 1-6 imide or C 1-6 etymynyl; and n is an integer from 2 to 10.

18. The lipid nanoparticles of claim 17, wherein the lipid nanoparticles further comprise nucleic acids.

19. The lipid nanoparticles of claim 18, wherein the nucleic acid comprises RNA.

20. The lipid nanoparticle of claim 19, wherein the RNA comprises mRNA.

21. The lipid nanoparticles according to any one of claims 18-20, wherein the nucleic acid encodes a protein.

22. The lipid nanoparticles of claim 21, wherein the protein is functional in hepatocytes or secreted by hepatocytes.

23. The lipid nanoparticles according to claim 21 or 22, wherein, after administration of the lipid nanoparticles to a subject, the activity or expression of the protein in the liver of the subject is at least 20 times higher than the activity or expression of the protein in the spleen of the subject.

24. The lipid nanoparticles according to any one of claims 18-23, wherein the nucleic acid or the protein encoded by the nucleic acid is a component of a gene editing system.

25. The lipid nanoparticles according to any one of claims 17-24, wherein R 1 and R 2 Each is C independently 1-3 alkyl.

26. The lipid nanoparticles according to claim 25, wherein R 1 and R 2 Each can be methyl, ethyl, or isopropyl.

27. The lipid nanoparticles according to claim 26, wherein R 1 and R 2 Each is a methyl group.

28. The lipid nanoparticles according to any one of claims 17-24, wherein R 1 and R 2 They combine with the nitrogen atoms they are attached to to form 3- to 8-membered saturated heterocyclic groups, in which nitrogen is the only heteroatom.

29. The lipid nanoparticles according to claim 28, wherein R 1 and R 2 They combine with the nitrogen atoms to form pyrrolidinyl or nitrogen-containing heterocyclic butyl groups.

30. The lipid nanoparticles according to any one of claims 17-29, wherein R 3 R 4 and R 5 Each is an independent cis-C with only one double bond. 6-12 Alkenyl group.

31. The lipid nanoparticles of claim 30, wherein the double bond is at the 2, 3, 4 or 5 position of the alkenyl group.

32. The lipid nanoparticles according to claim 31, wherein R 3 R 4 and R 5 They are both cis-non-3-en-1-yl or cis-oct-3-en-1-yl.

33. The lipid nanoparticles according to any one of claims 17-32, wherein R 6 R 8 and R 10 Each is independently hydrogen or C 1-20 alkyl.

34. The lipid nanoparticles according to claim 33, wherein R 6 R 8 and R 10 Each of them is hydrogen, methyl, ethyl, or butyl.

35. The lipid nanoparticles according to any one of claims 17-32, wherein R 6 R 8 and R 10 Each is C independently 1-20 Alkenyl group.

36. The lipid nanoparticles according to claim 35, wherein R 6 R 8 and R 10 Each is cis-dec-4-en-1-yl.

37. The lipid nanoparticles according to any one of claims 17-36, wherein R 7 R 9 and R 11 They are both hydrogen.

38. The lipid nanoparticles according to any one of claims 17-37, wherein R 3 R 6 and R 7 The total amount of carbon in it; R 4 R 8 and R 9 The total amount of carbon in; and R 5 R 10 and R 11 The total carbon content in each is independently between 7 and 12.

39. The lipid nanoparticles according to any one of claims 17-38, wherein R 12 and R 13 Each is hydrogen.

40. The lipid nanoparticles according to any one of claims 17-39, wherein X 1 X 2 and X 3 Each of them is either a covalent bond or a methylene group.

41. The lipid nanoparticles according to any one of claims 17-40, wherein n is 5, 6, 7 or 8.

42. The lipid nanoparticles according to claim 41, wherein n is 6 or 8.

43. The lipid nanoparticles according to claim 42, wherein n is 6.

44. The lipid nanoparticles according to any one of claims 17-43, wherein the lipid nanoparticles further comprise phospholipids or pharmaceutically acceptable salts thereof.

45. The lipid nanoparticles of claim 44, wherein the tails of the phospholipids are each saturated.

46. ​​The lipid nanoparticles of claim 45, wherein the phospholipid comprises 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

47. The lipid nanoparticles according to any one of claims 44-46, wherein the phospholipids or pharmaceutically acceptable salts thereof comprise 1 mol% to 20 mol% of the total lipids of the lipid nanoparticles.

48. The lipid nanoparticles according to any one of claims 17-47, wherein the ionizable lipid or a pharmaceutically acceptable salt thereof accounts for 30 mol% to 70 mol% of the total lipids of the lipid nanoparticles.

49. The lipid nanoparticles according to any one of claims 17-48, wherein the lipid nanoparticles further comprise cholesterol or a derivative thereof.

50. The lipid nanoparticles according to claim 49, wherein the cholesterol or its derivatives comprise 20 mol% to 60 mol% of the total lipids in the lipid nanoparticles.

51. The lipid nanoparticles according to any one of claims 17-50, wherein the lipid nanoparticles further comprise conjugated lipids.

52. The lipid nanoparticles of claim 51, wherein the conjugated lipid comprises polyethylene glycol (PEG)-lipid conjugates.

53. The lipid nanoparticles according to claim 51 or 52, wherein the conjugated lipids account for 0.1 mol% to 5 mol% of the total lipids of the lipid nanoparticles.

54. The lipid nanoparticles according to any one of claims 17-43, wherein the lipid nanoparticles comprise: The lipid nanoparticles contain 45 mol% to 60 mol% of ionizable lipids or pharmaceutically acceptable salts thereof, which constitute 45 mol% to 60 mol% of the total lipids present in the lipid nanoparticles. Phospholipids or pharmaceutically acceptable salts thereof, comprising 8 mol% to 12 mol% of the total lipids present in the lipid nanoparticles; Cholesterol or its derivatives comprising 30 mol% to 40 mol% of the total lipids present in the lipid nanoparticles; and The conjugated lipids comprise 0.5 mol% to 3 mol% of the total lipids present in the lipid nanoparticles.

55. The lipid nanoparticles according to any one of claims 17-43, wherein the lipid nanoparticles comprise: The lipid nanoparticles contain 52 mol% to 57 mol% of ionizable lipids or pharmaceutically acceptable salts thereof, which constitute 52 mol% to 57 mol% of the total lipids present in the lipid nanoparticles. Phospholipids or pharmaceutically acceptable salts thereof, comprising 10 mol% to 12 mol% of the total lipids present in the lipid nanoparticles; Cholesterol or its derivatives comprising 31 mol% to 34 mol% of the total lipids present in the lipid nanoparticles; and The conjugated lipids comprise 1 mol% to 3 mol% of the total lipids present in the lipid nanoparticles.

56. The lipid nanoparticles according to any one of claims 17-55, wherein, after the lipid nanoparticles are administered to a subject, the concentration of the ionizable lipids in the liver of the subject is less than 50% of the concentration of the ionizable lipids in the liver 24 hours after administration.

57. A pharmaceutical composition comprising: The lipid nanoparticles according to any one of claims 15-56, or the compound according to any one of claims 1-14; and Pharmaceutically acceptable carriers or pharmaceutically acceptable excipients.

58. A method for introducing nucleic acids into cells, the method comprising contacting the cells with lipid nanoparticles according to any one of claims 15-56.

59. The method of claim 58, wherein the cell is in a mammal.

60. The method of claim 59, wherein the cells comprise hepatocytes.

61. A method for in vivo delivery of nucleic acids, the method comprising administering lipid nanoparticles according to any one of claims 15-56 to a subject.

62. The lipid nanoparticles according to any one of claims 15-56, the compound according to any one of claims 1-14, or the pharmaceutical composition according to claim 57, for delivering nucleic acids to a subject.

63. Use of the lipid nanoparticles according to any one of claims 15-56, the compound according to any one of claims 1-14, or the pharmaceutical composition according to claim 57 in the preparation of a medicament for delivering nucleic acids into a subject.

64. A method for preventing or treating a disease or condition in a subject in need, the method comprising administering to the subject lipid nanoparticles according to any one of claims 15-56, a compound according to any one of claims 1-14, or a pharmaceutical composition according to claim 57.

65. The method of claim 64, wherein the disease or condition includes an infection, a liver disease or condition, or cancer.

66. The lipid nanoparticles according to any one of claims 15-56, the compound according to any one of claims 1-14, or the pharmaceutical composition according to claim 57, for the prevention or treatment of a disease or condition in a subject.

67. The lipid nanoparticles, compounds, or pharmaceutical compositions of claim 66, wherein the disease or condition includes infection, liver disease or condition, or cancer.

68. Use of the lipid nanoparticles according to any one of claims 15-56, the compound according to any one of claims 1-14, or the pharmaceutical composition according to claim 57 in the preparation of a medicament for the prevention or treatment of a disease or ailment of a subject.

69. The use according to claim 68, wherein the disease or condition includes infection, liver disease or condition, or cancer.

70. A method for editing genetic information in a cell, the method comprising contacting the cell with lipid nanoparticles according to claim 24.

71. The lipid nanoparticles according to claim 24, used for editing genomic information in cells.

72. Use of the lipid nanoparticles according to claim 24 in the preparation of a medicament for editing genomic information in cells.

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